1.1.1.2 alcohol dehydrogenase (NADP+) analysis the enzyme is utilized in a dehydrogenase-acetone NADP-regeneration system for the enzymatic preparative-scale production of 12-ketochenodeoxycholic acid, overview 1.1.1.6 glycerol dehydrogenase analysis glycerol dehydrogenase can be immobilised in a polycarbamoyl sulfonate-hydrogel and used as a sensor for glycerol 1.1.1.6 glycerol dehydrogenase analysis development of an integrated multienzyme electrochemical biosensor for the determination of glycerol in wines. The biosensor is based on the glycerol dehydrogenase/diaphorase bienzyme system. The enzyme system is immobilized together with the mediator tetrathiafulvalene on a 3-mercaptopropionic acid self-assembled monolayer-modified gold electrode by using a dialysis membrane 1.1.1.6 glycerol dehydrogenase analysis the recombinant chimeric fusion enzyme GDH-NOX has a potential application for quick glycerol analysis and dioxyacetone biosynthesis 1.1.1.6 glycerol dehydrogenase analysis assay for triglycerides. Triglycerides are hydrolysed to glycerol and fatty acids by lipoprotein lipase followed by the oxidation of glycerol to dihydroxyacetone with simultaneous production of NADH by glycerol dehydrogenase. Addition of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) to the reaction mixture removes NADH, allowing the reaction to complete while showing stoichiometric production of reduced WST-8. The reaction is linear up to 6.4mM, no interference by 2.5 g/l haemoglobin, 65 microM free bilirubin and 359 microM conjugated bilirubin is observed 1.1.1.6 glycerol dehydrogenase analysis GldA shows a strong intrinsic fluorescence at 320 nm, when excited at 280 nm. The fluorescence intensity decreases in the presence of NAD+, NADH, and dihydroxyacetone, the substrate and products for GldA, which allows to determine the dissociation constants for those molecules as 110.6 microM, 9,1 microM, 33.3 mM, respectively 1.1.1.6 glycerol dehydrogenase analysis accurate, simple and sensitive method for the quantitative analysis of triglycerides. Assay for triglycerides using glycerol dehydrogenase and a water-soluble formazan dye, WST-8 1.1.1.11 D-arabinitol 4-dehydrogenase analysis potential of using arabitol dehydrogenase from the non-virulent enteric bacterium, Escherichia colistrain C, as a plant selectable marker 1.1.1.12 L-arabinitol 4-dehydrogenase analysis enzymatic cycling assay for nicotinic acid adenine dinucleotide phosphate 1.1.1.17 mannitol-1-phosphate 5-dehydrogenase analysis application of CRISPRi-dCas9 technique for gene repression 1.1.1.18 inositol 2-dehydrogenase analysis determination of urinary myo-inositol by an improved enzymatic cycling method 1.1.1.25 shikimate dehydrogenase (NADP+) analysis usage of Escherichia coli shikimate dehydrogenase as sensor reaction for determination of the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae, quantitative measurements of physiological variables in the cytosolic compartment by GC-MS/MS, cytosolic NADPH/NADP ratio in batch experiments, overview. The steady state sensor reaction based cytosolic free NADPH/NADP ratio is 15.6 1.1.1.27 L-lactate dehydrogenase analysis CpLDH with APAD+ may be useful as a diagnostic tool for detection of protozoan parasite Cryptosporidium 1.1.1.27 L-lactate dehydrogenase analysis construction of multiplexed direct electron transfer-type lactate and glucose sensors using a fusion enzyme between L-lactate oxidase from Aerococcus viridans, A96L/N212K mutant, which is minimized in its oxidase activity and b-type cytochrome protein. The sensors achieve simultaneous detection of lactate and glucose without cross-talking error, with the detected linear ranges of 0.5-20 mM for lactate and 0.1-5 mM for glucose, sensitivities of 4.1 nA/mM x mm2 for lactate and 56 nA/mM x mm2 for glucose, and limit of detections of 0.41 mM for lactate and 0.057 mM for glucose 1.1.1.27 L-lactate dehydrogenase analysis reproducible and validated LDH assay optimized for several cell types for application in clinical medicine and biomedical sciences. Assay is cost effective and allows for experiment-specific optimization 1.1.1.30 3-hydroxybutyrate dehydrogenase analysis construction of a biosensor for amperometric sensing of beta-hydroxybutyrate using MXene nanosheets of type Ti3C2Tx modified with beta-hydroxybutyrate dehydrogenase. The MXene-based biosensor operates best at a potential of -0.35 V (vs. Ag/AgCl), displays a wide linear range (0.36 to 17.9 mM), a sensitivity of 0.480 microA per mM and cm, and a low detection limit (45 microM). The biosensor can be applied to the determination of beta-hydroxybutyrate in (spiked) serum samples 1.1.1.37 malate dehydrogenase analysis use of the thermostable enzyme from Vulcanithermus medioatlanticu DSM 14978 opens broad possibilities for the application of malate dehydrogenase as an analytical reagent and creation on biosensors for biochemical and cliniucal tests 1.1.1.40 malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) analysis squential fluorometric quantification of malic acid enantiomers in a single line flow-injection system using immobilized-enzyme reactors. An immobilized D -malate dehydrogenase (EC 1.1.1.83) reactor and an immobilized L-malate dehydrogenase (EC 1.1.1.40) reactor are introduced into the flowline in series. Sample and coenzyme (NAD+ or NADP+) are injected into the flow line by an open sandwich method. D -Malate is selectively oxidized by EC 1.1.1.83 when NAD+ is injected with a sample. When NADP+ is injected with a sample, L -malate is oxidized only by 1.1.1.40. NADH or NADPH produced by the immobilized-enzyme reactors is monitored fluorometrically at 455 nm 1.1.1.42 isocitrate dehydrogenase (NADP+) analysis simultaneous detection, quantitation and purification of glucose 6-phosphate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase by blue native gel electrophoresis 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] analysis enzyme can be used for glucose determination 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] analysis usage for quantitative determination of glucose in clinical tests and in the food industry 1.1.1.47 glucose 1-dehydrogenase [NAD(P)+] analysis use of glucose dehydrogenase in enzyme cycling method for measurement of allantoin in human serum 1.1.1.48 D-galactose 1-dehydrogenase analysis determination of lactose using a biosensor made of immobilized beta-galctosidase-galactose dehydrogenase fusion enzyme 1.1.1.48 D-galactose 1-dehydrogenase analysis determination of galactose in various hepatic diseases and galactosaemia in clinical biochemistry 1.1.1.50 3alpha-hydroxysteroid 3-dehydrogenase (Si-specific) analysis replacement of hsdA gene by the green fluorescent protein gene inserted downstream from the hsdA regulatory region and use of the resulting strain as fluorescence based biosensor system for steroid determination. With this cell-based system, testosterone can be determined in a range between 57 and 450 ng/ml, estradiol between 1.6 and 12.8 ng/ml, and cholesterol between 19.3 and 154.4 ng/nl. The sensitivity of this bioassay can be further increased by using only the cytosol of the mutant. With the resulting cell-free system testosterone is determined in a range between 28 and 219 pg/ml, estradiol between 0.029 and 0.430 fg/ml, and cholesterol between 9.7 and 77.2 fg/ml. The recovery ratio of the extraction is around 95% and the maximum fluorescence signals are obtained as early as after 30 min. Limitations of the established steroid biosensor system are quenching at higher steroid concentrations and the relatively high background of fluorescence 1.1.1.50 3alpha-hydroxysteroid 3-dehydrogenase (Si-specific) analysis engineering of 3alpha-hydrosteroid biosensor for androsterone determination by immobilizing the enzyme 3alpha-hydroxysteroid dehydrogenase in a composite electrode platform constituted of a mixture of multi-walled carbon nanotubes, octylpyridiniumhexafluorophosphate ionic liquid and NAD+ cofactor.This configuration allows the fast, sensitive and stable electrochemical detection of the NADH generated in the enzyme reaction. Reaction is linear for androsterone in the 0.5–10 microM concentration range. The detection limit achieved is 0.15 microM 1.1.1.B57 1-tetralone reductase [NADPH] analysis miniaturized assay for quantitative high-throughput screening, by monitoring NADPH oxidation as a decrease in A340 using a spectrophotometer 1.1.1.58 tagaturonate reductase analysis aldonate estimation 1.1.1.61 4-hydroxybutyrate dehydrogenase analysis dipstick assay for the detection of gamma-hydroxybutyrate in alcoholic beverages 1.1.1.67 mannitol 2-dehydrogenase analysis sensitive and specific photometric determination of mannitol in human serum 1.1.1.69 gluconate 5-dehydrogenase analysis enzymatic quantification of 5-keto-D-gluconate 1.1.1.83 D-malate dehydrogenase (decarboxylating) analysis sequential fluorometric quantification of malic acid enantiomers in a single line flow-injection system using immobilized-enzyme reactors. An immobilized D-malate dehydrogenase (EC 1.1.1.83) reactor and an immobilized L-malate dehydrogenase (EC 1.1.1.40) reactor are introduced into the flowline in series. Sample and coenzyme (NAD+ or NADP+) are injected into the flow line by an open sandwich method. D-Malate is selectively oxidized by EC 1.1.1.83 when NAD+ is injected with a sample. When NADP+ is injected with a sample, L -malate is oxidized only by 1.1.1.40. NADH or NADPH produced by the immobilized-enzyme reactors is monitored fluorometrically at 455 nm 1.1.1.85 3-isopropylmalate dehydrogenase analysis the cloned 3-isopropylmalate dehydrogenase gene can be used as a genetic marker in constructing vectors in Citrobacter freundii 1.1.1.85 3-isopropylmalate dehydrogenase analysis cloning of a fragment of DNA carrying the gene for 3-IMDH will be useful in the development of transformation methods in Candida albicans 1.1.1.85 3-isopropylmalate dehydrogenase analysis use of the cloned 3-isopropylmalate dehydrogenase gene for the development of a new host-vector system for cloning in Acetobacter aceti 1.1.1.93 tartrate dehydrogenase analysis determination of L-(+)-tartrate in wines and juices 1.1.1.93 tartrate dehydrogenase analysis quantification of L-tartrate in wine by a stopped-flow injection system with an immobilized enzyme reactor and fluorescence detection, the enzyme is immobilized on aminopropyl-controlled pore glass beads with glutaraldehyde 1.1.1.107 pyridoxal 4-dehydrogenase analysis the enzyme is useful in determination of vitamin B6 contents, method development, overview 1.1.1.108 carnitine 3-dehydrogenase analysis fluorometric determination of carnitine in serum with immobilized carnitine dehydrogenase and diaphorase 1.1.1.119 glucose 1-dehydrogenase (NADP+) analysis method for determination of D-glucose- and D-galactose levels in glycoconjugates. The NAD(P)H produced from the enzymatic oxidation of the monosaccharides reacts with a CuSO4-bathocuproinedisulfonic acid reagent to produce a color complex absorbing maximally at 486 nm. With galactose dehydrogenase and glucose dehydrogenase serving as the model enzymes, reaction analysis gives a linear plot from 2.5 to 250 nmol of sugar. Method has been applicated to sugar released by acid hydrolysis from lactose, porcine submaxillary mucin and raffinose was quantified 1.1.1.122 D-threo-aldose 1-dehydrogenase analysis determination of bound-fucose in biological materials by a coupled enzymatic method in a single buffer system 1.1.1.138 mannitol 2-dehydrogenase (NADP+) analysis determination of D-fructose in the presence of other sugars 1.1.1.146 11beta-hydroxysteroid dehydrogenase analysis high-throughput screening for potent and selective inhibitors of 11beta-hydroxysteroid dehydrogenase type I 1.1.1.146 11beta-hydroxysteroid dehydrogenase analysis use of microdialysis sampling coupled with liquid chromatography/electrospray ionization mass spectrometry to study 11beta-hydroxysteroid dehydrogenase type 1 catalyzed conversion of stable-isotope-labeled cortisone to cortisol in liver microsome. Results show species-specific reaction profiles, with a five times higher coversion rate in dog than in human and monkey liver microsome 1.1.1.149 20alpha-hydroxysteroid dehydrogenase analysis the enzyme is a marker for the murine X-zone 1.1.1.152 3alpha-hydroxy-5beta-androstane-17-one 3alpha-dehydrogenase analysis selective microquantitation of steroid substrates 1.1.1.159 7alpha-hydroxysteroid dehydrogenase analysis assay for stereospecific labeling of coenzymes NADP+ and NADPH 1.1.1.162 erythrulose reductase analysis assay development for microdetection of D-erythrulose in catabolic pathway analysis 1.1.1.205 IMP dehydrogenase analysis real-time reverse-transcription PCR assay for mRNA quantification of isoforms IMPDH1 and IMPDH2 in blood samples and cultured cells. Limits of detection and quantification are 10 and 1000 copies of cDNA per reaction, respectively 1.1.1.233 N-acylmannosamine 1-dehydrogenase analysis quantitative determination of N-acetylneuraminic acid 1.1.1.238 12beta-hydroxysteroid dehydrogenase analysis potential application in quantification of 12beta-hydroxyl groups in di- and trisubstituted bile acids 1.1.1.250 D-arabinitol 2-dehydrogenase analysis the specificity of the enzyme makes it useful for the development of a simple and specific method for the measurement of D-arabinitol, a metabolite of the pathogenic Candida spp. which has been described as a marker for disseminated candidiasis 1.1.1.261 sn-glycerol-1-phosphate dehydrogenase analysis structural and metabolic studies of bacterial and eucaryal phopholipids 1.1.1.270 3beta-hydroxysteroid 3-dehydrogenase analysis enzyme can be used for reconstitution of 4-methyl sterol demethylations of cholesterol biosynthesis from lanosterol 1.1.1.284 S-(hydroxymethyl)glutathione dehydrogenase analysis direct enzymatic assay of formaldehyde dehydrogenase permits screening of yeast colonies 1.1.1.284 S-(hydroxymethyl)glutathione dehydrogenase analysis the enzyme is useful for direct detection of formaldehyde in air by a novel NAD+- and glutathione-independent formaldehyde dehydrogenase-based biosensor, the sensor depends on the enzymatic conversion of the analyte to formic acid, method development, overview 1.1.1.284 S-(hydroxymethyl)glutathione dehydrogenase analysis the enzyme is useful in a formaldehyde-selective biosensor using NAD+- and glutathione-dependent recombinant formaldehyde dehydrogenase as a bio-recognition element immobilized on the surface of Si/SiO2/Si3N4 structure 1.1.1.284 S-(hydroxymethyl)glutathione dehydrogenase analysis usage of the enzyme in a bi-enzyme biosensor based on NAD+- and glutathione-dependent recombinant formaldehyde dehydrogenase activity and diaphorase activity for a formaldehyde assay, overview 1.1.1.333 decaprenylphospho-beta-D-erythro-pentofuranosid-2-ulose 2-reductase analysis developed of an assay method based on the visualization of mycobacterium replication within host cells and application for the search of compounds that are able to chase the pathogen from its hideout 1.1.1.341 CDP-abequose synthase analysis selection and use of primers to target defined regions of the abequose and paratose synthase genes responsible for biosynthesis of the oligosaccharide repeating units of the 0-antigenic lipopolysaccharide, in order to differentiate Salmonella serogroups. In a polymerase chain reaction assay utilizing these rjb-specific primers, all of the 40 salmonellae belonging to serogroups B, C2, and D plus A could accurately be identified among a total of 123 clinical isolates tested. No false-positive reactions were detected 1.1.1.374 UDP-N-acetylglucosamine 3-dehydrogenase analysis high-throughput chromatographic analysis of UDP–GlcNAc in a complex matrix of deproteinized cell extract 1.1.1.380 L-gulonate 5-dehydrogenase analysis YjjN can be applied for a quantitative L-galactonate and L-gulonate detection in a coupled reaction with diaphorase 1.1.1.414 L-galactonate 5-dehydrogenase (NAD+) analysis YjjN can be applied for a quantitative L-galactonate and L-gulonate detection in a coupled reaction with diaphorase 1.1.2.3 L-lactate dehydrogenase (cytochrome) analysis development of a amperometric biosensor selective to L-lactate, bioanalytical properties are very fast response and high sensitivity and selectivity 1.1.2.3 L-lactate dehydrogenase (cytochrome) analysis sensitive and stable visualization of enzyme activity in cell-free extracts or during purification by by formation of precipitates of Berlin blue 1.1.2.3 L-lactate dehydrogenase (cytochrome) analysis enzymatic oxidation of L-lactate catalyzed by flavocytochrome b2 and coupled with formazan production from nitrotetrazolium blue can be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry 1.1.2.3 L-lactate dehydrogenase (cytochrome) analysis lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells 1.1.3.2 L-lactate oxidase analysis development of a bienzyme fiberoptic sensor for flow injection analysis of L-lactate using lactate oxidase and peroxidase immobilized on a polyamide membrane. Hydrogen peroxide generated by lactate oxidase is substrate of peroxidase in presence of luminol. For the sensor strip, the detection limit is 250 pmol lactate, with 1.7% variation for 10 replicates using 6.25 nmol lactate 1.1.3.2 L-lactate oxidase analysis engineering the enzyme in order to minimize the effects of oxygen interference on sensor strips. Mutant A96L shows a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. After immobilization on a screen-printed carbon electrode and under argon or atmospheric conditions, the response current increases linearly from 0.05 to 0.5 mM L-lactate for both wild-type and mutant A96L. Under atmospheric conditions, the response of wild-type electrode is suppressed by 9-12% due to oxygen interference. The mutant maintains 56-69% of the response current at the same L-lactate level and minimizes the relative bias error to -19% from -49% of wild-type 1.1.3.4 glucose oxidase analysis coupling of the enzyme with Fenton's reagent used for the determination of glucose produced as a result of the hydrolysis of cellobiose catalyzed by beta-glucosidase 1.1.3.4 glucose oxidase analysis phosphate sensor consisting of glucose oxidase coimmobilized with glutaraldehyde with maltose phosphorylase and bovine serum albumin 1.1.3.4 glucose oxidase analysis enzyme immobilized in Bombyx mori silk fibroin membrane applied to glucose sensor 1.1.3.4 glucose oxidase analysis immobilized enzyme on polyacrylamide employed for the determination of glucose concentration in blood sera 1.1.3.4 glucose oxidase analysis biosensor system prepared for continuous flow analysis of enzyme activity 1.1.3.4 glucose oxidase analysis application in glucose biosensors. An unmediated, reagentless glucose biosensor is prepared with two polyethylenimine/glucose oxidase bilayers-modified pyrolytic graphite electrodes. A calibration linear range of glucose is 0.5-8.9 mM with a detection limit of 0.05 mM and sensitivity of 0.76 microA per mM 1.1.3.4 glucose oxidase analysis the enzym eis used in a model system to study physiological effects of hepatic H2O2 release on rat liver 1.1.3.4 glucose oxidase analysis the enzyme finds wide application in food industry and clinical analysis 1.1.3.4 glucose oxidase analysis the enzyme is useful in designing of biosensors for use in clinical, biochemical, and diagnostic assays 1.1.3.4 glucose oxidase analysis the enzyme might by useful in designing of biosensors for use in clinical, biochemical, and diagnostic assays 1.1.3.4 glucose oxidase analysis co-confined glucose oxidase and horseradish peroxidase bienzyme system as a biosensor for the detection of glucose gives a wider linear range of glucose than for free enzymes in solution 1.1.3.4 glucose oxidase analysis the enzyme is useful as biosensor for glucose detection 1.1.3.4 glucose oxidase analysis GOx is the main component in glucose biosensors for determination of glucose in industrial solutions and in body fluids such as blood and urine 1.1.3.4 glucose oxidase analysis te enzyme is used on electrode surfaces of biosensors 1.1.3.4 glucose oxidase analysis the enzyme GOx is applied in biosensor technologies 1.1.3.4 glucose oxidase analysis mutant glucose oxidase (B11-GOx) is obtained from directed protein evolution and wild-type enzyme. Higher glucose oxidation currents are obtained from B11-GOx both in solution and polymer electrodes compared to wild type enzyme. Improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with the mutant enzyme B11-GOx shows a faster electron transfer indicating a better electronic interaction with the polymer mediator. Promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells 1.1.3.B5 eugenol oxidase analysis development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method 1.1.3.6 cholesterol oxidase analysis platinum electrodes modified with thiolipid/lipid bilayer membranes. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. This electrode architecture may be useful in clinical sensing applications 1.1.3.6 cholesterol oxidase analysis the cholesterol oxidase-immobilized polyacrylonitrile hollow fiber may have an industrial or medical application for cholesterol determination or oxidation 1.1.3.6 cholesterol oxidase analysis the enzyme can be used as cholesterol biosensor co-immobilized with cholesterol esterase on oxygen electrodes, determination of total cholesterol in food samples 1.1.3.6 cholesterol oxidase analysis Cellulomonas enzyme is analytically reliable when used for serum cholesterol determination by the endpoint method. Its analytical performance is equivalent to Streptomyces enzymes and meets the analytical goals. It has an advantage over the other enzymes in that it does not ship in the frozen state 1.1.3.6 cholesterol oxidase analysis cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) can be immobilized onto tetraethylorthosilicate derived sol-gel films. These tetraethylorthosilicate sol-gel/ChEt/ChOx enzyme films can be used for the estimation of cholesterol oleate up to 780 mg/dl (mM). These TEOS sol-gel enzyme films provide selectivity towards glucose, lactate, uric acid and ascorbic acid 1.1.3.6 cholesterol oxidase analysis simple, reliable, fast and reproducible method to prepare cholesterol oxidase electrodes is described 1.1.3.6 cholesterol oxidase analysis the steady state amperometric measurements of free cholesterol are performed by an enzyme electrode which is developed through electrostatic immobilization of the cholesterol oxidase in poly(vinylferrocenium) perchlorate, PVF+ClO4-, film that is coated on a Pt electrode 1.1.3.6 cholesterol oxidase analysis the development of an enzyme-based sensor employing the enzyme ChOx has great potential as a simple and economical sensor system, engineering of the enzyme for electrochemical monitoring of cholesterol, overview 1.1.3.9 galactose oxidase analysis galactose oxidase adsorbed on, and covalently bound to, silica carriers is used for analytical determinations of D-galactose and galactose-containing sugars. Using a flowing oxygen electrode of the Clark-type, sensor system for enzymatic analysis of water solutions of galactose-containing carbohydrates is made. Measurements are taken both in the pulse and continuous modes of a substrate flowing through a column with an immobilized biocatalyst 1.1.3.9 galactose oxidase analysis galactose oxidase is an important component in electrochemical biosensors of galactose that are used for various biotechnology applications 1.1.3.9 galactose oxidase analysis the enzyme can be useful in biosensors 1.1.3.9 galactose oxidase analysis a photoelectrochemical biosensor for quantitative detection of galactose is obtained immobilizing galactose oxidase on TiO2 nanorod arrays modified F-doped tin oxide (FTO) electrode. The direct electron transfer to galactose oxidase is achieved. The generated photocurrent of the stable platform is significantly enhanced after the addition of galactose in solution and the photocurrent intensity shows linear relationship with the galactose concentration. CaCl2, uric acid and ascorbic acid have no interference with the detection of galactose. The sensor can be reused and applied to measure the concentration of galactose in lactose-free milk 1.1.3.10 pyranose oxidase analysis immobilization of enzyme on carbon nanotubes for application as enzymatic electrodes for enzyme-based biosensors and biofuel cells. The sensitivities of the covalent attachment, enzyme coating, and enzyme precipitate coating electrodes without 4-benzoquinone are 0.27, 0.76 and 3.7 mA/M/cm2, while covalent attachment, enzyme coating and enzyme precipitate coating electrodes in presence of 4-benzoquinone show 25, 25, and 60mA/M/cm2 of sensitivities, respectively. The maximum power densities of biofuel cells using covalent attachment, enzyme coating and enzyme precipitate coating electrodes without 4-benzoquinone are 41, 47 and 53 microW/cm2, while covalent attachment, enzyme coating and enzyme precipitate coating electrodes with 4-benzoquinone show 260, 330 and 500 microW/cm2, respectively 1.1.3.13 alcohol oxidase analysis enzyme may be useful for the colorimetric determination of methanol, ethanol, or other alcohols 1.1.3.13 alcohol oxidase analysis the enzyme is useful for construction of ethanol biosensors 1.1.3.13 alcohol oxidase analysis a dual biosensor analysis system based on alcohol oxidase and alcohol dehydrogenase for the simultaneous analysis of methanol–ethanol mixtures is developed. The alcohol dehydrogenase biosensor quantifies only the ethanol in the range 0.3-8 mmol/l without interference from methanol in concentrations as high as 100 mmol/l. The alcohol oxidase biosensor is able to respond to both analytes in the range 3-70 mmol/l for methanol and 15–110 mmol/l for ethanol. The concentration of ethanol and methanol from the sample is determined by processing analytical signals obtained from both biosensors 1.1.3.13 alcohol oxidase analysis amperometric sensor for ethanol based on one-step electropolymerization of thionine-carbon nanofiber nanocomposite containing alcohol oxidase. The ethanol biosensor can monitor ethanol ranging from 0.002 to 0.252 mM with a detection limit of 0.0017 mM. It displays a rapid response, an expanded linear response range as well as excellent reproducibility and stability 1.1.3.13 alcohol oxidase analysis an amperometric biosensor for ethanol monitoring is developed and optimised. The biosensor uses poly(neutral red), as redox mediator, which is electropolymerised on carbon film electrodes and alcohol oxidase from Hansenula polymorpha as recognition element, immobilised by cross-linking with glutaraldehyde in the presence of bovine serum albumin as carrier protein. The biosensor is used for the determination of ethanol in Portuguese red and white wines. No significant interferences were found from compounds usually present in wine 1.1.3.13 alcohol oxidase analysis AOD gene and isozyme structure may be used as a basis for reclassification of the methylotrophic yeasts 1.1.3.13 alcohol oxidase analysis selection procedure for isolation of the mutant forms of AOX, and their use as a suitable bioelement for biosensor technologies. The created biosensor based on mAOX (from the strain CA2) was characterised by a decreased affinity towards analyzed substrates and slightly increased Vmax. The operational stability of the mAOX-immobilised electrode is not affected and remains similar to an electrode based on the natural enzyme. The described methodology opens up the possibility for construction of biosensors appropriate for precise, rapid, and cheap analysis of target analytes, e.g. ethanol in real samples of wines, beers or fermentation cultures 1.1.3.13 alcohol oxidase analysis the enzyme is useful in alcohol biosensor applications 1.1.3.13 alcohol oxidase analysis fabrication of a self-powered ethanol biosensor comprising a beta-NAD+-dependent alcohol dehydrogenase bioanode and a bienzymatic alcohol oxidase and horseradish peroxidase biocathode. beta-NAD+ is regenerated by means of a toluidine blue modified redox polymer. The biofuel cell exhibits an open-circuit voltage of approximately 660 mV and can be used as self-powered device for the determination of the ethanol content in liquor 1.1.3.13 alcohol oxidase analysis the behaviour of commercially available AOx and ADH enzymes is studied in 12 different biosensor designs towards butanol-1 detection in liquid media. Four out of twelve proposed designs demonstrate a good signal reproducibility and linear response (up to 14.6 mM of butanol) under very low applied potentials (from -0.02 to -0.32 V) 1.1.3.15 (S)-2-hydroxy-acid oxidase analysis development of a lactate biosensor through immobilization of lactate oxidase in an albumin and mucin composed hydrogel by gluitaraldehyde cross-linking and trapping between two polycarbonate membranes. Hydrogen peroxide produced is detected on a platinum electrode. The response time of the sensor to 0.010 mM lactate requires 90 s to give a 100% steady-state response of 0.079 microA. Linear behavior is obtained between0.7 microM and 1.5 mM. The detection limit calculated from the signal to noise ratio was 0.7 microM 1.1.3.15 (S)-2-hydroxy-acid oxidase analysis construction of a D-Lactate electrochemical biosensor. The electrode for detection of D-lactate is prepared by immobilizing dye-linked D-LDH and multi-walled carbon nanotube within Nafion membrane. The electrode response to D-lactate is linear within the concentration range of 0.03-2.5 mM, and it shows little reduction in responsiveness after 50 days 1.1.3.17 choline oxidase analysis investigation of an acetylcholinesterase/choline oxidase-based amperometric biosensor as a liqid chromatography detector for acetylcholine determination in brain tissue 1.1.3.17 choline oxidase analysis two-enzyme sensor for determination of choline esters prepared by covalent co-immobilization of choline oxidase and butyrylcholinesterase 1.1.3.17 choline oxidase analysis the immobilized enzyme is used in amperometric biosensors for choline detection, method evaluation 1.1.3.17 choline oxidase analysis determination of lead ions by inhibition of choline oxidase enzyme using an amperometric choline biosensor. Choline oxidase is immobilized on a glassy carbon electrode modified with multiwalled carbon nanotubes through cross-linking with glutaraldehyde. In the presence of choline oxidase, choline is enzymatically oxidized into betaine at -0.3 V versus Ag/AgCl reference electrode, lead ion inhibition of enzyme activity causing a decrease in the choline oxidation current. Under the best conditions for measurement of the lowest concentrations of lead ions, the choline oxidase/multiwalled carbon nanotubes/glassy carbon electrode gives a linear response from 0.1 to 1.0 nM Pb2+ and a detection limit of 0.04 nM 1.1.3.17 choline oxidase analysis development of a metal composite material based on zirconium dioxide decorated gold nanoparticles (ZrO2 at AuNPs), copper (I) oxide at manganese (IV) oxide (Cu2O at MnO2) and immobilized choline oxidase (ChOx) onto a glassy carbon electrode (GCE) (ChOx/Cu2O at MnO2-ZrO2 at AuNPs/GCE) for enhancing the electro-catalytic property, sensitivity and stability of the amperometric choline biosensor. The ChOx/Cu2O at MnO2-ZrO2 at AuNPs/GCE displays a good electrocatalytic response to the oxidation of the byproduct H2O2 from the choline catalyzed reaction. The modified electrode also provides a wide linear range of choline concentration from 0.5 to 1000.0 microM with good sensitivity and low detection limit (0.3 microM). The apparent Michaelis-Menten constant is 0.08 mM with Imax of 0.67 microA. The choline biosensor presents high repeatability, good reproducibility, long time of use and good selectivity without interfering effects from possible electroactive species such as ascorbic acid, aspirin, amoxicillin, caffeine, dopamine, glucose, sucrose and uric acid 1.1.3.17 choline oxidase analysis facile and sensitive colorimetric biosensor based on DNAzyme-choline oxidase coupling used for the determination of choline. In this method, choline oxidase produces H2O2 and betaine in the presence of choline and oxygen, then, the DNAzyme converts colorless ABTS into green ABTS+ radicals. The linear range and the limit of detection of this biosensor are 0.1-25 microM and 22 nM. Choline measurement using this biosensor shows satisfactory selectivity and repeatability. Its recovery is 96.95-107.73% in biological samples 1.1.3.21 glycerol-3-phosphate oxidase analysis co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase on to aryl amine glass beads affixed on plastic strip for determination of triglycerides in serum 1.1.3.21 glycerol-3-phosphate oxidase analysis building of an improved amperometric triglyceride biosensor using lipase nanoparticles/glycerol kinase nanoparticles/glycerol 3-phosphate oxidase nanoparticles/pencil graphite electrode as the working electrode, Ag/AgCl as the standard electrode and Pt wirebas auxiliary electrode. The biosensor shows optimum response within 2.5 s at a pH 7.0 and temperature of 35°C. It measures current due to electrons generated at 0.1 V against Ag/AgCl, from H2O2, which is produced from triolein by coimmobilized enzyme nanoparticles. Analytical recovery of added triolein in serum is 98.01. The biosensor can be employed for determination of triglycerides in the serum of apparently healthy subject and persons suffering from hypertriglyceridemia 1.1.3.29 N-acylhexosamine oxidase analysis enzyme may by useful for measurement of N-acetyl-beta-D-glucosaminidase 1.1.3.38 vanillyl-alcohol oxidase analysis development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method 1.1.3.43 paromamine 6'-oxidase analysis development of a coupled enzyme assays including 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide and phenazine methosulfate, resulting in the production of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide formazan which can be monitored at 570 nm 1.1.3.44 6'''-hydroxyneomycin C oxidase analysis development of a coupled enzyme assays including 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide and phenazine methosulfate, resulting in the production of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide formazan which can be monitored at 570 nm 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) analysis application of mutant enzyme S231K as a glucose sensor constituent. The mutant has more than 8fold increase in its half-life during the thermal inactivation at 55 C compared with the wild-type enzyme and retains catalytic activity similar to the wild-type enzyme 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) analysis apoenzyme is used as a biological test system for the detection of very low amounts of pyrroloquinoline quinone 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) analysis enzyme is used as coupling enzyme for monitoring carbohydrate-transport reactions, the method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy-coupling mechanism or system with high-affinity constants for sugars 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) analysis because of its high turnover number, PQQ-GDH is proposed as an enzyme label for the development of sensitive electrochemical enzyme-amplified bioaffinity assays. It has, for example, been applied to the amperometric detection of DNA hybrids or sandwich DNA aptamers at the surface of a carbon electrode 1.1.5.2 glucose 1-dehydrogenase (PQQ, quinone) analysis its high stability at high temperature makes this enzyme potentially useful for applications in biosensors or biofuel cells 1.1.5.5 alcohol dehydrogenase (quinone) analysis construction and evaluation of an ethanol sensor based on the enzyme using direct electron-transfer processes between the polypyrrole entrapped quinohemoprotein alcohol dehydrogenase and a platinum electrode, overview 1.1.5.5 alcohol dehydrogenase (quinone) analysis the enzyme can be used in biosensors, method development, overview 1.1.5.5 alcohol dehydrogenase (quinone) analysis adhA expression is related to the ability to oxidize and grow on ethanol. Differential expression of pyrroloquinoline quinone–alcohol dehydrogenase could be a marker to analyse both growth and oxidation ability in some acetic acid bacteria, especially those of the genus Acetobacter 1.1.5.9 glucose 1-dehydrogenase (FAD, quinone) analysis possible use of the enzyme as amperometric glucose biosensor 1.1.5.9 glucose 1-dehydrogenase (FAD, quinone) analysis the enzyme can be used as O2-independent biosensor for glucoe detection 1.1.5.14 fructose 5-dehydrogenase analysis the enzyme is a satisfactory reagent for microdetermination of D-fructose 1.1.98.3 decaprenylphospho-beta-D-ribofuranose 2-dehydrogenase analysis developed of an assay method based on the visualization of mycobacterium replication within host cells and application for the search of compounds that are able to chase the pathogen from its hideout 1.1.99.4 dehydrogluconate dehydrogenase analysis enzymic microdetermination of 2-keto-D-gluconate 1.1.99.6 D-lactate dehydrogenase (acceptor) analysis construction of a D-Lactate electrochemical biosensor. The electrode for detection of D-lactate is prepared by immobilizing dye-linked D-LDH and multi-walled carbon nanotube within Nafion membrane. The electrode response to D-lactate is linear within the concentration range of 0.03-2.5 mM, and it shows little reduction in responsiveness after 50 days 1.1.99.6 D-lactate dehydrogenase (acceptor) analysis D-lactate content is of great interest for food analysis. A D-lactate sensing system using a thermostable dye-linked D-lactate dehydrogenase (Dye-DLDH) is developed. The electrode for detection of D-lactate is prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode is clearly observed upon exposure to D-lactate. The electrode response to D-lactate is linear within the concentration range of 0.03-2.5 mM, and it shows little reduction in responsiveness after 50 days 1.1.99.13 glucoside 3-dehydrogenase (acceptor) analysis amperometic enzyme electrode for sugar detection 1.1.99.18 cellobiose dehydrogenase (acceptor) analysis enzyme is used in highly selective amperometric biosensors 1.1.99.18 cellobiose dehydrogenase (acceptor) analysis cellobiose dehydrogense - electrode system for electrochemical applications 1.1.99.18 cellobiose dehydrogenase (acceptor) analysis development of biosensors, electrochemistry 1.1.99.18 cellobiose dehydrogenase (acceptor) analysis the enzyme has applications in lactose determination in food and as biosensor 1.1.99.18 cellobiose dehydrogenase (acceptor) analysis the enzyme is used in lactose determination in food 1.1.99.29 pyranose dehydrogenase (acceptor) analysis pyranose dehydrogenase is a promising candidate for enzymatic sensors of various sugars 1.2.1.3 aldehyde dehydrogenase (NAD+) analysis a reliable, enzyme-coupled assay for measuring glycerol dehydratase activity in crude-cell extract is developed using 1,2-propanediol as the substrate. In the assay, 1,2-propanediol is converted to propionaldehyde, which is quickly converted to 1-propionic acid by aldehyde dehydrogenase (with the production of NADH) or to 1-propanol by yeast alcohol dehydrogenase (with the consumption of NADH). The change in NADH concentration, as monitored at 340 nm spectrophotometrically, manifested as a straight line for 3 min, from which the glycerol dehydratase activity can be determined. Cells are assumed to have been disintegrated by physical methods (Bead Beater or French Press), not by chemical methods. The assay method should prove to be applicable to recombinant strains developed for the production of 3-hydroxypropionic acid and/or and/or 1,3-propanediol from glycerol 1.2.1.3 aldehyde dehydrogenase (NAD+) analysis spectrophotometric assay for the sensitive measurement of the glycerol dehydratase activity with a sub-nanomolar limit of detection. The assay method employs aldehyde dehydrogenase as a reporter enzyme, so the readout of the glycerol dehydratase activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-hydroxypropionaldehyde to 3-hydroxypropionic acid. The glycerol dehydratase assay is performed under the reaction conditions where the aldehyde dehydrogenase activity overwhelms the GDHt activity (i.e., 50fold higher activity of aldehyde dehydrogenase relative to glycerol dehydratase activity), affording sensitive detection of glycerol dehydratase with 360 pM limit of detection 1.2.1.5 aldehyde dehydrogenase [NAD(P)+] analysis ALDH is hereby proposed as a subtle nanoparticle determinant of kolaviron bioavailability and efficacy 1.2.1.12 glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) analysis GAPDH is a multi-functional protein that is used as a control marker for basal function, it is known to undergo cysteine oxidation under different types of cellular stress 1.2.1.46 formaldehyde dehydrogenase analysis presence of enzyme can be used as a selectable marker in DNA-mediated transformations 1.2.1.73 sulfoacetaldehyde dehydrogenase analysis the enzyme is useful for determination of sulfoacetaldehyde concentrations 1.2.1.104 pyruvate dehydrogenase system analysis sensitive and rapid assay procedures for human mitochondrial the pyruvate dehydrogenase (PDH) complex, the 2-oxoglutarate dehydrogenase (OGDH) complex and their 5 component enzymes, for use with crude tissue extracts 1.2.1.105 2-oxoglutarate dehydrogenase system analysis sensitive and rapid assay procedures for human mitochondrial the pyruvate dehydrogenase (PDH) complex, the 2-oxoglutarate dehydrogenase (OGDH) complex and their 5 component enzymes, for use with crude tissue extracts 1.2.3.3 pyruvate oxidase analysis rapid detection of phosphate using immobilized pyruvate oxidase 1.2.3.3 pyruvate oxidase analysis biosensor for inorganic phosphate 1.2.3.3 pyruvate oxidase analysis construction of an amperometric biosensor using nanoparticles of pyruvate oxidase, immobilized covalently onto pencil graphite electrode. The biosensor shows ideal working within 5 s under defined conditions of pH 6.0 and 30°C at an applied voltage of -0.1 V. Under standard assay conditions, a linear response is obtained between pyruvate concentration from 0.001 to 6000 microM and current. Lower detection limit is 0.58 microM, the biosensor can be used for over 210 days for the measurement of pyruvate in blood sera 1.2.3.3 pyruvate oxidase analysis use of nanoparticles of commercially available pyruvate oxidase covalently immobilized onto gold electrode as amperometric pyruvate biosensor for detection of pyruvate in serum. The biosensor shows optimum response within 7.5 s, at a potential of 0.28 V, pH 5.5 and 35°C and a lower detection limit of 0.67 microM. The analytical recovery of added pyruvate in sera is 99.0% and 99.5% within and between batches. The biosensor can be utilized for detection of total pyruvate level in sera of apparently healthy individuals and patients suffering from cardiogenic stress 1.2.3.4 oxalate oxidase analysis membrane inlet mass spectrometry assay to directly measure initial rates of carbon dioxide formation and oxygen consumption in the presence and absence of hydrogen peroxide 1.2.5.3 aerobic carbon monoxide dehydrogenase analysis construction of an enzyme-electrode based on carbon monoxide dehydrogenase (CODH) containing molybdenum (Mo) and copper (Cu), flavin adenine dinucleotide (FAD) and two different [2Fe-2S] clusters as cofactors, as a platform for dissolved CO concentration monitoring. The immobilized CODHs on Au electrode retain their catalytic activity and demonstrate changes to cyclic voltammetry and amperometry signals upon interactions with various dissolved CO. CODHs are capable of direct electron transfer without any mediator 1.2.7.1 pyruvate synthase analysis biochemical assay for activity, low-potential electrons are introduced by photochemical reduction of EDTA/deazaflavin and the generated pyruvate is trapped by chemical derivatization with semicarbazide. The product of CO2 fixation can be detected as pyruvate semicarbazone by HPLC-MS 1.2.7.4 anaerobic carbon-monoxide dehydrogenase analysis assay method for enzyme in complex with protein CooF which mediates electron transfer from enzyme to the CO-induced hydrogenase based on membranes containing high levels of CO-induced hydrogenase 1.2.7.5 aldehyde ferredoxin oxidoreductase analysis in situ generation of oxo-sulfidobis(dithiolene)tungsten(VI) complexes that model the proposed active-site structure for the AOR family of tungsten enzymes. The complexes are characterized by UV-vis, electrospray ionization mass spectrometry, IR, and resonance Raman spectroscopies and are found to mimic the coordination environment including the W=E (E=O, S) bond strengths and hydrolytic reactions of the tungsten center of the AOR family 1.2.7.8 indolepyruvate ferredoxin oxidoreductase analysis development of an effective affinity chromatography technique 1.2.7.11 2-oxoacid oxidoreductase (ferredoxin) analysis biochemical assay for activity, low-potential electrons are introduced by photochemical reduction of EDTA/deazaflavin and the generated pyruvate is trapped by chemical derivatization with semicarbazide. The product of CO2 fixation can be detected as pyruvate semicarbazone by HPLC-MS 1.3.1.1 dihydropyrimidine dehydrogenase (NAD+) analysis high performance liquid chromatography method for quantification of dihydrouracil to uracil ratio in plasma and application in screening for dihydropyrimidine dehydrogenase deficiency in patients treated with 5-fluorouracil. Plasma dihydrouracil to uracil ratio values are highly correlated with the plasma 5-fluorouracil-half-life values and are significantly associated with the toxic side effects, whereas, data set provided from genetic analysis of the coding sequences of the DPD gene are found to be insufficient to explain all the cases of the 5-fluorouracil-related toxicity pattern. Assay is suitable for routine clinical use for dihydropyrimidine dehydrogenase deficiency assessment in patients prior to 5-fluorouracil administration 1.3.1.2 dihydropyrimidine dehydrogenase (NADP+) analysis study on DPD enzyme expression using RT-PCR, immunohistochemistry, enzymatic activity and ELISA. Highest correlation is observed between protein expression measured by ELISA and enzyme activity, correlation of gene expression and ELISA is also significant 1.3.1.9 enoyl-[acyl-carrier-protein] reductase (NADH) analysis construction of tetracysteine-tagged enzyme variants carrying the tag at the N-terminus, C-terminus, or both N- and C-terminus. All the tetracysteine-tagged FabI enzymes have high enzyme activities while the enhanced green fluorescent protein-tagged FabI exhaustively loses the activity. The binding between 4',5'-bis(1,3,2-dithioarsolan-2-yl)fuorescein, i.e. FlAsH reagent and tetracysteine motif is stable against high pressure, high field strength, high temperature, and ultrasound. A capillary zone electrophoresis system equipped with a laser-induced fluorescence detector has a detection limit of 10-16 M for the labeled proteins 1.3.1.9 enoyl-[acyl-carrier-protein] reductase (NADH) analysis development of a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the enzyme's active site 1.3.1.19 cis-1,2-dihydrobenzene-1,2-diol dehydrogenase analysis Escherichia coli, which carry genes coding for benzene dioxygenase and benzene dihydrodiol dehydrogenase, can be used to monitor benzene pollution in environmental airsamples collected from an oil refinery. The procedures involving whole-cell bioassays determine the concentration of benzene through benzene dioxygenase activity, which allows for direct correlation of oxygen consumption, and through the benzene dihydrodiol dehydrogenase that causes catechol accumulation and restores NADH necessary for the activity of the first enzyme. The assay is sensitive enough to detect the benzene vapor at a concentration level of 0.01 mM in about 30 min. The assay is applicalble to on-line monitoring of benzene concentration, no particular treatment of environmental samples is required 1.3.1.22 3-oxo-5alpha-steroid 4-dehydrogenase (NADP+) analysis assay for evaluating 5alpha-reductase activity in large-scale clinical studies using the S9 fraction obtained by centrifugation of the homogenate supernatant at 9000 g for 30 min and gas chromatography-mass spectrometry 1.3.1.22 3-oxo-5alpha-steroid 4-dehydrogenase (NADP+) analysis radiosubstrate in vitro incubation method for the determination of 5alpha-reductase type 1 activity using rat liver microsomes as an enzyme source 1.3.1.33 protochlorophyllide reductase analysis POR is an ideal model for studying catalysis near the solvent glass transition 1.3.1.39 enoyl-[acyl-carrier-protein] reductase (NADPH, Re-specific) analysis sensitive assay method measuring the fluorescence decrease of NADPH as it is converted to NADP+ during the reaction, optimized for high-throughput screening 1.3.1.70 DELTA14-sterol reductase analysis partial purified enzyme suitable for reconstitution studies, reconstitution of isolated, soluble enzymes involved in cholesterol biosynthesis from lanosterol 1.3.1.103 2-haloacrylate reductase analysis monitoring the production of (S)-2-chloropropionate from 2-chloroacrylate as a model system for monitoring NADPH availability. A phosphofructokinase pfkA pfkB double-deletion strain shows the highest yield of 2-chloropropionic acid product. The flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis 1.3.1.103 2-haloacrylate reductase analysis monitoring the production of (S)-2-chloropropionate from 2-chloroacrylate as a model system for monitoring NADPH availability. The presence of transhydrogenase UdhA increases product yield and NADPH availability while the presence of transhydrogenase PntAB has the opposite effect. A maximum product yield of 1.4 mol product/mol glucose is achieved aerobically in a Pnt-deletion strain with UdhA overexpression, a 150% improvement over the wild-type strain 1.3.1.104 enoyl-[acyl-carrier-protein] reductase (NADPH) analysis development of a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the enzyme's active site 1.3.3.3 coproporphyrinogen oxidase analysis mass spectrometric assay for the two-step decarboxylative oxidation of coproporphyrinogen III to protoporphyrinogen IX catalyzed by CPO in mitochondria from human lymphocytes. The assay shows good reproducibility, uses simple workup by liquid-liquid extraction of enzymatic products, and employs commercially available substrates and internal standard. It was developed for use in clinical diagnostics of the inherited disorder hereditary coproporphyria 1.3.3.4 protoporphyrinogen oxidase analysis advantages of the continuous spectrofluorimetric assay over the discontinuous assay is of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples 1.3.3.4 protoporphyrinogen oxidase analysis advantages of the continuous spectrofluorimetric assay over the discontinuous assay is of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples, may be useful for assessing diminished PPO activities in variegate porphyria patient samples 1.3.3.5 bilirubin oxidase analysis enzyme activity in serum bilirubin assays can be monitored by capillary electrophoresis 1.3.3.5 bilirubin oxidase analysis immobilized onto aminopropyl and arylamine glass beads to form an enzyme bioreactor 1.3.3.5 bilirubin oxidase analysis sandwich-type enzyme-amplified amperometric detection of DNA with ambient O2 as the substrate at neutral pH in the presence of chloride 1.3.3.5 bilirubin oxidase analysis application of polyammonium cations to enzyme-immobilized electrode: application as enzyme stabilizer for bilirubin oxidase 1.3.3.5 bilirubin oxidase analysis application of poly[oxyethylene(dimethylimino)propyl-(dimethylimino)ethylene] as enzyme stabilizer for bilirubin oxidase immobilized electrode 1.3.3.5 bilirubin oxidase analysis BOD has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions 1.3.3.5 bilirubin oxidase analysis the ability to perform the oxygen reduction reaction under physiological conditions is the most interesting feature of the enzyme bilirubin oxidase in terms of the design of biocathodes for biofuel cell applications. The enzyme is commonly immobilized on a solid carrier to achieve the highest biocatalytic activity, stability, selectivity, and reusability. Optimization of the application of immobilized enzyme for direct electrocatalytic reduction of O2 1.3.3.6 acyl-CoA oxidase analysis useful for the determination of free fatty acids 1.3.3.6 acyl-CoA oxidase analysis amperometric propionate sensor 1.3.3.11 pyrroloquinoline-quinone synthase analysis the gene pqqC is a suitable molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads 1.3.7.5 phycocyanobilin:ferredoxin oxidoreductase analysis construction of a plasmid containing genes of apo-allophycocyanin alpha-subunit without chromophore and chromophore synthetases HO1, i.e. ferredoxin-dependent heme oxygenase, and PcyA, i.e. phycocyanobilin:ferredoxin oxidoreductase, and expression in Escherichia coli. Holo-allophycocyanin, i.e. allophycocyanin alpha-subunit with chromophore, can be synthesized by autocatalysis in Escherichia coli. Recombinant holo-allophycocyanin alpha-subunit shows the same spectral and fluorescent properties as phycocyanin and serves as a good substitute for native phycocyanin for fluorescent tagging. Recombinant allophycocyanin alpha-subunit can inhibit hydroxyl and peroxyl radicals more strongly than holo-allophycocyanin alpha-subunit and native allophycocyanin 1.3.7.8 benzoyl-CoA reductase analysis the detection of benzoyl-CoA reductase genes from bacterial pure cultures and environmental samples can be used to determine the genetic capability for anaerobic degradation of aromatic compounds and to monitor the anaerobic degradation of many different aromatic compounds in the environment. Sequence divergence of benzoyl-CoA reductase genes could be used to identify and distinguish among different bacterial populations degrading aromatic compounds in various environments. Microarray or real-time PCR amplification with specific primers for different types of benzoyl-CoA reductase genes could be applicable in environmental studies to determine which types are dominant and activated in particular environmental conditions and to evaluate the population response to variation in environmental factors. The first step in this approach is described 1.3.8.7 medium-chain acyl-CoA dehydrogenase analysis application of single exon and multiplex PCR protocol for sequencing based mutation screening of medium-chain acyl-CoA dehydrogenase and ornithine transcarbamylase genes. Both protocols give comparable resultswithout any re-design of the PCR primers or other optimization steps 1.3.98.1 dihydroorotate dehydrogenase (fumarate) analysis protocol to measure enzyme kinetic parameters based on isothermal titration calorimetry. Presence of dimethyl sulfoxide at 10%, v/v and Triton X-100 at 0.5%, v/v seems to facilitate the substrate binding process with a small decrease in KM value 1.3.99.6 3-oxo-5beta-steroid 4-dehydrogenase analysis determination of the concentration of chenodeoxycholic acid by an enzyme kit consisting of 3alpha-hydroxysteroid dehydrogenase, 3-keto-5beta-steroid-DELTA4-dehydrogenase, NAD+, nitroblue tetrazolium, diaphorase and some other reagents 1.3.99.23 all-trans-retinol 13,14-reductase analysis the mouse pluripotent P-19 cell metabolizes retinol to atRA and thus can be used in a cell-based screen for disruptors of the pathway. The disruption of the pathway is easily detected and quantitated, the P-19 cell provides an in vitro model system for identifying and exploring the mechanism of action of chemicals that interfere with the critical cellular pathway 1.3.99.29 phytoene desaturase (zeta-carotene-forming) analysis the engineered endogenous CrPDS L505F mutant is a dominant selectable marker for Chlamydomonas reinhardtii and possibly for other green algae 1.4.1.1 alanine dehydrogenase analysis determination of L-Ala 1.4.1.1 alanine dehydrogenase analysis alanine dehydrogenase from Streptomyces anulatus can be used as a bioreceptor in an ammonium biosensor that can detect ammonium ions in water samples. The sensor shows linear response in the range of 0.1-300mM NH4+ with the detection limit of 0.01 mM NH4+ and response time of 20 s. The sensor is showing good response at wide pH range (pH 5-11) and temperature range (20-50°C) suggesting its usage at ambient and non-ambient conditions. The sensor is successfully validated with Nessler's reagent method by using different water samples 1.4.1.1 alanine dehydrogenase analysis an enzymatic assay system to eliminate or measure D-Ala, which is reported to affect the taste of seafoods or sake, is constructed using alanine racemase (L-AlaR from Synechocystis sp. PCC6803) and L-alanine dehydrogenase (L-AlaDH from Phormidium lapideum). Using the assay system, the D-Ala contents of 7 crustaceans, i. e. mosa shrimp (Argis lar, Mosaebi), spiny lebbeid (Lebbeus groenlandicus, Oniebi), deepwater prawn (Pandalus eous,Amaebi), flathead lobster (Ibacus ciliates, Uchiwaebi), snow crab (Chionoecetes opilio, Zuwaigani), red snow crab (Chionoecetes japonicus, Benizuwaigani), and horsehair crab (Erimacrus isenbeckii, Kegani), are determined 1.4.1.2 glutamate dehydrogenase analysis evaluation of the C.Diff Quik Chek Complete Assay which tests for the presence of both glutamate dehydrogenase and toxins A and B. The assay allows 88% of specimens to be accurately screened as either positive or negative for the presence of toxigenic Clostridium difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant allows the easy, rapid, and highly sensitive and specific diagnosis of Clostridium difficile disease 1.4.1.2 glutamate dehydrogenase analysis gluD gene encoding glutamate dehydrogenase is highly conserved and glutamate dehydrogenase, which is used as marker for the presence of Clostridium difficile in fecal specimens, is readily expressed in vitro by all 77 Clostridium difficile ribotypes assayed. All ribotypes, including ARL 002, ARL 027, and ARL 106, are reactive in assays that detect Clostridium difficile glutamate dehydrogenase 1.4.1.2 glutamate dehydrogenase analysis the latex test-reactive protein is a glutamate dehydrogenase present in all isolates of Peptoclostridium difficile analyzed, suggesting that it is not related to pathogenicity 1.4.1.2 glutamate dehydrogenase analysis the protein that reacts in commercial latex tests for Clostridium difficile is a glutamate dehydrogenase 1.4.1.3 glutamate dehydrogenase [NAD(P)+] analysis development of a glutamate amperometric sol-gel biosensor. A carbon paste electrode is electrochemically modified with methylene green. NADP+ and glutamate dehydrogenase are coimmobilised in a sol-gel matrix. When coupled to a flow injection system, the biosensor shosd good electrocatalytic activity towards NADPH oxidation at a potential of + 0.3 V vs. Ag/AgCl. The biosensor yields a linear response from 50 microM to 10 mM glutamate, with a detection limit of 5 microM and reproducibility of results better than 2.3% 1.4.1.3 glutamate dehydrogenase [NAD(P)+] analysis simple and reliable staining procedure to detect GDH activity in plant tissues that can be used, with different purposes, to determine GDH expression in plant organs, tissues, extracts and also heterologous systems. The staining solution consists of Tris-HCl (pH 8.8), sodium glutamate, NAD, phenazine methosulfate and Nitro Blue Tetrazolium 1.4.1.4 glutamate dehydrogenase (NADP+) analysis preparation of glutamate sensors using NAD- and NADP-dependent glutamate dehydrogenase (GDH). Glutamate is determined from the anodic oxidation current of NADH and NADPH generated by the enzymatic reaction of GDH. Enzymes are deposited separately on carbon nanotube and reduced graphene oxide-modified glassy carbon electrodes 1.4.1.9 leucine dehydrogenase analysis postcolumn co-immobilized leucine dehydrogenase-NADH oxidase reactor for the determination of branched-chain amino acids by high-performance liquid chromatography with chemiluminescence detection 1.4.1.9 leucine dehydrogenase analysis assay of serum and urine for urea with use of urease and leucine dehydrogenase 1.4.1.9 leucine dehydrogenase analysis flow-injection determination of branched-chain L-amino acids with immobilized leucine dehydrogenase 1.4.1.9 leucine dehydrogenase analysis cheap and rapid determination of branched-chain amino and oxo acids 1.4.1.9 leucine dehydrogenase analysis high-performance liquid chromatographic determination of branched-chain alpha-keto acids in serum using immobilized leucine dehydrogenase as post-column reactor 1.4.1.15 lysine dehydrogenase analysis amperometric biosensor for L-lysine based on immobilized enzyme 1.4.1.18 lysine 6-dehydrogenase analysis highly specific determination of L-lysine concentration in serum 1.4.1.20 phenylalanine dehydrogenase analysis high yield synthesis of L-amino acids in presence of formate dehydrogenase: L-Phe from phenylpyruvate, L-Tyr from 4-hydroxyphenylpyruvate, L-Trp from indole-3-pyruvate, L-Met from 2-oxo-4-methylthiobutanoate, L-Val from 2-oxoisopentanoate, L-Leu from 2-oxoisohexanoate 1.4.1.20 phenylalanine dehydrogenase analysis the chimeric enzyme has a specific activity of 6% of that of the parental phenylalanine dehydrogenase 1.4.1.20 phenylalanine dehydrogenase analysis monitoring of phenylketonuria, colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase 1.4.1.20 phenylalanine dehydrogenase analysis enzymatic cycling assays for the determination of L-Phe and phenylpyruvate. Assay 1 couples glutamine transferase K with L-phenylalanine dehydrogenase. Assay 2 combines phenylanine dehydrogenase, L-amino acid oxidase, and catalase 1.4.1.20 phenylalanine dehydrogenase analysis production of optical and electrochemical sensors for phenylalanine whose molecular architecture can be controlled with precision using mixed stearic acid/phenylalanine dehydrogenase Langmuir films and their immobilization onto solid supports as Langmuir-Blodgett films. PheDH from the aqueous subphase enters the surfactant matrix up to an exclusion surface pressure of 25.3 mN/m, leading to stable and highly condensed mixed Langmuir monolayers. The enzyme fluidizes the stearic acid monolayers, reducing their maximum dipoles when condensed to their maximum, and disorganizes the alkyl chains of the fatty acid 1.4.1.21 aspartate dehydrogenase analysis development of a genetically encoded fluorescent protein construct for monitoring of L-Asp in vitro, and employment of aspartate dehydrogenase scaffold as a biorecognition element 1.4.1.21 aspartate dehydrogenase analysis usage of AspDH in the quantitative measurement of amino acids, 2-oxo acids, and ammonia or urea in studies involving clinical settings, bioprocess control, and nutrition 1.4.3.2 L-amino-acid oxidase analysis in-gel detection of L-amino acid oxidase activity. L-amino acid concentrations in the range of 2-5 mM lead to activity bands of similar intensities, while higher concentrations results in partial substrate inhibition. Complete supplement mixture containing adenine, uracil, and L-amino acids (Arg, Asp, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, Val, each at different concentrations) is optimized at concentrations of 0.1-0.5% in combination with 1 and 2 mM o-phenylendiamine and horseradish peroxidase concentrations of 0.1-0.8 U/ml 1.4.3.3 D-amino-acid oxidase analysis determination of D-amino acids 1.4.3.3 D-amino-acid oxidase analysis genotyping method development for establishing various pathologic-model animals under the complete care of their intrinsic DAO activity, which are useful for the screening of D-amino acids having physiological activity and/or diagnostic value 1.4.3.3 D-amino-acid oxidase analysis the enzyme is useful as a model FAD-containing protein 1.4.3.3 D-amino-acid oxidase analysis protocols for a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or alpha-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the alpha-keto acid production based on a chemical derivatization 1.4.3.3 D-amino-acid oxidase analysis sensitive assay method based on hydrogen peroxide production involving enzyme-coupled colorimetric assay with peroxidase 1.4.3.4 monoamine oxidase analysis kinetic assay for detection of MAO-A inhibitors in plant extracts using recombinant MAO-A expressed as GST-fusion protein in yesat 1.4.3.4 monoamine oxidase analysis on-line immobilized enzyme reactors can be used for the on-line screening of substances for MAO-A and substrate/inhibitor properties 1.4.3.4 monoamine oxidase analysis on-line immobilized enzyme reactors can be used for the on-line screening of substrances for MAO-A and substrate/inhibitor properties 1.4.3.4 monoamine oxidase analysis both (R)-(-)- and (S)-(+)-1-(1-[11C]methyl-1H-pyrrol-2-yl)-2-phenyl-2-(1-pyrrolidinyl)ethanone are very promising tracers for positron emission tomography of the MAO-A enzyme in brain. The carbon-11-labeling reaction is fairly simple and robust. Yields of more than 1 GBq are routinely obtained and with high specific activity of the final product. The metabolism of (R)-(-)- and (S)-(+)-1-(1-[11C]methyl-1H-pyrrol-2-yl)-2-phenyl-2-(1-pyrrolidinyl)ethanone is relatively slow in plasma of living pigs, in contrast to [11C]harmine, which is difficult to detect in plasma at times after 10 min. Parametric maps of [11C](R)-(-)- and (S)-(+)-1-(1-[11C]methyl-1H-pyrrol-2-yl)-2-phenyl-2-(1-pyrrolidinyl)ethanone binding are qualitatively very comparable to those of [11C]harmine 1.4.3.5 pyridoxal 5'-phosphate synthase analysis pyridoxamine phosphate oxidase assay applicable for measuring activity in erythrocytes 1.4.3.10 putrescine oxidase analysis specific measurement of putrescine with putrescine oxidase and aminobutyraldehyde dehydrogenase 1.4.3.10 putrescine oxidase analysis differential determination procedure for putrescine, spermidine and spermine 1.4.3.10 putrescine oxidase analysis enzyme can be used as biosensor for putrescine using dispersed multiwalled carbon nanotubes as ultra-microelectrodes ensuring a good electron transfer from the enzyme, method optimization, overview 1.4.3.11 L-glutamate oxidase analysis specific and sensitive determination of L-glutamate 1.4.3.11 L-glutamate oxidase analysis immobilization of L-glutamate oxidase and peroxidase for glutamate determination in flow injection analysis system 1.4.3.11 L-glutamate oxidase analysis hydrogel-immobilization of L-glutamate oxidases for a novel thick-film biosensor and application in food samples 1.4.3.11 L-glutamate oxidase analysis the enzyme can be used for detectioon of glutamate and H2O2 in a biosensor 1.4.3.11 L-glutamate oxidase analysis the enzyme is useful as a component of amperometric L-glutamate sensors used in the food industry and clinical biochemistry 1.4.3.14 L-lysine oxidase analysis L-lysine-alpha-oxidase is immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor is able to analyse L-lysine. The immobilised enzymes and amperometric biosensor can be used for substrate analysis and as a convenient tool for enzyme kinetic studies 1.4.3.19 glycine oxidase analysis development of a chemiluminescence biosensor for glyphoste detection by coupling the engineered enzyme to a spore-based system for H2O2 detection. The chemiluminescence intensity is linearly proportional to the glyphosate concentration within 0.09-75.00 mg/l 1.4.3.22 diamine oxidase analysis the enzyme can be used as biocatalytic component of an electrochemical biosensor for the determination of biogenic amines index in wine and beer samples 1.4.3.25 L-arginine oxidase analysis an amperometric biosensors for L-arginine determination is described that is based on L-arginine oxidase and peroxidase-like nanozymes 1.4.9.1 methylamine dehydrogenase (amicyanin) analysis immobilized enzyme used as histamine biosensor 1.4.99.B4 cis-4-hydroxy-D-proline dehydrogenase analysis estimation of the content of L-hydroxyprolines using coupling systems with metabolic enzymes of the trans-4-hydroxy-L-proline pathway (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and the trans-3-hydroxy-L-proline pathway (trans-3-hydroxy-L-proline dehydratase (T3LHypD) and DELTA1-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. A functional expression system of recombinant HypDH with a heterooligomeric structure is constructed in Escherichia coli cells. Enzymological characterization reveals that the beta-subunit acts as a catalytic subunit, and also that assembly with other subunit(s) improves the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline are successfully estimated within the ranges of 0.004-1 mM and 0.05-1 mM, respectively, and are consistent with the contents determined by HPLC. This enzymatic method is used to measure the content of trans-4-hydroxy-L-proline in the acid-hydrolysate of collagen, and blood plasma 1.5.1.3 dihydrofolate reductase analysis selectable and amplifiable gene marker for e.g. somatic cell hybridization studies 1.5.1.3 dihydrofolate reductase analysis development of a survival protein-fragment complementation assay based on dihydrofolate reductase. Proteins of interest are fused to complementary fragments of dihydrofolate reductase. If the proteins of interst interact physically, the dihydrofolate complementary fragments are brought together and fold into the native structure, reconstituting its activity 1.5.1.3 dihydrofolate reductase analysis development of a yeast protein fragment complementation assay system using dihydrofolate reductase and application for investigating eukaryotic protein-protein interaction in vivo. Fusion of human oncoprotein Ras and Ras-binding domain of Raf-1 to dihydrofolate reductase as a model and evaluation of interaction between these proteins 1.5.1.3 dihydrofolate reductase analysis heteroduplex tracking assay to detect dihydrofolate redctase L164-mutations in variants representing 1% of the parasites in an individual host 1.5.1.3 dihydrofolate reductase analysis use of multiple protein structure technique for structure-based drug discovery. Construction of receptor-based pharmacophores using multiple X-ray crystallographic structures. Models incorporate a fair degree of protein flexibility and are highly selective for known inhibitors over drug-like non-inhibitors 1.5.1.3 dihydrofolate reductase analysis study of protein dynamics, using a pump-probe method that employs pulsed-laser photothermal heating of a gold nanoparticle (AuNP) to directly excite a local region of the protein structure and transient absorbance to probe the effect on enzyme activity. Activity is accelerated by pulsed-laser excitation when the AuNP is attached close to a network of coupled motions in DHFR. No rate acceleration is observed when the AuNP is attached away from the network with pulsed excitation, or for any attachment site with continuous wave excitation 1.5.1.9 saccharopine dehydrogenase (NAD+, L-glutamate-forming) analysis the Spe-Sdh gene is specific to Basidiomycota, use of a specific fragment of the Spe-Sdh gene as a tool to unequivocally identify Basidiomycota isolates 1.5.1.17 alanopine dehydrogenase analysis a nonfluorescent negative stain to visualize the alanopine dehydrogenase isoenzyme pattern in crude-tissue homogenates from very small worms 1.5.1.20 methylenetetrahydrofolate reductase [NAD(P)H] analysis natural polymorphism I225V results in an unusual melting curve peak at 53.4°C instead of 51.6°C or 60.4°C in melting curve analysis after real-time polymerase chain reaction. Potential ability of melting analysis to identify new sequence variants 1.5.1.36 flavin reductase (NADH) analysis amperometric NADH biosensor system that employs the allosterically modulated reductase component of the 4-hydroxyphenylacetate hydroxylase in an adapted osmium(III)-complex-modified redox polymer film for analyte quantification. The activity of the reductase is enhanced upon binding of effector 4-hydroxy-phenylacetic acid, allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with 4-hydroxy-phenylacetic acid. Acceleration of NADH oxidation amplifies the response of the biosensor, with a 1.5fold increase in the sensitivity of analyte detection 1.5.1.42 FMN reductase (NADH) analysis Photobacterium leiognathi Lux G might contribute to the development of a NADH detection kit 1.5.1.53 methylenetetrahydrofolate reductase (NADPH) analysis sensitive radioenzymatic assay that measures directly the reductive catalysis of N ,N -methylenetetrahydrofolate. The radio-labeled substrate, [ C14]N ,N -methyl enetetrahydrofolate, is prepared by condensation of [C14]formaldehyde with tetrahydrofolate. Its stability is maintained for several months by storage at -80°C in a pH 9.5 buffer 1.5.3.1 sarcosine oxidase (formaldehyde-forming) analysis sarcosine oxidases can be applied in enzymatic measuring of creatinine 1.5.3.1 sarcosine oxidase (formaldehyde-forming) analysis improved amperometric biosensor for detection of creatinine based on immobilization of nanoparticles of creatininase, creatinase, and sarcosine oxidase onto glassy carbon electrode. The biosensor shows optimum response within 2 s at pH 6.0 in 0.1 M sodium phosphate buffer and 25°C, when operated at 1.0 V against Ag/AgCl. Recoveries of added creatinine in sera are 97.97% for 0.1 mM and 98.76% for 0.15 mM 1.5.3.13 N1-acetylpolyamine oxidase analysis assay method to measure N1-acetylpolyamine oxidase activity with N1,N11-didansylnorspermine as the substrate by high performance liquid chromatography. Among the dansylpolyamines, N1,N11-didansylnorspermine is a useful substrate for the development of the N1-acetylpolyamine oxidase assay method. Contaminating polyamines in biological samples do not interfere with the method 1.5.3.13 N1-acetylpolyamine oxidase analysis assay method to measure N1-acetylpolyamine oxidase activity with N1,N11-didansylnorspermine as the substrate by high performance liquid chromatography. Applied to cell extract from Chinese hamster ovary cell samples, the PAO activity can easily be detected with as little as 10 microg of protein, which corresponds to 1105 cells. The method is rapid, sensitive and useful. Contaminating polyamines in biological samples do not interfere with the method 1.5.3.17 non-specific polyamine oxidase analysis the seedling polyamine oxidase is used for development of colorimetric assay methods to determine total polyamine, i.e. spermidine and spermine, and spermine concentrations in whole blood of humans, respectively, mechanism, overview 1.5.3.23 glyphosate oxidoreductase analysis spectrophotometric method for determining glyphospate oxidoreductase activity in cell-free extract based on the rate of glyoxylate hydrazone formation 1.5.3.23 glyphosate oxidoreductase analysis use of quantitative competitive PCR to estimate the copy number of the synthetic gox gene as a transgene, and measure of its transcript levels in transformed canola lines. There is no direct relationship between copy number and gene expression level for the gene 1.5.3.25 fructosyl amine oxidase (glucosone-forming) analysis development of a continuous, coupled enzyme assay 1.5.8.2 trimethylamine dehydrogenase analysis microestimation of trimethylamine 1.5.99.B2 proline dehydrogenase (acceptor) analysis construction of a L-proline/O2 biofuel cell. On the anode, the electrocatalytic oxidation of L-proline by L-proline dehydrogenase is observed in the presence of ferrocenecarboxylic acid as mediator. On the cathode, electrocatalytic oxygen reduction is detected. The final biofuel cell using these electrodes achieves a current density of 6.00 microA/cm2 1.5.99.6 spermidine dehydrogenase analysis enzyme can be assayed in the crude extract by oxygen utilization or by the reaction of the product DELTA1-pyrroline with o-aminobenzaldehyde 1.5.99.13 D-proline dehydrogenase analysis an amperometric D-amino acid biosensor is prepared with a thermostable D-proline dehydrogenase and a carbon nanotube-ionic liquid gel 1.5.99.13 D-proline dehydrogenase analysis development of a D-amino acids electrochemical sensor based on immobilization of thermostable D-proline dehydrogenase within agar gel membrane. The D-Pro DH/agar immobilized electrode is applied to determination of D-amino acids in a human urine sample 1.6.2.2 cytochrome-b5 reductase analysis detailed analysis of biphasic rate of reduction of cytochrome b5 in membranes with evaluation of data in terms of two-dimensional random walk model. The initial rapid phase is completed within 10 msec and over 90% of cytochrome b5 are reduced in 40 msec 1.6.2.2 cytochrome-b5 reductase analysis potential use of the enzyme as biosensor 1.6.2.2 cytochrome-b5 reductase analysis microplate method based on a standard 96-well microplate format to simplify the quantification of NADH-CYB5R activity. Enzyme activity in 25 diagnosed cases of recessive congenital methemoglobinemia ranges from 6.09 to 10.07 IU/g Hb whereas normal control ranges from 13.42 to 21.58 IU/g Hb. Hemolysate samples have significant loss of activity when stored at 4°C and retain stable activity at -20°C for 1 week 1.6.2.4 NADPH-hemoprotein reductase analysis 96-well plate format assay to follow cytochrome c reduction 1.6.5.2 NAD(P)H dehydrogenase (quinone) analysis method to measure enzyme inhibition in intact cells based on the resorufin reductase activity of enzyme. Values for 50% inhibition of enzyme by several reported in vivo inhibitors is at least three orders of magnitude higher for intact cells than for cell lysates 1.6.5.2 NAD(P)H dehydrogenase (quinone) analysis the enzyme is a catalyst for 1,4-dihydronicotinamide adenine dinucleotide-dependent amperometric biosensors and biofuel cells. For NADH detection, a linear range between 0.005 mM and 1 mM, a limit of detection of 0.003 mM, and a high sensitivity can be reached 1.6.5.2 NAD(P)H dehydrogenase (quinone) analysis development of a functional affinity-based small-molecule probe composed of a potent small-molecule NQO1 inhibitor 4-hydroxy-3-[(2E)-3-(4-hydroxyphenyl)prop-2-enoyl]-2H-1-benzopyran-2-one as the recognition group, a linker and the fluorophores group FITC. The probe exhibits good inhibitory activity of NQO1 and can be used to label the protein in A549 cells at the micromolar level 1.6.99.1 NADPH dehydrogenase analysis enhanced specificity of histochemical enzyme reaction for nitric oxide synthase-1 in presence of 2 M urea 1.6.99.1 NADPH dehydrogenase analysis localization pattern of nitric oxide synthase through enzyme staining, intensity of staining differs in the optic lobes of male and female animals 1.7.1.1 nitrate reductase (NADH) analysis measuring in vitro nitrate reductase activity of assimilatory nitrate reductase enzymes from Mycolicibacterium smegmatis crude extract using both physiological and non-physiological electron donors. Product nitrite is converted to a water soluble red azo dye using sulfanilic acid and N-(1-naphthyl)ethylenediamine 1.7.1.1 nitrate reductase (NADH) analysis nitrate reductase is a suitable proxy of the effects of temperature changes on the ability of Ulva prolifera to uptake and metabolize nitrogen nutrients 1.7.1.2 Nitrate reductase [NAD(P)H] analysis enzymatic reduction as a method for NO3- determination in expired breath condensate as a way of monitoring inflammation and oxidative stress in the lungs 1.7.1.2 Nitrate reductase [NAD(P)H] analysis the amperometric nitrate reductase-phenosafranin bioelectrode is very convenient for the determination of nitrate in river water 1.7.1.2 Nitrate reductase [NAD(P)H] analysis detailed protocol for measuring relative nitrate reductase enzyme activity of tomato crude extracts. Product nitrite is converted to a water soluble red azo dye using sulfanilic acid and N-(1-naphthyl)ethylenediamine 1.7.1.2 Nitrate reductase [NAD(P)H] analysis measuring in vitro nitrate reductase activity of assimilatory nitrate reductase enzymes from Mycolicibacterium smegmatis crude extract using both physiological and non-physiological electron donors. Product nitrite is converted to a water soluble red azo dye using sulfanilic acid and N-(1-naphthyl)ethylenediamine 1.7.1.3 nitrate reductase (NADPH) analysis sequential injection analysis flow system for determination of nitrites and nitrates in human serum 1.7.1.3 nitrate reductase (NADPH) analysis measuring in vitro nitrate reductase activity of assimilatory nitrate reductase enzymes from Mycolicibacterium smegmatis crude extract using both physiological and non-physiological electron donors. Product nitrite is converted to a water soluble red azo dye using sulfanilic acid and N-(1-naphthyl)ethylenediamine 1.7.1.4 nitrite reductase [NAD(P)H] analysis microplate high-throughput nitrite reductase assay 1.7.1.6 azobenzene reductase analysis pro-fluorophore 1-methyl-1-([1-[(Z)-(methyl[4-[(E)-2-[6'-[(E)-[4-[methyl([4-[(1-methylpyrrolidinium-1-yl)methyl]-1H-1,2,3-triazol-1-yl]methyl)amino]phenyl]diazenyl]-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthen]-3'-yl]ethenyl]phenyl]iminio)methyl]-1H-1,2,3-triazol-4-yl]methyl)pyrrolidinium shows a green fluorescence upon reduction of the two diazo bonds suitable for the detection of azoreductases 1.7.1.16 nitrobenzene nitroreductase analysis fluorescence imaging of nitroreductase in zebrafish in vivo using a newnear-infrared fluorescence off-on probe. The probe is prepared by connecting 4-nitrobenzene as a quenching and recognizing moiety to a stable hemicyanine skeleton. The fluorescence off-on response of the probe is based on the enzyme-catalyzed reduction of the 4-nitrobenzene moiety, followed by the 1,6-rearrangement-elimination and the fluorophore release. The probe exhibits good analytical performance such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 14 ng/ml. The probe has been applied to visualize the distribution of nitroreductase in living zebrafish in vivo 1.7.1.16 nitrobenzene nitroreductase analysis immobilization of enzyme on polyethyleneimine-mediated silica formation, enzyme activity is significantly more stable than NbzA in solution. A microfluidic microreactor is suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics 1.7.2.1 nitrite reductase (NO-forming) analysis upon coimmobilization of cytochrome cd1 nitrite reductase Mhcd1 and cytochrome c552 on silver electrodes coated with self-assembled monolayers, cytochrome c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes an about 150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1 1.7.2.2 nitrite reductase (cytochrome; ammonia-forming) analysis development of a conductometric biosensor for nitrite detection using cytochrome c nitrite reductase immobilized on a planar interdigitated electrode. Under the optimum conditions at room temperature, the biosensor shows a fast response to nitrite with a linear range of 0.2–120 microM, a sensitivity of 0.194 microS per microM NO2?, and a detection limit of 0.05 microM. The biosensor also shows satisfactory reproducibility. When stored in potassium phosphate buffer at 4 ?C, the biosensor shows good stability over 1 month 1.7.2.2 nitrite reductase (cytochrome; ammonia-forming) analysis direct electrochemistry of the multihemic nitrite reductase using carbon nanotube dispersions deposited on pyrolytic graphite macroelectrodes. The multi-walled carbon nanotubes are most favourable for enzyme catalysis studied by cyclic voltammetry 1.7.2.2 nitrite reductase (cytochrome; ammonia-forming) analysis construction of pencil lead electrodes with ccNiR being simply drop casted onto the electrode. A functional and fully integrated voltammetric biosensor for nitrite quantification is obtained that allows to observe a decrease in the catalytic current due to cyanide addition. Under turnover conditions, the biosensor shows a linear response with the logarithm of cyanide concentration in the 5-76 microM (cyclic voltammetry) and 1-40 microM (square-wave voltammetry) ranges, with a sensitivity of 20-25% ln 1/[cyanide, microM] and a detection limit of 0.86-4 microM 1.7.2.2 nitrite reductase (cytochrome; ammonia-forming) analysis trapping of a putative intermediate by controlling the electrochemical potential at which reduction takes place. An [FeNO}]7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia. At low potentials the species is rapidly reduced and does not accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation 1.7.2.6 hydroxylamine dehydrogenase analysis use of hydroxylamine oxidoreductase to differentiate Nitrosomonas spp. and to identify autotrophic ammonia oxidizing bacteria. The ensembled use of single strand confirmation polymorphism and HAO enzyme zymogram in fingerprinting AOB provide better resolution and evenness, contributing significantly in autotrophic ammonia oxidizing bacteria diversity studies 1.7.3.3 factor-independent urate hydroxylase analysis a colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters based on hydrogen peroxide quantitation. The general advantages of the colorimetric assay are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material 1.7.3.3 factor-independent urate hydroxylase analysis modified colorimetric assay for uricase activity in flexible 96-well microtiter plates using the uricase/uric acid/horseradish peroxidase/4-aminoantipyrine/3,5-dichloro-2-hydroxybenzene sulfonate colorimetric reaction. The method is much more efficient than the conventional ones and greatly reduces assay time from 4 days to less than 20 h 1.7.3.3 factor-independent urate hydroxylase analysis development of an urate-selective microbial biosensor cells of the recombinant thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. The UOX producing cells are coupled to horseradish peroxidase and immobilized on graphite electrodes by physical entrapment behind a dialysis membrane. A high urate selectivity with a detection limit of about 8 microM is found 1.7.3.3 factor-independent urate hydroxylase analysis development of a high-throughput screening system of Bacillus fastidiosus uricase mutants, using 96-well plates and monitoring of uric acid by absorbance at 298 nm 1.7.3.3 factor-independent urate hydroxylase analysis specific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity. Uricase mutants whose activities are improved by more than 80% are recognized with higher sensitivity and specificity during screening a library of enzyme mutants expressed in Escherichia coli 1.7.3.3 factor-independent urate hydroxylase analysis uric acid can be detected in human sera with the enzyme with none of the tested uric acid analogs being a competitive substrate 1.8.1.B1 thioredoxin glutathione reductase analysis development of a cost sensitive classification model to evaluate the biological activity of inhibitors. Random forest analysis revealed 10 highly enriched scaffolds in the actives dataset. Models are evaluated using docking approaches 1.8.1.4 dihydrolipoyl dehydrogenase analysis development of a blue native-PAGE-based method for isolation of enzymatically active DLDH from animal tissues and visualization as well as quantification of its diaphorase activity using the NADH/nitroblue tetrazolium detection system 1.8.1.9 thioredoxin-disulfide reductase analysis thioredoxin reductase-1 may be an early indicator of acute exposure to low lead doses 1.8.1.15 mycothione reductase analysis rapid method for the relative quantification of mycothiol using high-sensitivity mass spectrometry with selected ion monitoring. The method uses only minimal sample cleanup, and does not require advanced chromatographic equipment or fluorescent compounds 1.8.3.1 sulfite oxidase analysis electroimmobilisation into polypyrrole film, use for amperometric detection of sulfite 1.8.3.1 sulfite oxidase analysis an enzyme-coupled electrode is successfully used for the determination of sulfite concentration in white wine and beer samples, enzyme electrode preparation and method evaluation 1.8.3.2 thiol oxidase analysis development of a fast and reliable qualitative plate test for screening secreted fungal sulfhydryl oxidases. Screening is based on the Ellman's reagent, i.e. 5,5'-dithiobis(2-nitrobenzoic acid) 1.8.3.2 thiol oxidase analysis development of a fast and reliable qualitative plate test for screening secreted fungal sulfhydryl oxidases. Screening is based on the Ellman's reagent, i.e. 5,5'-dithiobis(2-nitrobenzoic acid). Enzymes could be identified in Aspergillus tubingensis, Chaetomium globusum, Melanocarpus albomyces, Penicillium aurantiogriseum, Penicillium funiculosum, Coniophora puteana and Trametes hirsuta 1.8.3.3 glutathione oxidase analysis development of amperometric glutathione electrodes with immobilized enzyme for detection of H2O2 1.8.3.3 glutathione oxidase analysis development of a glutathione micro-enzyme sensor for amperometric detection in vivo, immobilization of enzyme on microelectrode tips 1.8.3.3 glutathione oxidase analysis development of an amperometric biosensor for determination of glutathione, by immobilizing covalently a glutathione oxidase onto the surface of gold coated magnetic nanoparticles modified Pt electrode. The electrode shows maximum response within 4 s, when polarizes at +0.4 V, pH 7.0 and 25°C. There is a linear relationship between electrode response and glutathione concentrations in the range 5.0-4000 microM with a detection limit of 0.1 microM. The biosensor shows 50% loss in its initial activity after its 150 uses over a period 4 months. Glutathione concentration in hemolysated erythrocytes as measured by the present biosensor is 2.8 mM in apparently healthy persons 1.8.4.10 adenylyl-sulfate reductase (thioredoxin) analysis development of a continuous spectrophotometric method for measuring APR activity by using sulfite-selective colorimetric or off-on fluorescent levulinate-based probes. The APR activity can be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product 1.8.4.11 peptide-methionine (S)-S-oxide reductase analysis rapid and specific microplate assay to monitor MsrA activity based on oxidation of dithiothreitol, whose color can be produced by reacting with Ellman's reagent 1.8.4.11 peptide-methionine (S)-S-oxide reductase analysis with MsrA adsorbed on glassy carbon and boron doped diamond electrodes surface, the first step reaction step corresponds to the oxidation of tyrosine and tryptophan residues and occurs for the same potential. The second step is histidine oxidation, and the third, at higher potentials, the second tryptophan oxidation. MsrA adsorbs on the hydrophobic carbon electrode surface preferentially through the three hydrophobic domains, C1, C2 and C3, which contain the tyrosine, tryptophan and histidine residues 1.8.99.2 adenylyl-sulfate reductase analysis the gene apsA is used for quantitative determination of the organism in wastewater, overview 1.8.99.2 adenylyl-sulfate reductase analysis luciferase-based method for measurement of 5'-adenylylsulfate reductase. The assay measures AMP production using an enzyme-coupled system that generates luminescence. The method can be used for both endpoint and continuous assays, utilizing pure enzyme preparations as well as crude protein extracts. The assay is suited to high-throughput sample analysis 1.9.6.1 nitrate reductase (cytochrome) analysis immobilization of whole cells and NAP enzyme for simultaneous bioremediation and bionanoparticles synthesis. The immobilized cells completely remove NO3- upon 192 h with Ag nanoparticles synthesis ranging from 23.26 to 58.14 nm. Immobilized NAP exhibits lower efficiency with 28.6% of NO3- elimination within 288 h and large aggregated Ag nanoparticles ranging from 94.44 nm to 172.22 nm 1.10.3.2 laccase analysis sensitive, rapid, and precise determination of phenols and their derivatives is important in environmental control and protection. An amperometric principle-based biosensor, employing immobilized laccase enzyme from Trametes versicolor, is developed for the detection of disubstituted methyl and methoxy phenols (industrial effluents). Evaluation of the influence of different enzyme immobilization techniques, on nylon membrane, on the performances of laccase-based Clark-type electrodes. The analytical properties and operating stabilities of the resulting biosensors are tested with different disubstituted methyl and methoxy derivatives of phenol substrates. Co-cross-linking method is superior to the other methods of immobilization in terms of sensitivity, limit of detection, response time, and operating stability. In co-cross-linking method of immobilization, laccase is mixed with bovine serum albumin as protein-based stabilizing agent and glutaraldehyde as crosslinking agent 1.10.3.3 L-ascorbate oxidase analysis quantitative determination of ascorbic acid with enzyme elctrode containing enzyme immobilized on nylon membrane 1.10.3.3 L-ascorbate oxidase analysis reagent for analysis of L-ascorbic acid in food 1.10.3.3 L-ascorbate oxidase analysis rapid and simple determination of L-ascorbate in food with immobilized enzyme 1.10.3.3 L-ascorbate oxidase analysis the enzyme is required in NO measurements to eliminates the interference of ascorbic acid and/or dehydroascorbic acid in the assays, overview 1.11.1.6 catalase analysis quality of catalase activity as a biomarker, the predictive quality is stress specific 1.11.1.B6 iodide peroxidase (vanadium-containing) analysis for iodoperoxidase activity measurement, the triiodide assay should only be used for qualitative applications. A quantitative spectrophotometric assay is based on the iodination of thymol blue but can only be used in the pH range 7-8 1.11.1.B6 iodide peroxidase (vanadium-containing) analysis to detect activity on polyacrylamide gels or in cross sections of algal part, o-dianisidine may be used. The probes are first incubated in a buffered solution containing 100 mM KBr or 100 mM KI, and 10 mM o-dianisidine, and the staining is started by addition of 2 mM H2O2. The formed HOBr or HOI will oxidize the organic dye resulting in polymerization and formation of a brown precipitate 1.11.1.7 peroxidase analysis application as enzyme immunoassays, diagnostic test kits, wastewater treatment and soil remediation 1.11.1.7 peroxidase analysis application of molecular absorption properties of horseradish peroxidase for self-indicating enzymatic interactions and analytical methods. This method can be applied for glucose determination where there is a low concentration (or an absence) of proteins 1.11.1.7 peroxidase analysis use of this enzyme as a biosensor to detect H2O2 in some food components such as milk or its derivatives 1.11.1.7 peroxidase analysis use of nanoporous TiO2 electrodes as a substrate for enzyme immobilization in order to enhance the electron transport across the semiconductor electrode-electrolyte interface. The immobilized HRP enzyme is stable and the overall electron transfer process is predominantly controlled by a diffusion process. The application for H2O2 biosensors is demonstrated by monitoring its reduction process using cyclic voltammetry 1.11.1.8 iodide peroxidase analysis development of an ELISA system for quantitative determination of TPO in biological fluids, based on the use of an analytical pair of monoclonal antibodies, one of which is immobilized on solid phase, the other being present in solution. The use of this system made it possible to determine the biochemical characteristics of immunoaffinity chromatography of the enzyme and propose a new method for obtaining a highly purified preparation of its protein 1.11.1.8 iodide peroxidase analysis evaluation of a rapid assay for TPO inhibition, using a stable fluorescent product (Amplex UltraRed). The assay consistently identifies the relative potency of strong to moderate inhibitors and chemicals known to be inactive. Results are less consistent for chemicals reported to be weak inhibitors of rodent TPO 1.11.1.9 glutathione peroxidase analysis activity is important in predicting tissue redox state 1.11.1.9 glutathione peroxidase analysis method of measuring of glutathione peroxidase activity using H2O2. At a concentration of reduced glutathione of 0.55 mM, the concentration of H2O2 of 0.192 mM is saturating for glutathione peroxidase. The duration of incubation does not exceed 60 sec, if 13% homogenate is used 1.11.1.11 L-ascorbate peroxidase analysis the ascorbate peroxidase procedure may be applied to ascorbate estimation in sources as black-currant juice, parsley leaf, broccoli influorescence, potato tuber, mouse liver and human urine 1.11.1.11 L-ascorbate peroxidase analysis APEX2, i.e. engineered mutant K14D/W41F/E112K/A134P of APX used for biotinylation of neighboring endogenous proteins, can be applicated for effective labeling in multiple Drosophila melanogaster tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle in different physiological conditions 1.11.1.11 L-ascorbate peroxidase analysis APEX2, i.e. engineered mutant K14D/W41F/E112K/A134P of APX, fused to a protein of interest covalently tags nearby proteins with biotin-phenol when H2O2 is added. High osmolarity and disruption of cell wall integrity permits live-cell biotin labeling in Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. APEX2 permits targeted and proximity-dependent labeling of proteins 1.11.1.12 phospholipid-hydroperoxide glutathione peroxidase analysis because the content of enzyme, irrespective of the cause of alteration, is correlated with fertility-related parameters, PHGPx can be considered a predictive measure for fertilization capacity 1.11.1.14 lignin peroxidase analysis different mechanisms for the bioelectrocatalysis of the enzyme depend on the chemical nature of the mediators and are of a special interest both for fundamental science and for application of the enzyme as solid-phase bio(electro)catalyst for decomposition/detection of of recalcitrant aromatic compounds 1.11.1.14 lignin peroxidase analysis evaluation of the effect of enzyme dosage, incubation time, and H2O2 addition profile on lignin activation by quantifying the phenoxy radicals formed using electron paramagnetic resonance spectroscopy. At optimal conditions, i.e. dose of 15 /g and continuous addition of H2O2, the content of phenoxy radicals is doubled as compared with an untreated control 1.11.1.16 versatile peroxidase analysis detection and quantification of soluble lignin transformation by measuring changes in the true colour of the system solution 1.11.1.16 versatile peroxidase analysis evaluation of the effect of enzyme dosage, incubation time, and H2O2 addition profile on lignin activation by quantifying the phenoxy radicals formed using electron paramagnetic resonance spectroscopy. At optimal conditions, i.e. dose of 15 /g and continuous addition of H2O2, the content of phenoxy radicals is doubled as compared with an untreated control 1.11.2.1 unspecific peroxygenase analysis use of enzyme for biosensor applications. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. After immobilization at a chitosan-capped gold-nanoparticle modified glassy carbon electrode, enzyme displays a pair of redox peaks with a midpoint potential of -278.5 mV vs. AgCl/AgCl at 1 M KCl for the Fe2+/Fe3+ redox couple of the heme-thiolate-containing protein. The signal is generated by the reduction of electrode-active reaction products e.g., p-benzoquinone and p-quinoneimine with electroenzymatic recycling of the analyte 1.11.2.2 myeloperoxidase analysis detection of myeloperoxidase activity in vivo by use of a paramagneitc substrate. The sensing probe is obtained by replacing the reducing substrate serotonin with 5-hydroxytryptophan. The resulting probe bis-hydroxytryptophan-gadolinium diethylenetriamine pentaacetic acid in vitro improves solubility in water, acts as a substrate, induces cross linking of proteins in the presence of myeloperoxidase,produces oxidation products which bind to plasma proteins and does not follow first order reaction kinetics. Bis-hydroxytryptophan-gadolinium diethylenetriamine pentaacetic acid is retained for up to five days in myeloperoxidase-containing sites and cleared faster than serotonin diethylenetriamine pentaacetic acid from enzyme-negative sites 1.13.11.2 catechol 2,3-dioxygenase analysis a highly sensitive microbial biosensor is constructed using the recombinant Escherichia coli BL21-C23O cell as bio-recognition element for catechol detection. Based on the synergistic effect of catechol 2,3-dioxygenase and nanoporous gold, the resulting microbial biosensor exhibits good performance for catechol detection with high sensitivity, strong anti-interference and good stability 1.13.11.3 protocatechuate 3,4-dioxygenase analysis use in flushing and scrubbing oxygen out of instruments such as stopped-flow spectrophotometers 1.13.11.4 gentisate 1,2-dioxygenase analysis high potential for use in biosensor technologies for monitoring the metabolism of tyrosine and salicylic acid, as well as a pollution of environment and different materials by polyaromatic compounds 1.13.11.27 4-hydroxyphenylpyruvate dioxygenase analysis colorimetric bacterial whole-cell screening system based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in Escherichia coli and subsequent production of a soluble melanin-like pigment. The addition of prototypical beta-triketone HPD inhibitors decreases pigment production in a dose-dependent manner with increasing inhibitor concentrations. The high-throughput screening assay shows plate uniformity, signal variability and spatial uniformity assessment 1.13.11.53 acireductone dioxygenase (Ni2+-requiring) analysis detection of one-bond 15N-13Calpha correlations in the vicinity of the paramagnetic Ni2+ via a double-quantum correlation experiment 1.13.11.63 beta-carotene 15,15'-dioxygenase analysis BCMO function in mammalian cells is analyzed by a retinoic acid receptor assay, which responds to the metabolic conversion of beta-carotene to retinoic acid in vivo, this tool can be used to screen more active BCMO for the industrial and pharmacological purpose of retinal production from beta-carotene 1.13.11.85 exo-cleaving rubber dioxygenase analysis development of an in vitro enzyme assay for oxidative rubber degradation based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone 1.13.12.5 Renilla-type luciferase analysis analysis of luciferase inhibitors by quantitative high-throughput screening and comparison of and structure-activity relationship using various luciferase-based detection reagents 1.13.12.5 Renilla-type luciferase analysis development of a bioluminiscent probe composed of peptide EYFP and luciferase for near-real-time single-cell imaging using bioluminescence resonance energy transfer BRET. The probe exhibits enhanced luciferase luminescence intensity and appropriate subcellular distribution when it is fused to targeting-signal peptides or histone H2AX. It allows high spatial and temporal resolution microscopy of living cells 1.13.12.5 Renilla-type luciferase analysis the use of control Renilla luciferase vectors as normalizers requires that they not be influenced by any variables in the experiment. The zinc finger transcription factor WT1 effect on luciferase activation varies from no significant effect in 293 and PC3 cells to strong enhancement in LNCaP cells treated with the androgen analog R1881. Hormone enhances WT1-mediated activation of luciferase and these interactions require an intact WT1 zinc finger DNA binding domain 1.13.12.5 Renilla-type luciferase analysis for analysis of the Hsp90 chaperone activity in complex with cochaperone Cdc37, split Renilla luciferase protein fragment-assisted complementation bioluminescence can be utilized to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/Cdc37 interaction in living cells 1.13.12.5 Renilla-type luciferase analysis Renilla luciferase is used in assay development for measurement of mitochondrial fusion, quantification via split-Renilla luciferase complementation in HeLa cells, validation of the Renilla luciferase reporter system for mitochondrial fusion, overview 1.13.12.5 Renilla-type luciferase analysis an advanced Fc-binding probe, FcUni-RLuc, is produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence HWRGWV is fused to Renilla luciferase, and the purified probe is employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells 1.13.12.5 Renilla-type luciferase analysis establishment and evaluation of an indirect autophagy flux assay based on monitoring the degradation of an autophagosome-associated fusion protein Rluc-LC3 by luminescence detection. The Rluc-LC3 assay is useful for the identification of genes, miRNAs, and small molecules that regulate autophagy flux in mammalian cells 1.13.12.5 Renilla-type luciferase analysis Renilla luciferase is a bioluminescent enzyme which is broadly used as a reporter protein in molecularbiosensors 1.13.12.5 Renilla-type luciferase analysis enhanced-sensitivity, synthetically facile reporter fusion to merge the bioluminescence output of Gaussia luciferase with the biotin-binding ability of tamavidin 2. The fusion construct enables direct bacterial expression of a reporter system incorporating two functionalities in a 1:1 stoichiometric relationship. Using a cold-shock expression system, highly concentrated construct can be obtained from standard culture volumes while retaining essentially native protein activity. The fusion construct can be included in a standard target-bridged assay design for the sensitive detection of miRNA targets 1.13.12.5 Renilla-type luciferase analysis luciferase can be functionally expressed in Staphylococcus aureus and can be used as a biosensor for the agr quorum sensing system which employs autoinducing peptides to control virulence. Luciferase is linked to the P3 promoter of the agr operon and biosensor strains are validated by evaluation of chemical agent-mediated activation and inhibition of agr. Use of Luciferase enables quantitative assessment of agr activity 1.13.12.6 Cypridina-luciferin 2-monooxygenase analysis intramolecular bioluminescence resonance energy transfer system consisting of a fusion protein of the enzyme and enhanced yellow fluorescent protein for investigating peptide processing in living cells 1.13.12.6 Cypridina-luciferin 2-monooxygenase analysis dual reporter assay using one luciferase for monitoring gene expression and a second as an internal control, based on secreted luciferases from Cypridina noctiluca and Gaussia princeps that do not require lysis or special equipment. The assay can be carried out sequentially, the activities are high and can be measured in a small volume and a simple procedure. Development of a one-tube reporter assay for the two enzymes 1.13.12.6 Cypridina-luciferin 2-monooxygenase analysis use of enzyme as a reporter in a screening system of Saccharomyces cerevisiae to improve secretion efficiency in yeast 1.13.12.6 Cypridina-luciferin 2-monooxygenase analysis Cypridina noctiluca luciferase is utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays 1.13.12.6 Cypridina-luciferin 2-monooxygenase analysis the secreted Cypridina luciferase (CLuc) is used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase is used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Tumor monitoring systems using dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals, e.g. BALB/cAJcl-nu/nu mice. Continuous non-invasive measurement of CLuc activity for tumor growth allows us to determine a suitable time to assess tumor spatial distribution and growth, thereby, reducing the animal stress and the experimental cost 1.13.12.6 Cypridina-luciferin 2-monooxygenase analysis the thermostable enzyme can be used for various research applications, including in vivo imaging and high throughput reporter assays 1.13.12.7 firefly luciferase analysis luminescence-based assays for ATP measurement in clinical chemistry and hygiene monitoring 1.13.12.7 firefly luciferase analysis development of a quantitative and highly sensitive luciferase-based assay for bacterial toxins, overview 1.13.12.7 firefly luciferase analysis expression of bioluminescent luciferase can be used for rapid and high throughput screening of drugs, e.g. quinolines, acting on Leishmania amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice, overview 1.13.12.7 firefly luciferase analysis analytical assay of metabolites like ATP, CoA, pyrophosphate, AMP 1.13.12.7 firefly luciferase analysis optical bioluminescence-based molecular imaging 1.13.12.7 firefly luciferase analysis Cypridina noctiluca luciferase is utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays 1.13.12.7 firefly luciferase analysis the secreted Cypridina luciferase (CLuc) is used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase is used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Tumor monitoring systems using dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals, e.g. BALB/cAJcl-nu/nu mice 1.13.12.7 firefly luciferase analysis an engineered, commercial thermostable luciferase is suitable for real-time monitoring of ATP release by bacteria, both in broth culture and on agar surfaces, which allows for the estimation of viable cell number by relating luminescence onset time to initial cell concentration. The method is able to rapidly detect the effect of antibiotics on bacterial cultures 1.13.12.7 firefly luciferase analysis luciferase-based determination of ATP/NAD(H) pools in combination with a microplate reader and the marine model bacterium Phaeobacter inhibens. Grey multiwell plates best balance sensitivity and crosstalk, and optimal incubation times are 5 min and 30 min for the ATP and NAD(H) assay, respectively, together allowing limits of detection of 0.042, 0.470 and 0.710 nM for ATP, NAD+, and NADH, respectively 1.13.12.9 phenylalanine 2-monooxygenase analysis a simple and rapid enzymic determination of L-Phe with L-phenylalanine oxidase (deaminating and decarboxylating) 1.13.12.24 calcium-regulated photoprotein analysis a fusion protein composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoaequorin (apoAQ) is expressed in Escherichia coli periplasm. Incubation with coelenterazine gives ZZ-aequorin which can be used as a reporter for detecting IgG. The measurable range of IgG coated on a 96-well plate is 1-1000 ng/ml 1.13.12.24 calcium-regulated photoprotein analysis fusion of aequorin to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer tomato-aequorin shows the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. Light output is sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tandem-dimer tomato-aequorin. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture are also recorded, and changes in fine neuronal projections are spatially resolved. It is possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tandem-dimer tomato-aequorin in muscle or the peritoneal cavity 1.13.12.24 calcium-regulated photoprotein analysis in presence of O2 and coelenterazine, apoaequorin forms an aequorin 2-hydroperoxide. When pretreated with dithiothreitol, photoirradiation of the aequorin 2-hydroperoxide leads to formation of the dithiothreitol-modified cysteine residues located near the chromophore to sulfinic acid or sulfenic acid 1.13.12.24 calcium-regulated photoprotein analysis mutant D119A is suitable for measuring Ca2+ inside the endoplasmic reticulum, the mitochondria, endosomes and the outside of live cells 1.13.12.24 calcium-regulated photoprotein analysis use of the enzyme as an instrument to monitor the development of high specific DNA aptamers 1.14.11.2 procollagen-proline 4-dioxygenase analysis assay method for screening the inhibitors of prolyl 4-hydroxylase to treat fibrosis. The assay uses rat hepatic stellate HSC-6 cells and cobalt chloride as a substitue for a hypoxic condition, the procedure takes three days after treatment with agents 1.14.11.9 flavanone 3-dioxygenase analysis an HPLC method for the determination of phenylalanine ammonia-lyase, flavanone 3-hydroxylase and flavonol synthase enzyme activity is proposed. This method is based on the determination of the compounds produced and consumed on the enzymatic reaction in just one chromatographic analysis. Optimisation of the method consideres kinetic studies to establish the incubation time to perform the assay. The method is an approach to measure the activities of the three enzymes simultaneously increasing the rapidity, selectivity and sensitivity over other exiting methods 1.14.11.11 hyoscyamine (6S)-dioxygenase analysis the enzyme is a molecular marker for PCR-based screening approach for tropane alkaloids producing endophytic fungi 1.14.11.16 peptide-aspartate beta-dioxygenase analysis isolation of human single-chain Fv fragments directed against human aspartyl-asparaginyl beta-hydroxylase. Antibodies show significant binding to recombinant enzyme in ELISA, tumor cell lines, and tumor tissues. They target different domains of the enzyme. A goat-anti-human IgG saporin conjugate can be delivered into tumor cells by antibody 6-22 and elicits cytotoxicity toward the tumor cells in vitro 1.14.11.17 taurine dioxygenase analysis development of a colorimetric assay method for determination of taurine in commercially available beverages and some biological samples using the taurine dioxygenase. Taurine determination in food control, biological research, and diagnoses based on urinary taurine concentration 1.14.11.29 hypoxia-inducible factor-proline dioxygenase analysis assay for measuring the substrate hydroxylation-coupled decarboxylation of radioactive 2-oxoglutarate to radioactive carbon dioxide as a fast, efficient, and diverse method 1.14.11.29 hypoxia-inducible factor-proline dioxygenase analysis specific protocols for the knockdown and inhibition of the HIF prolyl hydroxylases 1-3 and the asparagine hydroxylase factor-inhibiting HIF using RNA interference and hydroxylase inhibitors, respectively 1.14.11.30 hypoxia-inducible factor-asparagine dioxygenase analysis specific protocols for the knockdown and inhibition of the HIF prolyl hydroxylases 1-3 and the asparagine hydroxylase factor-inhibiting HIF using RNA interference and hydroxylase inhibitors, respectively 1.14.11.33 DNA oxidative demethylase analysis development of a fluorogenic probe design (MAQ) that is directly responsive to ALKBH3 repair activity. MAQ uses the fluorescence-quenching properties of 1-methyladenine, removal of the alkyl group results in an more than 10fold light-up signal. The probe is specific for ALKBH3 over its related homologue ALKBH2 and can be used to identify and measure the effectiveness of enzyme inhibitors. The probe functions efficiently in cells, allowing imaging and quantitation of ALKBH3 activity by microscopy and flow cytometry 1.14.11.51 DNA N6-methyladenine demethylase analysis development of assay method using a methylation-sensitive restriction endonuclease 1.14.11.53 mRNA N6-methyladenine demethylase analysis development of a single-quantum-dot-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of m6A can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the quantum dot surface, enabling the occurrence of FRET between the quantum dot and Cy5 1.14.11.66 [histone H3]-trimethyl-L-lysine9 demethylase analysis KDM4A possesses the potential to act as an oxygen sensor in the context of chromatin modifications, with possible implications for epigenetic regulation in hypoxic disease states 1.14.11.69 [histone H3]-trimethyl-L-lysine36 demethylase analysis KDM4A possesses the potential to act as an oxygen sensor in the context of chromatin modifications, with possible implications for epigenetic regulation in hypoxic disease states 1.14.12.3 benzene 1,2-dioxygenase analysis the enzyme is used in a whole-cell assay as biosensor for benzene concentration and vapopur, overview 1.14.12.11 toluene dioxygenase analysis whole cell bioassay for the detection of benzene, toluene, ethyl benzene, and xylenes (BTEX) 1.14.12.17 nitric oxide dioxygenase analysis heterologous expression of Escherichia coli flavohemoglobin within a lentiviral delivery system boosts endogenous cellular consumption of NO, thus providing a simple and efficacious approach to studying mammalian NO-biology 1.14.13.1 salicylate 1-monooxygenase analysis biosensor system for determining salicylate in body fluids 1.14.13.1 salicylate 1-monooxygenase analysis an amperometric biosensor is developed for the interference-free determination of L-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by L-glutamate dehydrogenase (EC 1.4.1.3) in combination with salicylate hydroxylase (EC 1.14.13.1). The enzymes are entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane 1.14.13.1 salicylate 1-monooxygenase analysis two types of amperometric ATP biosensors are developed by using the coimmobilization of salicylate hydroxylase (SHL, EC 1.14.13.1), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), and hexokinase (HEX, EC 2.7.1.1) on a Clark-type oxygen electrode and on a screenprinted electrode. The principles of the determination schemes are as follows: HEX transfers the phosphate group from ATP to glucose to form glucose-6-phosphate. G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NAD+. The product, NADH initiates the irreversible decarboxylation and hydroxylation of salicylate by SHL to consume dissolved oxygen and generate catechol. This results in a detectable signal on a Clark-type electrode due to the SHL-enzymatic consumption of oxygen, or a detectable signal on a screen-printed electrode due to the SHL-enzymatic generation of catechol in the measurement of ATP. Both sensors show high performance characteristics with broad detection ranges, short measuring times, and good specificities 1.14.13.1 salicylate 1-monooxygenase analysis usage of the enzyme in a fluorescence assay that detects 3-hydroxybutyrate and cholesterol in the nanomolar range and is more sensitive than the current SHL-dehydrogenase amperometric sensors, making it applicable to the construction of a fiber-optic fluorescence biosensor for clinical diagnostic uses 1.14.13.2 4-hydroxybenzoate 3-monooxygenase analysis a bienzyme-based Clark electrode is developed for the interference-free determination of L-glutamate. This sensor is based on the specific dehydrogenation by L-glutamate dehydrogenase (EC 1.4.1.3) in combination with p-hydroxybenzoate hydroxylase (EC 1.14.13.2). The enzymes are entrapped by a poly(carbamoyl) sulfonate hydrogel on a Teflon membrane 1.14.13.9 kynurenine 3-monooxygenase analysis comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine and application to measure kynurenine monooxygenase inhibition in cell and tissue extracts, as well as cellular assays 1.14.13.22 cyclohexanone monooxygenase analysis application of an aerobic, high-throughput growth selection platform in Escherichia coli for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance 1.14.13.22 cyclohexanone monooxygenase analysis effective halo-based selection method for mutant proteins using the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole) 1.14.13.22 cyclohexanone monooxygenase analysis protocol for linker design for beneficial effects on expression levels, enzyme stability and/or enzyme performance. Analysis of the effect of the length of a glycine-rich linker for the epsilon-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system 1.14.13.24 3-hydroxybenzoate 6-monooxygenase analysis 3-hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.14.13.24) from Rhodococcus jostii RHA1 is used as a lipid-containing reference protein for enzyme 4-hydroxybenzoate 1-hydroxylase, 4HB1H (EC 1.14.13.64). Models for Cp_4HB1H from Candida parapsilosis strain CBS604, Fo_4HB1H-like from Fusarium oxysporum, and VibMO1 from Boreostereum vibrans (UniProt ID A0A167KUL3) are generated using Rhodococcus jostii Rj_3HB6H (PDB ID 4bk1) and Pseudomonas putida Pp_SALH (PDB ID 5evy) as template structures, overview 1.14.13.39 nitric-oxide synthase (NADPH) analysis by using enzyme NADPH-d histochemistry, a marker for NOS activity, the effect of leptin on NO-producing cells in the arcuate nucleus (ARC) and paraventricular nucleus (PVN) of male Wistar rats is assessed 1.14.13.231 tetracycline 11a-monooxygenase analysis construction of a sensitive fluorescent reporter system to detect and characterize the activity of enzymes that act upon the antibiotic, tetracycline and its derivatives. In this system, expression of the lux operon is regulated by the tetracycline repressor, TetR, which is expressed from the same plasmid under the control of an arabinose-inducible promoter. Addition of very low concentrations of tetracycline derivatives, well below growth inhibitory concentrations, result in luminescence production. Introduction of a plasmid expressing TetX, a tetracycline-inactivating enzyme, causes a marked loss in luminescence due to enzyme-mediated reduction in the intracellular tetracycline concentration 1.14.13.231 tetracycline 11a-monooxygenase analysis construction of an enzyme-based biosensor for sensitive determination of tetracycline using TetX2 immobilized on modified glassy carbon electrodes. The electrode as a biosensing interface based on the oxygen reduction peak current resuls in a linear range response from 0.5 to 5 microM with a detection limit of 18 nM 1.14.13.236 toluene 4-monooxygenase analysis use of selective inhibitor phenylacetylene to differentiate toluene-degrading isolates from an aquifer 1.14.13.243 toluene 2-monooxygenase analysis enzymativc biosensor for in situ measurement of toluene using toluene ortho-monooxygenase, while an optical fiber with an oxygen-sensitive ruthenium-based phosphorescent dye serves as the transducer. The biosensor has a limit of detection of 3 microM, a linear signal range up to 100 microM, and a response time of 1 h 1.14.14.3 bacterial luciferase analysis enzyme can be used to monitor changes in gene expression as a reporter system in slow-growing mycobacteria, i.e. Mycobacterium tuberculosis strain H37Ra, determination of recombinant enzyme decay rate 1.14.14.3 bacterial luciferase analysis expression of the bacterial luciferase system in mammalian cells for generation of bioreporters for in vivo monitoring and diagnostics technology, method evaluation and optimization, overview 1.14.14.3 bacterial luciferase analysis high-throughput, homogeneous, bioluminescent assay for Pseudomonas aeruginosa gyrase inhibitors and other DNA-damaging agents based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinoline inhibition 1.14.14.3 bacterial luciferase analysis the enzyme is used as a reporter system tool for analysis of promoter and gene expression activity, overview 1.14.14.3 bacterial luciferase analysis bioluminescent assay based on a system of coupled enzymatic reactions catalyzed by bacterial luciferase and NADH:FMN-oxidoreductase to monitor toxicity and antioxidant activity of bioactive compounds such as fullerenols, perspective pharmaceutical agents, nanosized particles, water-soluble polyhydroxylated fullerene-60 derivatives. Fullerenols suppress bioluminescent intensity at concentrations above 0.01 g/l and above 0.001 g/l for C60O2-4(OH)20-24 and Fe0.5C60(OH)xOy, respectively 1.14.14.3 bacterial luciferase analysis sensitive and selective bacterial luminescence method for the detection of pyruvic acid based on lactate dehydrogenase and the bacterial luciferase-FMN:NADH oxidoreductase bioluminescence in vitro. NADH involved in the LDH reaction system can be quantitatively analyzed by the bioluminescence system. A good linear relationship between the luminescence intensity and pyruvic acid concentration is observed within the range of 0.00014–0.001 mol/l, and the pyruvic acid detection limit is 0.000085 mol/l 1.14.14.3 bacterial luciferase analysis a minimized cascade for Lux with greater ease of use, utilizes a chemoenzymatic reaction with biomimetic nicotinamide 1-benzyl-1,4-dihydronicotinamide in place of the flavin reductase reaction in the Lux system. The minimized cascade reaction can be applied to monitor bioluminescenceof the Lux reporter in eukaryotic cells effectively, and can achieve higher efficiencies than the system with flavin reductase 1.14.14.3 bacterial luciferase analysis fusion of circularly permuted Venus, a bright variant of yellow fluorescent protein, to the C-terminus of subunit LuxB to induce bioluminescence resonance energy transfer (BRET). By using decanal as added substrate, color change and ten-times enhancement of brightness is achieved in Escherichia coli upon expression. Expression of the Venus-fused luciferase in human embryonic kidney cell lines or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhances the autonomous bioluminescence 1.14.14.18 heme oxygenase (biliverdin-producing) analysis glial HO-1 expression might be an early marker of non-hemorrhagic cell injury in the striatum, time course of induction, overview 1.14.14.18 heme oxygenase (biliverdin-producing) analysis live-cell activity assay based on heterologous coexpression in Escherichia coli of heme oxygenase and its mutants and a fluorescent biliverdin biosensor 1.14.14.19 steroid 17alpha-monooxygenase analysis assay based on direct electrochemistry of CYP17A1 entrapped in didodecyldimethyl ammonium bromide-modified electrode under aerobic conditions in the supporting electrolyte solution 1.14.14.23 cholesterol 7alpha-monooxygenase analysis development of a straightforward dual-reporter bioluminescent assay to simultaneously monitor isoform CYP7A1 transcriptional regulation and cell viability in Chlamydia pneumoniae and human cytomegalovirus infected human hepatoblastoma Hep-G2 cells 1.14.14.33 ethylenediaminetetraacetate monooxygenase analysis design of a system of primers to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. The system can be effectively used for detecting the emoA gene in representatives of Alpha- and Gammaproteobacteria 1.14.14.36 tyrosine N-monooxygenase analysis direct electrochemical investigation of plant cytochrome P450s by nanodisc technology. Full length CYP79A1, CYP71E1 and NADPH P450 oxidoreductase of the dhurrin pathway are reconstituted individually in nanoscale lipid patches, nanodiscs, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 reveal reversible redox peaks with average midpoint potentials of 80 mV and 72 mV vs. Ag/AgCl, respectively. NADPH P450 oxidoreductase yields two pairs of redox peaks with midpoint potentials of 90 mV and ?300 mV, respectively. The average heterogeneous electron transfer rate constant is calculated to be 1.5 per s 1.14.14.37 4-hydroxyphenylacetaldehyde oxime monooxygenase analysis direct electrochemical investigation of plant cytochrome P450s by nanodisc technology. Full length CYP79A1, CYP71E1 and NADPH P450 oxidoreductase of the dhurrin pathway are reconstituted individually in nanoscale lipid patches, nanodiscs, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 reveal reversible redox peaks with average midpoint potentials of 80 mV and 72 mV vs. Ag/AgCl, respectively. NADPH P450 oxidoreductase yields two pairs of redox peaks with midpoint potentials of 90 mV and -300 mV, respectively. The average heterogeneous electron transfer rate constant is calculated to be 1.5 per s 1.14.14.47 nitric-oxide synthase (flavodoxin) analysis development of a robust enzyme assay for measuring NOS activity and inhibition 1.14.15.1 camphor 5-monooxygenase analysis the enzyme is useful in whole cell biocatalyst systems 1.14.15.3 alkane 1-monooxygenase analysis a certain type of alkB gene is present in almost everymember of the genus Rhodococcus. The alkB gene type may be applicable for differentiating closely related Rhodococcus species, properly assigning environmental isolates to existing Rhodococcus species, and assessing whether a new Rhodococcus isolate represents a novel species of the genus 1.14.15.4 steroid 11beta-monooxygenase analysis use of primary culture of hippocampal neurons isolated from fetus as a model system to study the regulation of the enzymes responsible for corticoid synthesis 1.14.15.6 cholesterol monooxygenase (side-chain-cleaving) analysis examination of protein-membrane interactions between P450scc and its redox partners on 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes containing cholesterol (20%), using a quartz crystal microbalance with dissipation monitoring 1.14.15.9 spheroidene monooxygenase analysis construction of a simple and cost-effective sensor independent from specific reagents and instruments for monitoring qualities of marine aquacultural fields. A sensor strain consisting of a crtA deleted host strain of Rhodovulum sulfidophilum, in which the CrtA gene is reintroduced downstream of the promoter for the dimethyl sulfide dehydrogenase (Ddh) gene cluster, is constructed to detect dimethyl sulfide in the nursery of lavers based on the conversion of the carotenoid colors 1.14.16.2 tyrosine 3-monooxygenase analysis the enzyme is useful for determination of neuronal loss after brain treatment with neurotoxic compounds, overview 1.14.16.4 tryptophan 5-monooxygenase analysis a Dugesia japonica TPH antibody is a useful marker for serotonergic neurons in planarians 1.14.17.3 peptidylglycine monooxygenase analysis enzyme PAM-dependent amidation has the potential to signal oxygen levels in the same range as the hypoxia-inducible factor (HIF) system 1.14.18.2 CMP-N-acetylneuraminate monooxygenase analysis enzyme is used for the detection of human cancer like colon cancer, melanoma, retinoblastoma cell lines and tissues, germ cell tumours and breast cancer by detection of N-glycolylneuraminic acid-containing glycolipids 1.14.18.3 methane monooxygenase (particulate) analysis gene pmoA, which encodes the key subunit of the pMMO enzyme is commonly used as functional biomarker for surveying aerobic methane or ammonia oxidizers in the environment 1.14.19.54 1,2-dehydroreticuline synthase analysis [1-2H, 13C]-(R,S)-reticuline is enzymatically converted into [1-13C]-dehydroreticuline. Release of the hydrogen atom in position C-1 of the isoquinoline alkaloid during the oxidative conversion can be exploited as a sensitive assay system for the enzyme 1.14.19.59 tryptophan 6-halogenase analysis development of a quantitative halogenase assay based on a Suzuki-Miyaura cross-coupling towards the formation of a fluorescent aryltryptophan and optimization for application in crude Escherichia coli lysate 1.14.20.6 flavonol synthase analysis an HPLC method for the determination of phenylalanine ammonia-lyase, flavanone 3-hydroxylase and flavonol synthase enzyme activity is proposed. This method is based on the determination of the compounds produced and consumed on the enzymatic reaction in just one chromatographic analysis. Optimisation of the method consideres kinetic studies to establish the incubation time to perform the assay. The method is an approach to measure the activities of the three enzymes simultaneously increasing the rapidity, selectivity and sensitivity over other methods 1.14.20.10 L-tyrosine isonitrile desaturase/decarboxylase analysis the isonitrile functional group has a characteristic Raman resonance (observed 2121 per cm for rhabduscin) enabling observation of its cellular localization 1.14.99.39 ammonia monooxygenase analysis the functional gene amoA is used to compare the diversity of ammonia oxidizing bacteria in the water column and sediment–water interface of the two freshwater lakes Plusssee and Schoehsee and the Baltic Sea 1.14.99.39 ammonia monooxygenase analysis gene amoA, which encodes the key subunit of the AMO enzyme is commonly used as functional biomarker for surveying aerobic methane or ammonia oxidizers in the environment 1.14.99.53 lytic chitin monooxygenase analysis development of a chitinase-coupled assay method and an assay based on consumption of ascorbate. There is no H2O2 production in the presence of colloidal chitin at 4 mg/ml 1.14.99.53 lytic chitin monooxygenase analysis method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity 1.14.99.54 lytic cellulose monooxygenase (C1-hydroxylating) analysis assay based on adsorption of the fluorescent dye SYTO-62, adapted to identify and localize LPMO-catalyzed formation of carboxyl groups on a cellulose surface model substrate which provides amorphous and crystalline regions alternating on a nano-flat cellulose surface 1.14.99.54 lytic cellulose monooxygenase (C1-hydroxylating) analysis a fast, robust, and sensitive spectrophotometric activity assay based on a peroxidase activity of LPMO, using 2,6-dimethoxyphenol and H2O2. The high molar absorption coefficient of the formed Product coerulignone displays a high molar absorption coefficinet that makes the assay sensitive and allows reliable activity measurements of LPMO in concentrations of approx. 0.5-50 mg/l 1.14.99.54 lytic cellulose monooxygenase (C1-hydroxylating) analysis development of a beta-glucosidase-assisted method to quantify the release of C1-oxidized glucooligosaccharides from cellulose 1.14.99.54 lytic cellulose monooxygenase (C1-hydroxylating) analysis feeding the dissolved reagents into the reaction system during measurements with obtaining a simultaneous response in the oxygen consumption rate allows in situ monitoring the LPMO inhibition and activation by EDTA and Cu2+ ions as well as studying other effects on the enzymatic reaction 1.14.99.56 lytic cellulose monooxygenase (C4-dehydrogenating) analysis a fast, robust, and sensitive spectrophotometric activity assay based on a peroxidase activity of LPMO, using 2,6-dimethoxyphenol and H2O2. The high molar absorption coefficient of the formed Product coerulignone displays a high molar absorption coefficinet that makes the assay sensitive and allows reliable activity measurements of LPMO in concentrations of approx. 0.5–50 mg/L 1.14.99.60 3-demethoxyubiquinol 3-hydroxylase analysis development of a mouse model with decreased coenzyme Q, CoQ, transgsgene expression of COQ7/CLK-1. The mice show a concomitant decrease in CoQ9, mitochondrial respiratory enzyme activity and the generation of reactive oxygen species in the mitochondria 1.15.1.1 superoxide dismutase analysis assay system based on the inhibitory effect of superoxide dismutase on the spontaneous autoxidation of 6-hydroxydopamine 1.16.3.1 ferroxidase analysis enzyme can be used in analytical biochemistry, especially for the construction of enzyme sensors, enzymes immobilized in enzyme-containing membranes coating oxygen sensitive electrodes and serve for a specific amperometric determination of their substrates in biological materials 1.17.1.4 xanthine dehydrogenase analysis use of rosy mutant strains to probe structure and function of xanthine dehydrogenase 1.17.1.9 formate dehydrogenase analysis enzyme may be used for direct spectrophotometric assay of formate 1.17.4.2 ribonucleoside-triphosphate reductase (thioredoxin) analysis development of a rapid freeze-quench apparatus for the preparation of millisecond quench time rapid freeze-quench samples which can be packed into small sample tubes used for continuous-wave and pulsed high-frequency electron paramagnetic resonance spectroscopy 1.17.9.1 4-methylphenol dehydrogenase (hydroxylating) analysis the enzyme is used as a tool to characterize catabolic differences between phenol- and p-cresol-degrading Pseudomonas fluorescens strains PC18 and PC24, overview 1.17.99.11 3-oxo-DELTA1-steroid hydratase/dehydrogenase analysis the substrate preference of AtcABC enables the application of atcABC genes as molecular biomarkers to assess anaerobic androgen biodegradation in the environment 1.18.6.2 vanadium-dependent nitrogenase analysis development of A method for removing other compounds which inhibit color development and for compensating for the interference produced by creatine. ThE method avoids time-consuming microdiffusion and routinely makes available the efficiency of ATP hydrolysis coupled to substrate reduction (ATP/2e ratio) with N2 as a reducible substrate 1.21.99.5 tetrachloroethene reductive dehalogenase analysis reductive dehalogenation assay can be used for the sensitive and rapid quantification of the corrinoid cofactor 1.97.1.1 chlorate reductase analysis proteomics-based methods are excellent tools for monitoring expression of perchlorate and chlorate-reducing enzyme systems in pure and mixed cultures, including environmental samples 2.1.1.1 nicotinamide N-methyltransferase analysis development of a NNMT activity assay using ultra-high-performance hydrophilic interaction chromatography. It allows for rapid separation of the reaction products, coupled with quadrupole time-of-flight mass spectrometric detection for enhanced sensitivity and enabling high-throughput sample analysis 2.1.1.2 guanidinoacetate N-methyltransferase analysis nonradioactive method for measuring enzyme activity in lymphoblasts by using HPLC with UV detector 2.1.1.2 guanidinoacetate N-methyltransferase analysis development of a fast, less invasive, and valid method to measure GAMT activity in lymphocytes using LC-MS/MS. GAMT activity decreases when the venous blood sample is older. Blood isolated after 48 h is not reliable to use for lymphocyte analysis for GAMT enzymatic analysis 2.1.1.4 acetylserotonin O-methyltransferase analysis the standard radio-chemical assay is successful for pineal HIOMT only, whereas specific homogenization buffers and HPLC are required to detect retinal activity 2.1.1.5 betaine-homocysteine S-methyltransferase analysis synthesis of a series of S-substituted derivatives of homocysteine as potential inhibitors of human recombinant BHMT, some of these compounds are very potent inhibitors, having IC50 values in the nanomolar range 2.1.1.6 catechol O-methyltransferase analysis high-performance liquid chromatography determination of methionine adenosyltransferase activity using catechol-O-methyltransferase-coupled fluorometric detection, a nonradioactive, sensitive, rapid, and specific method for the determination of methionine adenosyltransferase activity 2.1.1.6 catechol O-methyltransferase analysis design of electrochemical biosensor systems for the detection of specific DNA sequences in PCR-amplified nucleic acids related to the catechol-O-methyltransferase Val108/158Met polymorphism based on intrinsic guanine signal 2.1.1.8 histamine N-methyltransferase analysis guinea-pig HNMT has 292 amino acid residues, its sequences are more similar to the human and pig sequences than to the mouse and rat sequences, respectively 2.1.1.11 magnesium protoporphyrin IX methyltransferase analysis a continuous enzyme-coupled spectrophotometric assay for ChlM from Synechocystis sp. PCC 6803 is reported 2.1.1.13 methionine synthase analysis assay using HPLC with fluorescence detection 2.1.1.14 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase analysis development of a robust fluorescence-based activity assay suitable for high-throughput screening 2.1.1.17 phosphatidylethanolamine N-methyltransferase analysis development of A system that transports phosphatidylethanolamine to the mitochondria where it is methylated to phosphatidylcholine and phosphatidylcholine is transported back to other organelles and supports the growth of the mutants defective in phosphatidylcholine synthesis 2.1.1.17 phosphatidylethanolamine N-methyltransferase analysis simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and can be separated from water soluble S-[methyl-3H]adenosyl-L-methionine by organic extraction 2.1.1.28 phenylethanolamine N-methyltransferase analysis enzyme serves as a marker for tissues and cells producing epinephrine 2.1.1.28 phenylethanolamine N-methyltransferase analysis method to measure tissue contents and activity of phenylethanolamine N-methyltransferase, utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry. The method is based on measurement of deuterium-labeled epinephrine produced from reaction of norepinephrine with deuterium-labeled S-adenosyl-Lmethionine as the methyl donor. In addition to enzyme activity the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine is linear over the range of 0.1 to 5000 pM, with 0.5 pM epinephrine representing the lower limit of quantification 2.1.1.34 tRNA (guanosine18-2'-O)-methyltransferase analysis Arg-41 is the catalytic center, Lys-90, Arg-166, Arg-168, and Arg-176 are the initial sites of tRNA binding, Arg-8, Arg-19, and Lys-32 are required in the tRNA binding site for continuation the catalytic cycle, Arg-11-His-71-Met-147 interaction is involved in the structural element in release of S-adenosyl-L-homocysteine, His-34 plays a role in the assisted phosphate binding site, Arg-109 has an unknown function 2.1.1.34 tRNA (guanosine18-2'-O)-methyltransferase analysis dissociation of tRNA from the tRNA-enzyme complex after the methyl-transfer reaction is caused by the dissociation of enzyme subunits 2.1.1.34 tRNA (guanosine18-2'-O)-methyltransferase analysis reveals a fold typical of members of the SpoU clan of proteins, a subfamily of the alpha/beta-knot superfamily, with alpha-helical extensions at the N- and C-termini that are likely to be involved in tRNA binding 2.1.1.34 tRNA (guanosine18-2'-O)-methyltransferase analysis through the log phase, the content of the TrmH protein is not changed obviously, but in the stationary phase, the content of the TrmH protein is slowly reduced 2.1.1.35 tRNA (uracil54-C5)-methyltransferase analysis genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), to identify the relevant enzymatic targets. FICC-Seq is a robust method for accurate and reliable detection of relevant enzymatic target sites 2.1.1.37 DNA (cytosine-5-)-methyltransferase analysis synthesis of acyclic nucleosides containing a 5-fluorouracil nucleobase, FC-ac1, and FC-ac2 to study the reaction mechanism. The oligodeoxynucleotides containing ODN-FC-ac1 and ODN-FC-ac2 show an increased flexiblilty. A negative correlation is observed between the flexibility and ODN-DNMT complex formation. A weaker nucleobase stacking effect due to the increase in flexibility at the acyclic modification site may provide a higher flip out effect, while the local flexibility would influence the scanning and recognition steps 2.1.1.53 putrescine N-methyltransferase analysis convenient and accurate enzyme-coupled colorimetric PMT assay, based on the conversion of S-adenosyl-L-homocysteine by 5'-methylthioacenosine/S-adenosylhomocysteine nucleosidase and S-ribosylhomocysteine lyase 2.1.1.56 mRNA (guanine-N7)-methyltransferase analysis cap methylation is a principal target of the antifungal activity of sinefungin 2.1.1.56 mRNA (guanine-N7)-methyltransferase analysis enzyme is composed of catalytic vD1 (498–844) and stimulatory vD12 subunits, yeast is a powerful system for suppressor analysis of interacting poxvirus proteins, screen of amino acids in vD1-C, at which mutations restore methyltransferase function in conjunction with defective vD12 proteins, reference to the crystal structure of the microsporidian cap methyltransferase suggests that distinct functional classes of suppressors are selected 2.1.1.56 mRNA (guanine-N7)-methyltransferase analysis second Trypanosoma brucei RNA (guanine N-7) cap methyltransferase, named TbCgm1, TbCmt1 is homologous with TbCgm1, consisting of a C-terminal (guanine N-7) methyltransferase domain and an N-terminal guanylyltransferase domain, which contains signature motifs found in the nucleotidyl transferase superfamily 2.1.1.56 mRNA (guanine-N7)-methyltransferase analysis yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. Sinefungin effectively suppressed N7-MTase in the yeast system. The results validate the yeast assay system for inhibitor screening yet also demonstrate the difference between cell-based and in vitro biochemical assays. The yeast system is applied to the screening of 3000 natural product extracts 2.1.1.57 methyltransferase cap1 analysis characterization of the side chain pKa for a single lysine analogue within a 316-residue protein containing 21 lysines and 1678 carbon atoms at natural isotope abundance, achieved by the single reactive cysteine of a K175C mutant of VP39 modified to S-(beta-aminoethyl)cysteine (gamma-thialysine) using freshly prepared (13C)aziridine at room temperature 2.1.1.57 methyltransferase cap1 analysis sequence homology relative to Dengue virus 2'OMTase 2.1.1.63 methylated-DNA-[protein]-cysteine S-methyltransferase analysis ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, may significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells 2.1.1.63 methylated-DNA-[protein]-cysteine S-methyltransferase analysis N-terminal domain plays a critical structural role in maintaining an active configuration of the C-terminal domain, N-hAGT and C-hAGT domains can protect from N-methyl-N'-nitro-N-nitrosoguanidine in Escherichia coli GWR-109 cells 2.1.1.63 methylated-DNA-[protein]-cysteine S-methyltransferase analysis overexpression of human MGMT provides protection against the toxic effects of N-methyl-N'-nitro-N-nitrosoguanidine 2.1.1.63 methylated-DNA-[protein]-cysteine S-methyltransferase analysis strategy of loop insertion to alter enzyme specificity shall be general and applicable to other classes of proteins, the isolated AGT mutant can be applied in molecular imaging, where the mutant and parental AGTs are used to label two different AGT fusion proteins with different fluorophores in the same living cell or in vitro, allows establishment of fluorescence-based assays to detect protein-protein interactions and measure enzymatic activities 2.1.1.67 thiopurine S-methyltransferase analysis determination of thiopurine S-methyltransferase activity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquid chromatographic technique. This method minimzes sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional refferal laboratory 2.1.1.67 thiopurine S-methyltransferase analysis monitoring of 6-thiopurine S-methyltransferase activity is especially important when patients are treated with 6-thiopurine drugs (drugs in treatment of acute lymphoblastic leukemia), since severe bone marrow toxicity may be induced if patients have deficient 6-thiopurine S-methyltransferase activity. The HPLC-based method enables the rapid screening of 6-6-thiopurine S-methyltransferase activities in patients treated with 6-thiopurines 2.1.1.67 thiopurine S-methyltransferase analysis monitoring of TPMT activity may be of benefit to improve thiopurine therapy 2.1.1.72 site-specific DNA-methyltransferase (adenine-specific) analysis computational analysis of a published Dam knockout microarray alongside other publicly available 2.1.1.72 site-specific DNA-methyltransferase (adenine-specific) analysis Dam alters the Ca2+ regulation of Yop secretion but does not affect the temperature regulation of Yop/Ysc expression, in Dam-overproducing strains transcription of the lcrGV and yopN-tyeA operons is slightly upregulated, but LcrG is absent from lysates, while the amounts of YopN and TyeA are not changed, clpXP expression increases after Dam overproduction and the ClpP protease then degrades LcrG, thereby releasing a block in type III secretion 2.1.1.72 site-specific DNA-methyltransferase (adenine-specific) analysis structural model of the type IC M.EcoR124I DNA methyltransferase, comprising the HsdS subunit, two HsdM subunits, the cofactor S-adenosyl-L-methionine and the substrate DNA molecule 2.1.1.72 site-specific DNA-methyltransferase (adenine-specific) analysis the dimer stabilizes and enhances site-specific DNA-binding by the MTase, because the DNA-binding site may require an interfacial structure formed by both monomers, role of Type II MTases as defense systems against phage 2.1.1.72 site-specific DNA-methyltransferase (adenine-specific) analysis the first Gua is recognized by K9, removal of which abrogates the first base-pair recognition, the flipped target Ade binds to the surface of EcoDam in the absence of S-adenosyl-L-methionine, which illustrates a possible intermediate in the base-flipping pathway, the orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120 2.1.1.100 protein-S-isoprenylcysteine O-methyltransferase analysis highly sensitive capillary electrophoresis method for monitoring of the enzymic activity 2.1.1.100 protein-S-isoprenylcysteine O-methyltransferase analysis approaches to establish the quantitative structure–activity relationship of indoloacetamides as inhibitors of ICMT 2.1.1.104 caffeoyl-CoA O-methyltransferase analysis downregulating caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa lines neither reduces syringyl lignin units nor wall-bound ferulate, inconsistent with a role for this enzyme in 3-O-methylation of syringyl monolignol precursors and hydroxycinnamic acids 2.1.1.107 uroporphyrinogen-III C-methyltransferase analysis red fluorescent compounds are associated with the recombinant mature SUMT which are identified as sirohydrochlorin and trimethylpyrrocorphin by spectroscopic analysis, slightly alter the protein secondary structure 2.1.1.107 uroporphyrinogen-III C-methyltransferase analysis uroporphyrinogen III methyltransferase (UMT) is a reporter owing to the catalytic products in the cells that emit strong red fluorescence under UV light. The gene encoding the functional barley UMT (bUMT) is engineered by error-prone PCR and the application of UMT as a red fluorescent reporter in Escherichia coli is broadened. A variant, termed mbUMT, is selected and emitts stronger cell fluorescence than the wild type bUMT expressed in different Escherichia coli strains, under different promoters and induction conditions respectively. The constructed mbUMT with a C-terminal ssrA tag is degraded in cells by the protease ClpXP encoded by Escherichia coli chromosome, whereas the bUMT is expressed as active aggregates. Before they are exported to the periplasm, both proteins catalyze the substrate in the cytoplasm and emit cell fluorescence. The results suggest that the evolved bUMT is a better candidate to monitor in vivo degradation by Escherichia coli ClpXP 2.1.1.117 (S)-scoulerine 9-O-methyltransferase analysis transient RNA silencing of scoulerine 9-O-methyltransferase expression by double stranded RNA in Coptis japonica protoplasts. Utility of gene silencing based on in vitro synthesized dsRNA to study function and behavior of endogenous enzymes 2.1.1.142 cycloartenol 24-C-methyltransferase analysis in hydroponically-grown Nicotiana benthamiana, using a virus-induced gene silencing system, the transcript levels of 3-hydroxy-3-methylglutaryl-CoA reductase 1 (HMGR1), cycloartenol synthase 1 (CAS1), sterol side chain reductase 2 (SSR2) and S-adenosyl-L-Met-dependent C-24 sterol methyltransferase 1 (SMT1) have been reduced in leaf, stem and root tissues in approximately 2 weeks 2.1.1.160 caffeine synthase analysis method to produce a recombinant caffeine synthase from guarana in Escherichia coli and its purification by affinity chromatography 2.1.1.160 caffeine synthase analysis protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase in cell suspensions of Coffea arabica L. as well as its activity 2.1.1.165 methyl halide transferase analysis use of an MHT-expressing Escherichia coli to report on gene expression in a microbe within a moist soil under a range of growth conditions in a lab setting. The gas reporting approach is applied to monitor Escherichia coli conjugation within an agricultural soil and examine how hydration affects horizontal gene transfer 2.1.1.180 16S rRNA (adenine1408-N1)-methyltransferase analysis development of a fluorescence-based binding assay for 30S-NpmA interaction 2.1.1.261 4-dimethylallyltryptophan N-methyltransferase analysis ergot alkaloid pathway reconstruction in Aspergillus nidulans is an approach used to better understand the biosynthesis of these mycotoxins 2.1.1.348 mRNA m6A methyltransferase analysis radioactivity-based assays using 3H-S-adenosyl-L-methionine for kinetic characterization of m6A-RNA modifications by METTL3-14 complex and m6A-RNA demethylase ALKBH5. Assay conditions are suitable for screening for ligands in a 384-well format with Z' factors of 0.78 and 0.77, respectively 2.1.1.355 [histone H3]-lysine9 N-trimethyltransferase analysis development of a microplate biotin/avidin peptide methylation assay, which is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of histone lysine methyltransferases and other protein methyltransferases and is also well suited for high-throughput applications 2.1.1.355 [histone H3]-lysine9 N-trimethyltransferase analysis sequence context of modified residue affects G9a activity and modification in the proximal amino acids influences methylation 2.1.1.356 [histone H3]-lysine27 N-trimethyltransferase analysis three-dimensional solution structure of vSET bound to S-adenosyl-L-homocysteine and a histone H3 peptide containing mono-methylated lysine 27, only invariant active site residue tyrosine 105 in vSET facilitates methyl transfer from S-adenosyl-L-homocysteine to the substrate lysine by aligning intermolecular interactions in the lysine access channel of the enzyme 2.1.1.364 [histone H3]-lysine4 N-methyltransferase analysis ab initio quantum mechanical/molecular mechanical molecular dynamics simulations with the umbrella sampling method to determine free energy profiles for histone lysine methylation catalyzed by SET7/9 and its corresponding uncatalyzed reaction in aqueous solution, activation free energy barrier for the methyl transfer reaction catalyzed by SET7/9 is 22.5 kcal/mol, which agrees with the experimental value of 20.9 kcal/mol very well, SET7/9 lowers the barrier for the methyl-transfer reaction step by 8.4 kcal/mol compared with the uncatalyzed reaction 2.1.1.364 [histone H3]-lysine4 N-methyltransferase analysis multiple ab initio quantum mechanical/molecular mechanical free energy calculations and molecular dynamics simulations for the investigation of the methyl-transfer reaction catalyzed by SET7/9 2.1.1.364 [histone H3]-lysine4 N-methyltransferase analysis development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. The use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay 2.1.2.1 glycine hydroxymethyltransferase analysis molecular dynamics simulations and interaction energy analysis for compounds designed as potential selective inhibitors of Plasmodium falciparum SHMT based on the conformational and dynamics differences observed between the residues Asp146 and Glu137 in the active sites of human SHMT and Plasmodium falciparum SHMT, respectively 2.1.2.1 glycine hydroxymethyltransferase analysis protein sequence of the two isoforms, the short form SHMT-S and the long form SHMT-L, show 59% identity 2.1.2.1 glycine hydroxymethyltransferase analysis SHMT1 is a zinc-inducible gene, provides first mechanism for the regulation of folate-mediated one-carbon metabolism by zinc 2.1.3.1 methylmalonyl-CoA carboxytransferase analysis use of enzyme as a molecular marker for specific detection of Propionibacterium freudenreichii in human microbiota. Rapid quantification of bacteria by real time PCR using enzyme operon 2.1.3.3 ornithine carbamoyltransferase analysis after administration of carbon tetrachloride, allyl alcohol, D-galactosamine, lipopolysaccharide, and concanavalin A, the significant increase in the serum levels of the markers is faster in type-I arginase and ornithine carbamoyltransferase than aspartate aminotransferase and alanine aminotransferase. The extent of the increase at the peak is always higher in type-I arginase and ornithine carbamoyltransferase than in spartate aminotransferase and alanine aminotransferase 2.2.1.1 transketolase analysis low cost, rapid colorimetric transketolase assay, able to detect value above 8% bioconversion using non-alpha-hydroxylated aldehydes as acceptor substrates. The assay is significantly faster and more convenient to use than HPLC and can be used with a range of aliphatic and aromatic aldehydes. In addition, analysis of the alpha,alpha'-dihydroxyketone produced in the bioconversion can be quantified using this assay system with high-throughput. Furthermore, this method has the potential to be used to screen other chemical reactions or bioconversions leading to the formation of products possessing a 2-hydroxyketone motif 2.2.1.1 transketolase analysis a rapid microplate-based approach for measuring the denaturation curves by intrinsic tryptophan fluorescence for simple monomeric and two-state unfolding proteins like transketolase 2.2.1.1 transketolase analysis coupling of a transketolase reaction that converts D-fructose 6-phosphate to D-erythrose 4-phosphate, which can then be converted to 4-phosphate D-erythronate using erythrose-4-phosphate dehydrogenase, a reaction that reduces NAD+ to NADH and can be easily followed spectrophotometrically 2.2.1.1 transketolase analysis establishment of a rapid microplate-based HPLC assay for transketolase, for rapidly determining substrate and product concentration suitable for optimisation of biocatalytic process conditions and screening directed evolution libraries, which can be used to determine transketolase activity with a throughput of up to 1200 samples per day, whereas the well-to-well variation from HPLC measurement is just 1.9% for the lowest activities measured 2.2.1.1 transketolase analysis highly effective, stable and sensitive method for measuring TKT activity, incorporating xylulokinase, which induces generation of xylulose 5-phosphate from xylulose, from Saccharomyces cerevisiae into conventional TKT assay 2.2.1.1 transketolase analysis tetrazolium red-based colorimetric assay to screen for transketolase activity with a range of aldehyde acceptors. The assay is able to detect >8% bioconversion using non-alpha-hydroxylated aldehydes as acceptor substrates and is significantly faster and more convenient to use than chromatographic procedures 2.2.1.1 transketolase analysis development of a gas chromatography-based method to screen enzyme activity and stereoselectivity on a wide range of polyol substrates. Method shows reproducibility, sensitivity and range of detection. In combination with HPLC screening, it can be used efficiently to test mutant libraries obtained by directed evolution methods 2.2.1.6 acetolactate synthase analysis Vis/NIR spectroscopy combined with LS-SVM, i.e. least squares-support vector machine, can be successfully applied for the determination of acetolactate synthase activity and protein content of rapeseed leaves. Vis/NIR spectroscopy is a promising detection technique for the on field applications in oilseed rape 2.2.1.7 1-deoxy-D-xylulose-5-phosphate synthase analysis development of a sensitive assay suitable for plant extracts that is based on the decarboxylation of labeled pyruvate and detection of 13CO2 by isotope ratio mass spectrometry 2.3.1.1 amino-acid N-acetyltransferase analysis due to the close phylogenetic relationship and similar biochemical properties of xanthomonad NAGS-K and mammalian NAGS the enzyme from Xanthomonas campestris could become a good model for mammalian NAGS in structural, biochemical and biophysical studies 2.3.1.1 amino-acid N-acetyltransferase analysis assay using UPLC-MS/MS in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d5 acid) dilution for the quantitative detection of N-acetylglutamate produced by NAGS 2.3.1.4 glucosamine-phosphate N-acetyltransferase analysis development of a fluorescence assay where reactive thiols of CoA, generated from the acyl transfer reaction, are monitored using monobromobimane 2.3.1.5 arylamine N-acetyltransferase analysis development and evaluation of genotoxicity screening sensors measuring DNA damage from metabolsim of arylamines and based on ultrathin films containing DNA and N-acetyltransferase on pyrolytic graphite electrodes. N-acetyltransferase in the film catalyzes the conversion of the arylamine 2-aminofluorene to 2-acetylaminofluorene by acetyl coenzyme A-dependent N-acetylation. DNA damage is measured subsequently using ctaalytic voltammetric oxidation with Ru(bpy)32+ 2.3.1.5 arylamine N-acetyltransferase analysis the NAT enzyme activates an arylhydroxamic acid functionality into a nitrenium ion that reacts fast, covalently, and under neutral conditions with nucleophilic residues of neighboring proteins. Strong labeling is only observed with an arylhydroxamic acid bearing an electron donating substituent. Clear labeling is achieved on a subcellular level in living cells that are transfected with a genetically targeted NAT to the nucleus or the cytosol 2.3.1.6 choline O-acetyltransferase analysis real-time multiplex PCR technique can be carried out in a single tube and can differentiate between the polymorphic sites of the ChAT gene associated with Alzheimer’s disease 2.3.1.7 carnitine O-acetyltransferase analysis specific activity is higher than in commercial preparations from other sources, enzyme prepared from yeast may be used for determination of carnitine in biological materials, this preparation is free of acetyl-CoA-deacetylase activity 2.3.1.7 carnitine O-acetyltransferase analysis in medicine and biochemical practice, the enzyme isolated from breast muscle is used analytically 2.3.1.7 carnitine O-acetyltransferase analysis enzyme in pure form facilitates assay of carnitine and its acyl derivatives in tissues 2.3.1.7 carnitine O-acetyltransferase analysis determination of free and total carnitine in plasma by an enzymatic reaction and spectrophotometric quantitation spectrophotometric determination of carnitine. the detection limit is 0.0017 mM/l in plasma 2.3.1.7 carnitine O-acetyltransferase analysis paper chromatography-based assay for the detection of acetylcholine and L-acetylcarnitine. The assay can be used to measure both choline acetyltransferase and carnitine acetyltransferase activity in the same samples 2.3.1.9 acetyl-CoA C-acetyltransferase analysis assay based on the specific absorbance at 303 nm of the Mg2+-enolate complex of acetoacetyl-Coa 2.3.1.13 glycine N-acyltransferase analysis determination of kinetic parameters of recombinant human enzyme using the traditional colorimetric method and a HPLC-ESI-MS/MSmethod 2.3.1.20 diacylglycerol O-acyltransferase analysis dehydration is an effective method of storage of samples destined for estimation of the microsomal enzyme activity 2.3.1.22 2-acylglycerol O-acyltransferase analysis development of a MGAT enzymatic assay of human intestinal microsomes using a high-throughput mass spectrometry-based detection system 2.3.1.22 2-acylglycerol O-acyltransferase analysis development of LC/ESI/MS/MS-based fat absorption assay using 1-oleoyl-glycerol-d5 and U13C-triglyceride to assess the ability of MGAT2 inhibitors to inhibit fat absorption in CD1 mice by a meal tolerance test 2.3.1.24 sphingosine N-acyltransferase analysis fluorescent CERS assay with quantification using thin-layer chromatography or high-performance liquid chromatography 2.3.1.26 sterol O-acyltransferase analysis use of inhibitor K-604, selective for isoform ACAT-1, to measure the individual enzymatic activities of isoforms ACAT-1 and ACAT-2 in semniferous tubule 2.3.1.28 chloramphenicol O-acetyltransferase analysis valuable tool in studies of eukaryotic gene expression, quantitative aspects of the use of bacterial chloramphenicol acetyltransferase as a reporter system in the yeast Saccharomyces cerevisiae 2.3.1.28 chloramphenicol O-acetyltransferase analysis application of chloramphenicol acetyltransferase as a new and convenient selectable marker for stable nuclear transformation as well as potential chloroplast transformation of Cyanidioschyzon merolae 2.3.1.42 glycerone-phosphate O-acyltransferase analysis peroxisomal enzyme can be used as marker enzyme for peroxisomal membrane due to insensitivity to glycerol-3-phosphate 2.3.1.45 N-acetylneuraminate 7-O(or 9-O)-acetyltransferase analysis methods for assaying the activity of sialate-O-acetyltransferases are presented 2.3.1.45 N-acetylneuraminate 7-O(or 9-O)-acetyltransferase analysis the non-radioactive ELISA can quickly detect sialate-O-acetyltransferase, and thus, may become a suitable tool for acute lymphoblastic leukemia-monitoring in larger scale 2.3.1.48 histone acetyltransferase analysis methods for the analysis of histone acetyltransferase activity in vitro 2.3.1.50 serine C-palmitoyltransferase analysis useful as a model to elucidate the reaction mechanism 2.3.1.57 diamine N-acetyltransferase analysis colorimetric assay for monitoring enzyme SSAT1 activity in zebrafish using a 412-nm absorption spectrum in the presence of Ellman's reagent. The method is cost-effective and simple and also applicable to detect the cellular enzyme activity 2.3.1.59 gentamicin 2'-N-acetyltransferase analysis rapid analysis of antibiotic concentration in serum 2.3.1.67 1-alkylglycerophosphocholine O-acetyltransferase analysis procedure for identification of LPCAT2-specific inhibitors via high-throughput screening 2.3.1.75 long-chain-alcohol O-fatty-acyltransferase analysis development of an enzymic assay by indirect measurement of fatty alcohol and fatty acyl-CoA esterification catalyzed by the WS/DGAT enzyme using the coupled reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with free CoA liberated during the esterification reaction 2.3.1.78 heparan-alpha-glucosaminide N-acetyltransferase analysis direct method to assay HGSNAT enzymatic activity using fluorescent BODIPY-glucosamine, i.e. 1-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl-glycylamino]-beta-D-glucosamine, as a substrate 2.3.1.78 heparan-alpha-glucosaminide N-acetyltransferase analysis direct method to assay HGSNAT enzymatic activity using fluorescent BODIPY-glucosamine as a substrate. The assay is specific and potentially applicable for the biochemical diagnosis of mucopolysaccharidosis III type and high-throughput screening for HGSNAT inhibitors 2.3.1.82 aminoglycoside 6'-N-acetyltransferase analysis apication of the aacA-aphD gene as an efficient marker for plastid transformation. It produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. When tethered to plastid expression signals, the aac6-aph2 gene provides sufficiently strong resistance to tobramycin and gentamicin to select plastid transformants in tobacco 2.3.1.84 alcohol O-acetyltransferase analysis method to quantify the activity of alcohol acetyltransferase using a SnO2-based sensor of electronic nose, on the basis of its higher sensitivity to the reducing alcohol than the oxidizing ester. The method has been successfully applied to determine the alcohol acetyltransferase activity of a cider yeast strain 2.3.1.85 fatty-acid synthase system analysis targeted liquid chromatography-mass spectroscopy method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship screening cascade. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values 2.3.1.85 fatty-acid synthase system analysis simple mass spectrometry-based assay that affords monitoring of FASN activity and its product specificity. Purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, at defined time points the reaction mixture is spiked with an internal non-esterified palmitic acid standard and extracted, and the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. The assay supports essentially noise-free detection and absolute quantification of de novo synthetized 13C-labeled non-esterified fatty acids 2.3.1.86 fatty-acyl-CoA synthase system analysis development of a high-throughput cellular bioluminescent reporter screen for inhibitors of the FASII pathway 2.3.1.87 aralkylamine N-acetyltransferase analysis colorimetric method for rapid detection of biogenic amines based on arylalkylamine N-acetyltransferase with distinct advantages including simple handling, high speed, low cost, good sensitivity and selectivity. The enzyme can specially recognize biogenic amines and catalyse their acetylation in the presence of AcCoA, and even in the presence of amino acids and aminoglycoside antibiotics having similar structures 2.3.1.94 6-deoxyerythronolide-B synthase analysis kinetic assay for the megasynthase that stoichiometrically relates NADPH consumption to polyketide production 2.3.1.95 trihydroxystilbene synthase analysis use of expression of stilbene synthases in Nicotiana benthamiana via Agrobacterium tumefaciens-mediated transient expression as a rapid and direct approach to perform functional analysis of stilbene synthases 2.3.1.95 trihydroxystilbene synthase analysis analysis of resveratrol levels in both berry skins and leaves in 95 grapevine accessions. Trans-resveratrol content ranges from 0.03 to 68.4 microg/g fresh weight in berry skins and from 0.04 to 11.69 microg/g fresh weight in leaves. Eight loci are significantly associated to resveratrol content in a wide grapevine germplasm collection. Three simple sequence repeat loci in berry skins with positive effects are mapped onto Chromosome 16. These loci are close to STS17 or STS27 2.3.1.97 glycylpeptide N-tetradecanoyltransferase analysis the automated assay can be used for proteomic studies to determine the myristoylation state of any protein 2.3.1.105 alkylglycerophosphate 2-O-acetyltransferase analysis TCA-precipitation method for the determination of 1-alkyl-sn-glycero-3-phosphate:acetyl-CoA acetyltransferase in human renal tissue 2.3.1.135 phosphatidylcholine-retinol O-acyltransferase analysis ARPE-19 cell system is appropriate for studying the visual cycle enzymes 2.3.1.183 phosphinothricin acetyltransferase analysis an immunoassay method is developed to quantitatively detect phosphinothricin-N-acetyltransferase encoded by the Bialaphos resistance gene in genetically modified pepper 2.3.1.183 phosphinothricin acetyltransferase analysis the electrochemical DNA biosensor is used to the detection of the PAT gene sequence, which is one of screening detection genes ot the transgenic plants 2.3.1.183 phosphinothricin acetyltransferase analysis use of pat as marker gene for the screening of genetically modified crops 2.3.1.183 phosphinothricin acetyltransferase analysis use of phosphinothricin acetyltransferase gene as marker for the screening of genetically modified crops 2.3.1.211 bisdemethoxycurcumin synthase analysis engineering of an in vivo reporter assay for phenylalanine ammonia-lyase efficiency in Escherichia coli based on a plant type III polyketide biosynthetic pathway. The candidate phenylalanine ammonia-lyases are coexpressed with 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase from Oryza sativa. A microplate-based assay is used to measure the titer of dicinnamoylmethane 2.3.1.217 curcumin synthase analysis method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. By applying this method, and constructing a dendrogram based on these markers, seven Curcuma species are clearly distinguishable and Curcuma longa specimens are geographically distinguishable 2.3.1.217 curcumin synthase analysis method to detect expression differences between species in detail, based on RNA sequencing analysis. The difference in the contents of curcuminoids among the species can be explained by the changes in the expression of genes encoding diketide-CoA synthase, and curcumin synthase at the branching point of the curcuminoid biosynthesis pathway 2.3.1.218 phenylpropanoylacetyl-CoA synthase analysis method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. By applying this method, and constructing a dendrogram based on these markers, seven Curcuma species are clearly distinguishable and Curcuma longa specimens are geographically distinguishable 2.3.1.218 phenylpropanoylacetyl-CoA synthase analysis method to detect expression differences between species in detail, based on RNA sequencing analysis. The difference in the contents of curcuminoids among the species can be explained by the changes in the expression of genes encoding diketide-CoA synthase, and curcumin synthase at the branching point of the curcuminoid biosynthesis pathway 2.3.1.259 N-terminal methionine Nalpha-acetyltransferase NatF analysis development of a proteolysis-based assay, named PROMPT, i.e. PROtease assay for Membrane Protein Topology, to determine the topology of protein C-termini 2.3.1.301 mycobacterial beta-ketoacyl-[acyl carrier protein] synthase III analysis discontinuous assay for the FabH enzyme that eliminates the need for the washing steps or specialty scintillation proximity assay beads and the preparation of acyl carrier proteins. The assay involves the reduction of radiolabled long-chain beta-ketoacyl CoA product to its dihydroxy derivative, which partitions into a nonpolar phase for quantitation, while the reduced radiolabeled substrate derivative remains in the aqueous phase 2.3.1.302 hydroxycinnamoyl-CoA:5-hydroxyanthranilate N-hydroxycinnamoyltransferase analysis method to measure hydroxycinnamoyl-CoA:5-hydroxyanthranilate N-hydroxycinnamoyltransferase reaction rates in real time using 5,5'-dithio-bis-(2-nitrobenzoic acid) (i.e. DTNB) to spectrophotometrically quantify, in real time, the release of free CoA during the transferase reaction 2.3.1.304 poly[(R)-3-hydroxyalkanoate] polymerase analysis deveopment of a phaE biosensor as a a biomarker for microorganisms that contain class III PHA synthase. The phaE biosensor has a high specificity for polyhydroxyalkanoate-producing haloarchaea. The lowest amount of genomic DNA of Haloquadratum walsbyi detected by the biosensor is approximately 250 fg. The phaE biosensor can be applied for screening of polyhydroxyalkanoate-producing haloarchaea from environmental samples 2.3.2.2 gamma-glutamyltransferase analysis assays for gamma-glutamyl transferase (GGT1) activity in blood are widely used in a clinical setting to measure tissue damage 2.3.2.2 gamma-glutamyltransferase analysis gamma-glutamyltransferase can be used as a marker related with oxidative stress 2.3.2.2 gamma-glutamyltransferase analysis GGT level is a good predictor of low coronary flow reserve at the receiver-operating characteristic curve 2.3.2.2 gamma-glutamyltransferase analysis plasma layer stratification might occur in primary lithium-heparin tubes for a limited number of routine clinical chemistry tests, introducing a statistically significant bias in the measurement of gamma-glutamyltransferase, lactate dehydrogenase, triglycerides and C-reactive protein in the upper vs. the bottom section. When delayed testing is necessary for these parameters, plasma should be separated after centrifugation and appropriately mixed before delayed/repeated analysis or aliquoting 2.3.2.2 gamma-glutamyltransferase analysis analytical method for the selective determination and speciation of S-nitrosoglutathione and its metabolite S-nitrosocysteinylglycine, based on liquid chromatography separation coupled to on-line enzymatic hydrolysis of S-nitrosoglutathione by commercial GGT. The limit of quantitation for S-nitrosoglutathione and S-nitrosocysteinylglycine in plasma ultrafiltrate is 5 nM, with a precision of 1-6% within the 5-1500 nM dynamic linear range. The method is applied to evaluate the recovery of exogenous S-nitrosoglutathione after addition of aliquots to human plasma samples presenting with different total GGT activities. By inhibiting GGT activity in a time-dependent manner, the recovery of S-nitrosoglutathione is inversely correlated with plasmatic levels of endogenous GGT 2.3.2.2 gamma-glutamyltransferase analysis simple, robust and highly sensitive assay based on an alpha-phenylthio-containing glutathione peptide mimic gamma-glutamyl-D,L-phenylthioglycylglycine that eliminates thiophenol upon GGT-catalyzed hydrolysis of the c-glutamyl peptide bond 2.3.2.3 lysyltransferase analysis a method to obtain a stable enriched membrane fraction containing MprF, and the techniques necessary to quantitatively monitor its activity in vitro and in vivo is reported 2.3.2.6 lysine/arginine leucyltransferase analysis a novel method to quantify L/F transferase activity by matrix assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF, is reported 2.3.2.13 protein-glutamine gamma-glutamyltransferase analysis specific tests for measuring the activities of TGase 1 and factor XIII based on their ability to incorporate biotinylated peptides onto spermine-conjugated wells. The rapid and sensitive colorimetric assay shows high sensitivity when TGase isozymes are assayed using their identified preferred substrate peptide. The limit of detection for factor XIII and TGase 1 is as low as 0.01 mU/ml. In each case, good linearity is obtained 2.3.2.B14 L,D-transpeptidase analysis synthesis of fluorescently tagged peptidoglycan analogs of L,D-transpeptidase substrates to directly interrogate Ldt function in live cells. Structural analogs of nascent peptidoglycan are metabolically incorporated into the peptidoglycan scaffold by Ldts. Subtle modifications such as amidation of iso-Glu can control peptidoglycan crosslinking 2.3.2.15 glutathione gamma-glutamylcysteinyltransferase analysis a HPLC method for the analysis of the activity of phytochelatin synthase is developed 2.3.2.17 N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysyl-(N6-glycyl)-D-alanyl-D-alanine-diphosphoundecaprenyl-N-acetylglucosamine:glycine glycyltransferase analysis definition of a consensus sequence of the femA gene and analysis of interspecies variations for staphylococci species-specific identification 2.3.2.26 HECT-type E3 ubiquitin transferase analysis application of ubiquitin-activity-based probes such as ubiquitin-vinyl methylester, ubiquitin-propargylamide to label HECT domains. E2-Ub-activity-based probes can label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1 2.3.2.26 HECT-type E3 ubiquitin transferase analysis development of ubiquitin variants that inhibit or activate HECT E3 enzymes. Ubiquitin variant inhibitors block the E2-binding site, and activators occupy a ubiquitin-binding exosite. Ubiquitin variants reveal regulation mechanisms among NEDD4 subfamily HECTs and are useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids. Ubiquitin variant activators bind to the N-lobe exosite and differentially modulate related HECT E3 ligases 2.3.2.27 RING-type E3 ubiquitin transferase analysis development of a structure decomposition method that utilizes network analysis and computational thermodynamic measures of fold stability changes upon amino acids alterations 2.3.2.31 RBR-type E3 ubiquitin transferase analysis co-crystal structure of HOIP RBR domain with single-domain antibody, use as a platform for soaking of ligands that target the active site cysteine of HOIP 2.3.2.31 RBR-type E3 ubiquitin transferase analysis development of activity-based probes by reengineering of a ubiquitin-charged E2 conjugating enzyme using tosyl-substituted doubly activated genes and application in profiling the transthiolation activity of the RBR E3 ligase Parkin in vitro and in cellular extracts 2.3.2.31 RBR-type E3 ubiquitin transferase analysis development of chemical probes, ubiquitin C-terminal fluorescein thioesters UbMES and UbFluor, to qualitatively and quantitatively assess the activity of the RBR E3 ligase PARKIN in a simple experimental setup and in real time using fluorescence polarization. UbFluor quantitatively detects naturally occurring activation states of parkin caused by Ser65 phosphorylation and phosphorylated ubiquitin 2.3.2.32 cullin-RING-type E3 NEDD8 transferase analysis development of peptides to probe the protein-protein interaction between the cullin-2 scaffold protein and the adaptor subunit elongin BC within the context of the von Hippel-Lindau complex. Peptides MSLKPRVV and MS-cyclohexylglycine-KPRVV can disrupt the native interaction between the von Hippel-Landau complex and Cul2 2.3.3.1 citrate (Si)-synthase analysis cellular stress marker 2.3.3.8 ATP citrate synthase analysis coupled fluorometric assay for EC 2.3.1.1 based on coupling coenzyme A production to the oxidation of NADH via ATP-citrate lyase and malate dehydrogenase 2.3.3.10 hydroxymethylglutaryl-CoA synthase analysis a visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase is established 2.4.1.1 glycogen phosphorylase analysis a highly sensitive and convenient assay for glycogen phosphorylase activity by analysing its chainlengthening action on a fluorogenic maltooligosaccharide derivative in a glucose-1-phosphate-rich medium. A maltotetraosyl residue comprising the non-reducing-end of a pyridylaminated maltooligosaccharide is indispensable for the chain-lengthening action of phosphorylase, and pyridylaminated maltohexaose is the most suitable substrate. Pyridylaminated maltoheptaose produced by the chain elongation reaction can be isolated and quantified at 10 fmol. Method has about 1000 times greater sensitivity than the spectrophotometric phosphate assay 2.4.1.1 glycogen phosphorylase analysis use of phosphorylase and tissue ATPase as biomarkers for exposure of aquatic organisms to cypermethrin intoxication 2.4.1.8 maltose phosphorylase analysis usage of the enzyme for a conductometric specific phosphate biosensor, method development and evaluation, overview 2.4.1.12 cellulose synthase (UDP-forming) analysis antibodies against cotton Gossypium hirsutum Ces A3 are used to identify and localize Ces A proteins in Micrasterias denticulata 2.4.1.14 sucrose-phosphate synthase analysis sucrose-phosphate synthase is a biochemical marker of high sucrose accumulation in sugarcane 2.4.1.15 alpha,alpha-trehalose-phosphate synthase (UDP-forming) analysis trehalose-6-phosphate synthase is an intrinsic selection marker for plant transformation 2.4.1.16 chitin synthase analysis the chs3 gene encoding the chitin synthase 3 is used for synthetically lethality screening for mutation determination, overview 2.4.1.17 glucuronosyltransferase analysis after infection of Sf9 insect cells with increasing amounts of recombinant baculovirus, encoding either UGT1A9 or UGT2B7, the correlation between glucuronidation rates and degree of infection follows different trends, depending on whether activity is the actual activity measured or is corrected for UDP-glucuronosyltransferase expression level. Above a certain low level of infection, further increases in infection ratios lead to a large decline in normalized activity, presumably due to the presence of full-length but inactive enzyme in the sample. Inaccuracies in comparison of normalized activity between different batches of a recombinant UDP-glucuronosyltransferase can be reduced by lowering the degree of infection of the insect cells, in combination with careful monitoring of UDP-glucuronosyltransferase expression. Poly-His-containing peptides, fused to the UDP-glucuronosyltransferase C-terminus, allow sensitive immunodetection of expressed enzymes with monoclonal antibodies. A minor increase in the Km values has been detected in the His-tagged UDP-glucuronosyltransferases, but no changes in parameters such as the kinetic model and the effects of albumin addition 2.4.1.20 cellobiose phosphorylase analysis quantitative determination of cellobiose in presence of glucose or glucose-1-phosphate 2.4.1.25 4-alpha-glucanotransferase analysis investigation of molecular characteristics, microstructures, and physicochemical properties of modified starch gels prepared from partial enzyme treatments of the corn and rice starch pastes. Unlike the native and partially modified normal starches, the native and partially modified waxy starches can not form gels strong enough for textural analysis after 24 h for gel setting. The partially modified normal starches show specific apparent amylose contents and maximum iodine absorption wavelength, as well as the tri-modal molecular weight profiles and flatter side-chain distributions. The partially modified normal starch gels possess fractured surfaces with discontinuous crystalline fibrous assembly, which result in more brittle, rigid, and resilient gels compared with the native gels 2.4.1.27 DNA beta-glucosyltransferase analysis use of recombinant enzyme to estimate global 5-hydroxymethyl cytosine content in a variety of genomic DNAs. Most of the genomic DNAs derived from vertebrate tissue and cell lines contain 5-hydroxymethyl cytosine. DNA from mouse, human, and bovine brains displays 0.5?0.9% of the total nucleotides as 5-hydroxymethyl cytosine, which is higher compared to the levels found in other tissues. A comparison between cancer and healthy tissue genomes suggests a lower percentage of 5-hydroxymethyl cytosine in cancer 2.4.1.34 1,3-beta-glucan synthase analysis simple and sensitive method for characterization of enzyme products by analysis of newly synthesized polysaccharides by 13C-nuclear magnetic resonance 2.4.1.34 1,3-beta-glucan synthase analysis glucan synthase activity assay based on size-exclusion chromatography coupled with pulsed-amperometric detection and radiation counting (SEC-PAD-RC), which allows for the simultaneous characterization of the amount and length of the polymer product 2.4.1.35 phenol beta-glucosyltransferase analysis development of a high-throughput assay for screening recombinant UDP-glucose-dependent glycosyltransferases and analysis of the activity with xenobiotics 2,4,5-trichlorophenol, 4-nitrophenol, 2-chloro-4-trifluoromethylphenol, 1-naphthol, triclosan and expression profiling of UDP-glucose-dependent glycosyltransferases following treatment with the herbicide safener fenclorim 2.4.1.41 polypeptide N-acetylgalactosaminyltransferase analysis development of a bump-hole chemical reporter system for studying GalNAc-T activity in vitro. GalNAc-T2 ire rationally engineered to contain an enlarged active site (hole) and probed with a collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach is applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate 2.4.1.41 polypeptide N-acetylgalactosaminyltransferase analysis development of isogenic cell-model systems withTet-On inducible expression of GalNAc-T genes GALNT2 and GALNT11 in a knockout background in HEK-293 cells to identify O-glycopeptides differentially affected by induction of GalNAc-T2 or -T11 2.4.1.43 polygalacturonate 4-alpha-galacturonosyltransferase analysis development of enzyme-bound solid-phase support containing long-pectic oligogalacturonides anchored to the resin material via disulfide-containing cleavable linker, enzyme is reversibly bound, for performance of solid-phase biosynthetic reaction to investigate the pectin oligomer biosynthesis, product identification by MALDI-TOF mass spectrometry, overview 2.4.1.B62 small GTPase glucosyltransferase analysis development of a rapid and simple method for toxin A removal from culture filtrates 2.4.1.B62 small GTPase glucosyltransferase analysis evaluation of the C.Diff Quik Chek Complete Assay hich tests for the presence of both glutamate dehydrogenase and toxins A and B. The assay allows 88% of specimens to be accurately screened as either positive or negative for the presence of toxigenic Clostridium difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant allows the easy, rapid, and highly sensitive and specific diagnosis of Clostridium difficile disease 2.4.1.B64 glucosyltransferase Waag analysis activity assay for WaaG using 14C-labeled UDP-glucose and lipopolysaccharide purified from a WaaG deletion strain of Escherichia coli. Addition of the lipids phosphatidylglycerol and cardiolipin, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increase activity 2.4.1.69 type 1 galactoside alpha-(1,2)-fucosyltransferase analysis determination of GDP-L-fucose:galactoside 2'-fucosyltransferase in human serum employing o-nitrophenyl-beta-D-galactopyranoside, phenyl-2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside, or phenyl beta-D-thiogalactopyranoside as acceptors. The method can simultaneously monitor possible competing reactions which may interfere with determination of alpha-2-L-fucosyltransferase activity 2.4.1.152 4-galactosyl-N-acetylglucosaminide 3-alpha-L-fucosyltransferase analysis synthesis of fluorescein-isothiocyanate-labeled and carboxyfluorescein-labeled NDP-beta-L-fucose derivatives, and application in labeling of different glycoproteins with the aid of fucosyltransferases. The fluorescein-isothiocyanate-labeled fucose is the best of these substrates, and the bacterial enzyme FucT tolerates the fluorescent substrates better than human fucosyltransferases 2.4.1.165 N-acetylneuraminylgalactosylglucosylceramide beta-1,4-N-acetylgalactosaminyltransferase analysis the enzyme is a marker of the colonic cell maturation 2.4.1.186 glycogenin glucosyltransferase analysis development of assay method with n-dodecyl-beta-D-maltoside as substrate 2.4.1.198 phosphatidylinositol N-acetylglucosaminyltransferase analysis PIGA proteins possess characteristic motifs that can be used for identifying PIG-A proteins from newly sequenced genomes. Statistical as well as phylogenetic analysis demonstrates that PIG-A proteins evolved from glycosyltransferases, PIG-A proteins from archaeabacteria and primitive eukaryotes are closer to bacterial GT4 glycosyltransferases than to eukaryotic PIG-A proteins and should be classified as such rather than as 'true' PIG-A protein 2.4.1.212 hyaluronan synthase analysis a rapid, continuous, and convenient three-enzyme coupled UV absorption assay is developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase. Activity is measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products are characterized by gel electrophoresis. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media 2.4.1.212 hyaluronan synthase analysis quantification of newly synthesized hyaluronan by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. The method measures HAS activity in the plasma membrane fraction and also in the cytosolic membranes. The technique is used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1, platelet-derived growth factor BB, and tunicamycin on HAS activities 2.4.1.242 NDP-glucose-starch glucosyltransferase analysis based on a generated monoclonal antibody, a high throughput ELISA is developed that allows the quick idenification of wheat lines carrying the 7A allele of GBSS1 with minute amounts of sample 2.4.1.252 GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase analysis method for real-time detection of recombinant GumH enzymatic activity using a Quartz Crystal Microbalance with dissipation monitoring, QCM-D. Monitoring the real-time cellobiose-diphosphate-polyprenol carrier lipid-QCM-D transducer in the presence of GDP-man and GumH enzyme shows a mass increase, indicating the transfer of mannose. The determination of mannosyltransferase function is validated by a HPLC method developed for determination of GDP produced by enzymatic reaction 2.4.1.255 protein O-GlcNAc transferase analysis application of a high-throughput OGT assay to a library of peptides 2.4.1.255 protein O-GlcNAc transferase analysis metabolic labeling method to introduce the diazirine photocross-linking functional group onto O-GlcNAc residues in mammalian cells. Cells are engineered to produce diazirine-modified UDP-GlcNAc (UDP-Glc-NDAz), which is transferred to substrate proteins by endogenous OGT, producing O-GlcNDAz. Modified proteins can be covalently cross-linked to their binding partners, providing information about O-GlcNAc-dependent interactions 2.4.1.255 protein O-GlcNAc transferase analysis peptide microarray approach to discover novel OGT substrates and study its specificity 2.4.1.255 protein O-GlcNAc transferase analysis single-well OGT enzyme assay that utilizes His6-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-nitrilotriacetic acid plate OGT assay facilitates discovery of OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants 2.4.1.303 UDP-Gal:alpha-D-GlcNAc-diphosphoundecaprenol beta-1,3-galactosyltransferase analysis the synthetic substrate GlcNAcalpha-PO3-PO3-(CH2)11-O-phenyl is used to develop an assay for UDP-Gal:GlcNAc-R galactosyltransferase 2.4.1.313 protein O-mannose beta-1,3-N-acetylgalactosaminyltransferase analysis detection and/or imaging of O-GlcNAc as well as potential sites for O-GlcNAc modification on biological samples. Occupied modification sites are detected using in vitro incorporation of azido-GalNAc by B3GALNT2, and unoccupied sites are detected by in vitro incorporation of azido-GlcNAc by O-GlcNAc transferase (OGT), via click chemistry 2.4.1.345 phosphatidyl-myo-inositol alpha-mannosyltransferase analysis development of a cell-free assay. Membranes from Mycobacterium smegmatis overexpressing the pimA gene incorporate mannose from GDP-[14C]Man into di- and tri-acylated phosphatidylinositol mono-mannosides 2.4.2.1 purine-nucleoside phosphorylase analysis determination of enzymic activity using commercially available substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside and photometric assessment of the reaction 2.4.2.1 purine-nucleoside phosphorylase analysis development of a set of empirical scoring functions and its application to evaluate binding affinities and docking results 2.4.2.1 purine-nucleoside phosphorylase analysis electrochemical biosensor to assay purine nucleoside phosphorylase activity based on the facilitation of sliver nanoparticles, which are modified on an electrode surface, to the electron transfer reactivity of guanosine and guanine. Assay displays a broad linear range of 4-20 units/ml with a detection limit of 0.1 unit/ml. Additional development development of a rapid UV–vis spectroscopy assay method for PNP activity 2.4.2.4 thymidine phosphorylase analysis methods to measure thymidine and deoxyuridine concentrations and thymidine phosphorylase activity in biological samples. Thymidine phosphorylase activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate thymidine, either spectrophotometrically or by HPLC-UV. The protocols allow the detection of thymidine phosphorylase dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy, MNGIE 2.4.2.8 hypoxanthine phosphoribosyltransferase analysis the branched bi-enzyme system with xanthine oxidase is an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine, overview 2.4.2.9 uracil phosphoribosyltransferase analysis biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay 2.4.2.26 protein xylosyltransferase analysis measurement of fibrosis marker xylosyltransferase I activity by HPLC electrospray ionization tandem mass spectrometry. The simple and robust LC-MS/MS assay permits the rapid and accurate determination of XT-I activity in human serum 2.4.2.28 S-methyl-5'-thioadenosine phosphorylase analysis methylthioadenosine phosphorylase protein expression may be a predictive marker of interferon therapy resistance in patients with melanoma and disease progression 2.4.2.30 NAD+ ADP-ribosyltransferase analysis establishment of an immortalized PARP-1-/- murine endothelial cell line HYKO6 as a tool to study PARP-1-mediated endothelial cell dysfunction 2.4.3.9 lactosylceramide alpha-2,3-sialyltransferase analysis high-throughput scintillation proximity assay to detect GM3S activity to screen GM3S inhibitors. Methods for detecting the activity of GM3S and sialyltransferase ST3Gal3 are established, through direct measurement of the enzyme products using an automatic rapid solid-phase extraction system directly coupled to a mass spectrometer 2.5.1.2 thiamine pyridinylase analysis improvements in a spectrophotometric thiaminase I activity assay that measures the disappearance of 4-nitrothiophenol providing scalable sample processing protocols and a 96-well microtiter plate format. Organisms devoid of thiaminase I, based upon previous work, show no activity with this assay. In addition, activity is found in a variety of fishes and one fern species from which this enzyme has not previously been reported 2.5.1.18 glutathione transferase analysis acetylcholinesterase and glutathione S-transferase activities in Daphnia magna are used in the standardized Daphnia magna immobility toxicity test for toxic substances e.g. in water quality monitoring, overview 2.5.1.18 glutathione transferase analysis immobilized GST mutant Gln53Ala is used to assemble a biosensor for malathion 2.5.1.32 15-cis-phytoene synthase analysis psy gene of tea cultivars is closely correlated to accumulation of carotenoids which are precursors of tea flavour volatiles, thus the expression strength of psy gene can be used as an indicator for screening quality of tea cultivars 2.5.1.46 deoxyhypusine synthase analysis DHS activity can be determined by coupling the first phase reaction with the NADH-Glo assay in which the generation of luminescence is dependent on NADH derived from the DHS partial reaction. The non-radioactive DHS/NADH-Glo coupled assay is highly specific, sensitive and reproducible and can be configured for high throughput screening of small molecule libraries 2.5.1.47 cysteine synthase analysis development of a primary cell-based phenotypic screening in both the presence and absence of L-cysteine and selection hits that show differential amebicidal effects between the two conditions 2.5.1.58 protein farnesyltransferase analysis procedure for labeling designed ankyrin repeat proteins (DARPins) engineered with a C-terminal CVIA sequence using an azide-containing FPP analog by yeast PFTase and procedures to subsequently conjugate the labeled DARPins to a TAMRA fluorophore 2.5.1.63 adenosyl-fluoride synthase analysis the enzyme is applied as a catalyst for the efficient incorporation of [18F]-fluoride into [18F]-59-fluoro-59-deoxyadenosine, [18F]-59-fluoro-59-deoxyinosine and [18F]-5-fluoro-5-deoxyribose for positron emission tomography, PET, applications, useful for imaging tumors, monitoring the distribution of drugs and identifying cell and receptor degeneration in the brain 2.5.1.63 adenosyl-fluoride synthase analysis the fluorinase enzyme from Streptomyces cattleya is applied as a catalyst for the efficient incorporation of [18F]-fluoride into [18F]-59-fluoro-59-deoxyadenosine, [18F]-59-fluoro-59-deoxyinosine and [18F]-5-fluoro-5-deoxyribose for positron emission tomography, PET, applications, useful for imaging tumors, monitoring the distribution of drugs and identifying cell and receptor degeneration in the brain, coupled enzyme system, overview 2.5.1.78 6,7-dimethyl-8-ribityllumazine synthase analysis development and characterization of an in vivo applicable magnetic resonance positive contrast agent by conjugating Gd(III)-chelating agent complexes to lumazine synthase isolated from Aquifex aeolicus (AaLS). The r1 relaxivity of Gd(III)-DOTA-AaLS-R108C is 16.49 mM/s and its r1/r2 ratio is 0.52 at the magnetic field strength of 7 T. The results of 3D MR angiography demonstrate the feasibility of vasculature imaging within 2 h of intravenous injection of the agent and a significant reduction in T1 values observed in the tumor region 7 h post-injection in the SCC-7 flank tumor model. Gd(III)-DOTA-AaLS-R108C can serve as a potential theranostic nanoplatform at high magnetic field strength 2.5.1.79 thermospermine synthase analysis simple method to separate spermine and thermospermine in extracts from plant tissues of less than 500 mg. Polyamines extracted from plant tissues are benzoylated, and the derivatives are completely resolved by high-performance liquid chromatography on a C18 reverse-phase column. With this method, the thermospermine contents in various tissues of Arabidopsis thaliana have been monitored. Stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves 2.5.1.136 2-acylphloroglucinol 4-prenyltransferase analysis HPLC assay for the prenylation reactions, measuring enzyme activities measured in protein extracts from hop flowerbudsflowers and young cones 2.5.1.137 2-acyl-4-prenylphloroglucinol 6-prenyltransferase analysis HPLC assay for the prenylation reactions, measuring enzyme activities measured in protein extracts from hop flowerbudsflowers and young cones 2.6.1.2 alanine transaminase analysis sequential online capillary electrophoresis assay to study enzyme inhibitors. Evolution of the inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time 2.6.1.7 kynurenine-oxoglutarate transaminase analysis development of a coupled fluorescence assay for human KATII with short assay time 2.6.1.7 kynurenine-oxoglutarate transaminase analysis development of a sensitive, high-throughput, label-free RapidFire mass spectrometry assay for kynurenine aminotransferase. The method is directly applicable to KATII enzymes from different animal species, allowing to select proper animal model(s) to evaluate human KATII inhibitors 2.6.1.7 kynurenine-oxoglutarate transaminase analysis the enzyme is a key enzyme for studying the nephrotoxic mechanism of some xenobiotics and the formation of chemopreventive compounds in the mouse kidney 2.6.1.13 ornithine aminotransferase analysis development of two new continuous, coupled assays for ornithine-delta-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multi-well plates for convenience and high throughput. The first assay is based on the reduction of DELTA1-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1, which results in the concomitant oxidation of NADH to NAD+. This procedure is three times more sensitive than previous methods, and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor PLP of OAT by an unamplified modification of the commercially available Amplex® Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT 2.6.1.15 glutamine-pyruvate transaminase analysis the enzyme is a key enzyme for studying the nephrotoxic mechanism of some xenobiotics and the formation of chemopreventive compounds in the mouse kidney 2.6.1.16 glutamine-fructose-6-phosphate transaminase (isomerizing) analysis development of an assay for glucosamine 6-phosphate synthase that measures the production of glucosamine 6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230 nm or quantifying the free thiol with 5,5'-dithio-bis(2-nitrobenzoic acid), i.e. Ellman’s reagent in a discontinuous manner. Simple assay method, which can be adapted to 96-well microtiter plate format 2.6.1.16 glutamine-fructose-6-phosphate transaminase (isomerizing) analysis assay of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment 2.6.1.30 pyridoxamine-pyruvate transaminase analysis determination method for individual natural vitamin B6 compounds. Compounds are specifically converted into 4-pyridoxolactone by pyridoxal 4-dehydrogenase and coupling reactions involving pyridoxine 4-oxidase, pyridoxal 4-dehydrogenase, or pyridoxamine-pyruvate aminotransferase and pyridoxal 4-dehydrogenase. Application of method for analysis of food samples 2.6.1.42 branched-chain-amino-acid transaminase analysis development of a coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants 2.6.1.44 alanine-glyoxylate transaminase analysis development of an indirect glycolate cytotoxicity assay using CHO cells expressing glycolate oxidase and various normal and mutant forms of AGT 2.6.1.62 adenosylmethionine-8-amino-7-oxononanoate transaminase analysis radiochemical assay for the bifunctional enzyme that monitors the formation of acid stable [14C]-DTB from acid-labile 14CO2 in the presence of appropriate substrates and cofactors 2.6.1.62 adenosylmethionine-8-amino-7-oxononanoate transaminase analysis simple, cheap, and sensitive microplate fluorescence assay for 7,8-diaminopelargonic acid aminotransferase activity, allowing linear detection of 7,8-diaminopelargonic acid in the range of 20 nM to 50 microM. The method relies on the direct detection in the enzymatic reaction mixture of the vicinal diamine 7,8-diaminopelargonic acid derivatized with ortho-phthalaldehyde and 2-mercaptoethanol. The assay was validated with the known inhibitor desmethyl-KAPA, i.e. 8-amino-7-oxopelargonic acid, and adapted to microplate for high-throughput screening 2.6.1.62 adenosylmethionine-8-amino-7-oxononanoate transaminase analysis analytical method to measure the enantiomeric excess of substrate 8-amino-7-oxononanoic acid, based on the derivatization of its amine function, by orthophtalaldehyde and N-acetyl-L-cysteine, followed by high pressure liquid chromatography separation. Using this methodology and enantiopure samples of 8-amino-7-oxononanoic acid, it appears that racemization of 8-amino-7-oxononanoic acid occurs rapidly with half-lives from 1 to 8 h, not only in 4 M HCl but also in the usual pH range, from 7 to 9 2.6.1.62 adenosylmethionine-8-amino-7-oxononanoate transaminase analysis simple, cheap, and sensitive microplate fluorescence assay, allowing linear detection of 7,8-diaminopelargonic acid in the range of 20 nM to 50 microM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine 7,8-diaminopelargonic acid derivatized with ortho-phthalaldehyde and 2-mercaptoethanol. The assay is validated with inhibitor 8-amino-7-oxopelargonic acid and adapted to microplate 2.6.1.62 adenosylmethionine-8-amino-7-oxononanoate transaminase analysis microplate fluorescence assay for 7,8-diaminopelargonic acid aminotransferase that is simple, cheap, and sensitive, allowing linear detection of 7,8-diaminopelargonic acid in the range of 20 nM to 50 microM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine 7,8-diaminopelargonic acid derivatized with ortho-phthalaldehyde and 2-mercaptoethanol 2.6.1.72 D-4-hydroxyphenylglycine transaminase analysis development of a simple, inexpensive, accurate and precise kinetic method for the quantitation of 2-oxoglutarate (AKG) in cardioplegic solution and athletic supplements. The assay relies on an enzymatic transamination of AKG and D-4-hydroxyphenylglycine to form 4-hydroxybenzoylformate and L-glutamate using D-phenylglycine aminotransferase. The method shows good linearity (r2 = 0.9994) over an AKG concentration range of 0.020-0.160 mM. The limits of detection and quantitation are 0.00409 and 0.01362 mM, respectively. The detection is not interfered with by excipients in the samples 2.7.1.1 hexokinase analysis detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay 2.7.1.1 hexokinase analysis high-throughput assay for screening small molecule collections to identify inhibitors of the Plasmodium falciparum hexokinase. The assay employs an ADP-GloTM reporter system in a 1536-well plate format, is robust with a signal-to-background of 3.4, a percent coefficient of variation of 6.8 and a Z'-factor of 0.75 2.7.1.3 ketohexokinase analysis development of a three-dimensional chemical-feature-based QSAR pharmacophore model by using Discovery Studio v2.5 to identify inhibitors, and screening of chemical databases for validated pharmacophore hypothesis. Hit compounds show good hydrogen bonding interactions with the keyamino acids such as Arg108, Asp258, Gly41, Gly255, Asn42 and water molecules W12, W27, respectively 2.7.1.11 6-phosphofructokinase analysis assay for phosphofructokinase-1 using capillary electrophoresis based on the separation and detection by ultraviolet absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation is enhanced by the addition of Mg2+ to the separation buffer. the assay for directly monitors the enzyme-catalyzed reaction 2.7.1.20 adenosine kinase analysis the coexpression of adenosine kinase TbAK and adenosine transporter gene TbAT1 in Saccharomyces cerevisiae ado1 ade2 double mutants proves to be a convenient tool for testing nucleoside analogues for uptake and activation by Trypanosoma brucei adenosine salvage enzymes 2.7.1.22 ribosylnicotinamide kinase analysis simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of nicotinamide phosphoribosyltransferase EC 2.4.2.12, quinolinate phosphoribosyltransferase EC 2.4.2.19, nicotinate phosphoribosyltransferase EC 6.3.4.21 and nicotinamide riboside kinase in whole-cell extracts and biological fluids yielding an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes 2.7.1.23 NAD+ kinase analysis studies of enzyme activation by calmodulin, calmodulin-like proteins, calcium-binding proteins, and calmodulin mutants 2.7.1.23 NAD+ kinase analysis enzyme-dependent assay for detection and quantification of calmodulin inhibitors 2.7.1.23 NAD+ kinase analysis in-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis. This technique allows the monitoring of the activity and expression of this kinase in various biological systems 2.7.1.24 dephospho-CoA kinase analysis dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters. The DPCK method can be applied accurately to pools of CoA metabolites isolated from cellular material 2.7.1.52 fucokinase analysis development of an alternative way of labeling of fucosylated structures by metabolic engineering for the identification of fucosylated glycoproteins, using a chemoenzymatic approach. In this approach, the activities of Bacteroides fragilis 9343 L-fucokinase/guanosine-5'-diphosphate-Fuc pyrophosphorylase and human alpha1,3-fucosyltransferase 9 are combined in a Namalwa cellular model, the system can be applied to labeling of alkyne-modified fucosylated glycoproteins, e.g. for labeling of alkynyl fucose containing glycans, N-linked glycosylation site mapping and fucosylated glycoprotein identification by IGOT-LC-MS analysis, overview 2.7.1.59 N-acetylglucosamine kinase analysis the kinase is applied for high-sensitive detection of the amino sugars in cell wall preparations by radioactive phosphorylation 2.7.1.74 deoxycytidine kinase analysis deoxycytidine kinase from B-cell chronic lymphocytic leukaemia lymphocytes can be detected by use of anti-phospho-ser74 antibodies 2.7.1.78 polynucleotide 5'-hydroxyl-kinase analysis a method for real-time monitoring of the activity and kinetics of T4 polynucleotide kinase by use of a singly fluorophore-labeled DNA-hairpin smart probe coupled with lambda exonuclease cleavage is described 2.7.1.78 polynucleotide 5'-hydroxyl-kinase analysis assay for the detection of T4 polynucleotide kinase/phosphatase activity based on a terminal transferase-aided photoinduced electron transfer strategy. The method is highly sensitive, the T4 PNKP detection limit is 0.01 U/ml 2.7.1.78 polynucleotide 5'-hydroxyl-kinase analysis fluorometric method for the determination of the activity of T4 polynucleotide kinase phosphatase PNKP. A partially doublestranded DNA substrate is dephosphorylated on addition of T4 PNKP to generate the long dsDNA, which acts as a template for synthesizing copper nanoclusters. The assay has an analytical range from 0.07 U/ml to 15 U/ml and a detection limit of 0.06 U/ml 2.7.1.78 polynucleotide 5'-hydroxyl-kinase analysis label-free fluorescent assay for T4 polynucleotidekinase/phosphatase PNKP activity using poly(thymine)-templated fluorescent copper nanoparticles as a fluorescent indicator. A T-rich hairpin primer with a 3'-phosphoryl end, serves as both the substrate for T4 PNKP and DNA template for the formation of fluorescent copper nanoparticles. Upon hydrolysis by T4 PNKP, the resulting hairpin primer with a 3'-hydroxyl end is elongated to form a long double-strand product by DNA polymerase, which prohibits the formation of fluorescent copper nanoparticles 2.7.1.86 NADH kinase analysis the enzyme is useful for amplification of NADH in presence of excess NAD+ and is applicable to sensitive measurement of NAD+ dependent dehydrogenase or ist substrate 2.7.1.95 kanamycin kinase analysis competitive assay that mimics in vivo nucleotide triphosphate concentrations and usage by the enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enables the determination of partitioning of phosphate flux from nucleotide triphosphate donors to antibiotics 2.7.1.107 diacylglycerol kinase (ATP) analysis generation of a soluble mutant selection assay based on a library of random diacylglycerol kinase delta1 mutants of sterile alpha-motif and murine dihydrofolate reductase as the selectable marker 2.7.1.136 macrolide 2'-kinase analysis competitive assay that mimics in vivo nucleotide triphosphate concentrations and usage by the enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enables the determination of partitioning of phosphate flux from nucleotide triphosphate donors to antibiotics 2.7.1.138 ceramide kinase analysis enzymatic assay to quantify mass levels of long chain ceramides in cellular lipid extracts 2.7.1.153 phosphatidylinositol-4,5-bisphosphate 3-kinase analysis low-cost bioassay that readily measures phosphatidylinositol 3-kinase inhibition. The in vivo assay is based on the fact that the overproduction of phosphatidylinositol 3-kinase is toxic in yeast, and uses the ability of commercial phosphatidylinositol 3-kinase inhibitors to rescue cell growth. The use of 0.003% sodium dodecyl sulfate and the elimination of the Snq2 detoxification pump, optimize the bioassay by enhancing its sensitivity. From 9600 extracts tested, 0.6% lead to a recovery of yeast growth reproducibly, selectively, and in a dose-dependent manner 2.7.1.171 protein-fructosamine 3-kinase analysis convenient assay for the determination of FN3K activity in erythrocytes, which can be performed in routine laboratories 2.7.1.181 polymannosyl GlcNAc-diphospho-ditrans,octacis-undecaprenol kinase analysis strategy for improving poor 3.5 A resolution initial phases by density modification and cross-crystal averaging with an additional data set to build a crude model 2.7.1.227 inositol phosphorylceramide synthase analysis assays using CHAPS- or taurodeoxycholate-washed membranes are reproducible, completely dependent on added acceptor substrate C6-7-nitro-2-1,3-benzoxadiazol-4-yl-ceramide, and more than 95% dependent on added donor substrate phosphatidylinositol. Product formation is linear with respect to both enzyme concentration and time, and transfer efficiency is improved more than 20fold as compared to assays using crude membranes 2.7.1.227 inositol phosphorylceramide synthase analysis expression of isoform IPCS2 in a yeast-based assay to screen for inhibitors 2.7.1.227 inositol phosphorylceramide synthase analysis high-throughput screening of inhibitors via recombinant expression in Saccharomyces cerevisiae lacking IPC synthase activity 2.7.1.235 lipopolysaccharide core heptose(I) kinase analysis chemiluminescence-based enzyme-linked immunosorbent assay for the detection of WaaP kinase activity. The assay displays high sensitivity, uses dephosphorylated LPS as a substrate, and monoclonal antibody 7-4 that specifically recognizes phosphate substituents in the LPS core 2.7.2.3 phosphoglycerate kinase analysis computational docking method for analysis of the docking of both aromatic and aliphatic bisphosphonates to the Trypanosoma brucei PGK 2.7.2.3 phosphoglycerate kinase analysis first quantitative analysis of the binding of alkyl bisphosphonates to yeast PGK, with activities being predicted within, on average, a factor of 4 over a 2500x overall range in activity 2.7.2.3 phosphoglycerate kinase analysis first quantitative analysis of the inhibition of human phosphoglycerate kinase by aromatic bisphosphonates by 3D-QSAR (CoMFA, CoMSIA) and pharmacophore modeling methods 2.7.2.3 phosphoglycerate kinase analysis development of a sensitive sandwich ELISA using an immuno-affinity-purified chicken polyclonal antibody for capturing PGK1 and an immuno-affinity-purified rabbit polyclonal antibody for detecting it. The sandwich ELISA can be used to quantify PGK1 levels in conditioned media of cell cultures for in vitro PGK1 export studies and in human serum for potential use ascreen for cancer 2.7.2.3 phosphoglycerate kinase analysis PGK1 might be a potential protein biomarker of intracellular oxidative status 2.7.2.3 phosphoglycerate kinase analysis in situ PCR is useful for detecting PGK-1 gene in paraffin-embedded sections under optimized conditions of both PCR cycle number and proteinase K concentration 2.7.3.3 arginine kinase analysis evaluation of a region of the nuclear gene that encodes arginine kinase for phylogenetic analysis of the Brachyura 2.7.4.1 ATP-polyphosphate phosphotransferase analysis specific and sensitive assessment of polyphosphate in mycorrhizal system of Tagetes patula inoculated with Archaeospora leptoticha with a polyphosphate kinase/luciferase system 2.7.4.1 ATP-polyphosphate phosphotransferase analysis usage of polyphosphate kinase gene ppk1 as a high-resolution genetic marker to study population structure in activated sludge of Candidatus Accumulibacter phosphatis 2.7.4.1 ATP-polyphosphate phosphotransferase analysis usage of the gene ppk as reporter gene used in monitoring of gene expression in mammalian cells, method development involving 31P-magnetic resonance spectrocopy or 31P-resonance imaging, overview 2.7.4.1 ATP-polyphosphate phosphotransferase analysis enzyme-linked assay in which His-tagged PPK2 is immobilized on a plate and then biotinylated aptamer inhibitors are added 2.7.4.3 adenylate kinase analysis method for simultaneous detection of adenylate kinase isoforms directly on gel or nitrocellulose after separation by denaturing electrophoresis and electroblotting. Method allows for quantitative dection of enzyme activity from amny sources in both its reaction courses 2.7.4.3 adenylate kinase analysis coarse grained model for the interplay between protein structure, folding and function which is applicable to allosteric or non-allosteric proteins. High strain energy is correlated with localized unfolding during the functional transition. Competing native interactions from the open and closed form can account for the large conformational transitions. Local unfolding may be due, in part, to competing intra-protein interactions 2.7.4.3 adenylate kinase analysis detection of adenylate kinase isoforms directly on gel or nitrocellulose after separation by denaturing electrophoresis and electroblotting. Method allows to clarify the apparent molecular weights of most of those enzymes that follow the reaction of AMP plus MgATP and is alos quantitative. GTP:AMP phosphotransferase are not detectable 2.7.4.33 AMP-polyphosphate phosphotransferase analysis sensitive method for detecting AMP by using polyphosphate(polyP)-AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii in conjugation with firefly luciferase. The method allows detection of AMP over the concentration range of 0.3-100 pmol per assay 2.7.7.1 nicotinamide-nucleotide adenylyltransferase analysis development of a bioluminescent assay using Photinus pyralis ATP-dependent luciferase and luciferin for NMNAT1 in a 384-well plate format, for screening of forward and reverse reactions 2.7.7.1 nicotinamide-nucleotide adenylyltransferase analysis NMNAT can be applied to the colorimetric NMN or NaMN assays, which employ either adenylation of NMN to NAD by NMNAT or adenylation of NaMN to deamido-NAD (NaAD) by NMNAT followed by amidation of NaAD to NAD by NAD synthetase (NADS, EC 6.3.1.5) or an NAD cycling reaction using 12alpha-hydroxysteroid dehydrogenase (12a-HSD, EC 1.1.1.176) and diaphorase (DI, EC 1.6.99.3) to accumulate reduced 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium. The enzymatic cycling method enables detection of 0.5 mM (12.2 nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer 2.7.7.6 DNA-directed RNA polymerase analysis assay for RNA polymerase activity that uses the dye RiboGreen to detect transcripts by fluorescence and is thus free of the expense, short shelf life, and high handling costs of radioisotopes. The method is relatively quick and can be performed entirely in microplate formate, allowing for the processing of dozens to hundreds of samples in parallel 2.7.7.7 DNA-directed DNA polymerase analysis the enzyme is moderately processive. It can substitute for Taq in polymerase chain reaction (PCR) and can bypass DNA lesions that normally block Taq. Such properties make the Dpo4-like enzymes ideally suited for the polymerase chain reaction amplification of damaged DNA samples. By using a blend of Taq and Dpo4-like enzymes a polymerase chain amplicon is obtained from ultraviolet-irradiated DNA that is largely unamplifyable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in polymerase chain reactions, augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications 2.7.7.15 choline-phosphate cytidylyltransferase analysis CTP:phosphocholine cytidylyltransferase enzyme assay that employs separation of non-radioactive CDP-choline from CTP. The assay was tested using a truncated version of rat CCTalpha by measuring CDP-choline product formation, and the HPLC method can be applied to glycerol 3-phosphate cytidylyltransferase and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively 2.7.7.18 nicotinate-nucleotide adenylyltransferase analysis NMNAT can be applied to the colorimetric NMN or NaMN assays, which employ either adenylation of NMN to NAD by NMNAT or adenylation of NaMN to deamido-NAD (NaAD) by NMNAT followed by amidation of NaAD to NAD by NAD synthetase (NADS, EC 6.3.1.5) or an NAD cycling reaction using 12alpha-hydroxysteroid dehydrogenase (12a-HSD, EC 1.1.1.176) and diaphorase (DI, EC 1.6.99.3) to accumulate reduced 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium. The enzymatic cycling method enables detection of 0.5 mM (12.2 nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer 2.7.7.B22 transposase analysis mutant K212R/P214R/G251R/A338V, i.e. Tn5-059, displays a lowered GC insertion bias. Tn5-059 reduces AT dropout and increases uniformity of genome coverage in both bacterial genomes and human genome. Tn5-059 generates a higher library diversion when compared to Nextera v2 for human exomes. When used for human exomes, Tn5-059 delivers consistent library insert size over a range of input DNA, allowing up to a tenfold variance from the 50 ng input recommendation 2.7.7.B22 transposase analysis Tn5 transposase is capable of direct tagmentation of RNA/DNA hybrids in vitro. This activity can be used to replace the traditional library construction procedure of RNA sequencing. Results of transposase-assisted RNA/DNA hybrids Co-tagmEntation are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis 2.7.7.23 UDP-N-acetylglucosamine diphosphorylase analysis quick and sensitive enzyme detection assay based on the diphosphatase-coupled colorimetric method with malachite green, and assay for glucosamine-1-phosphate N-acetyltransferase activity of the bifunctional enzyme, both applicable in microtitter plates 2.7.7.24 glucose-1-phosphate thymidylyltransferase analysis microtiter plate–based colorimetric assay for RmlA enzyme activity, based on hydrolysis of the diphosphate formed and detection of phosphate by malachite green reagent 2.7.7.24 glucose-1-phosphate thymidylyltransferase analysis modified continuous spectrophotometric assay for facile detection of diphosphate released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides 2.7.7.31 DNA nucleotidylexotransferase analysis DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates. Amino- and nitrophenyl-modified dNTPs are good substrates giving 3'-end stretches of different lengths depending on the nucleotide and concentration. d[7-Deaza-7-(3-nitrophenyl)]GTP is efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide, resulting in a considerable enhancement of voltammetric signals due to the nitro group reduction. Discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes is easily accomplished, and tumour suppressor p53 protein is able to recognize a specific binding site within tail-labelled DNA substrates 2.7.7.31 DNA nucleotidylexotransferase analysis reliable and accurate 3'-end miRNA-labeling method for microarray detection by terminal-deoxynucleotidyl transferase TdT. Using its ability to add polynucleotides at a RNA receptor molecule by using deoxycytidine triphosphate, miRNA is successfully labeled by adding fluorescence deoxycytidine triphosphates to its 3'-end. The TdT-labeling method can detect as little as 0.04 fmol of synthetic small RNA, and produce precise and accurate measurements that span a linear dynamic range from 0.04 to 5 fmol of synthetic small RNA 2.7.7.31 DNA nucleotidylexotransferase analysis construction of a nonhomologous end-joining assay vector NAV, containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I-SceI generates a double-strand break in NAV that excises mKate2 and ccdB. Repair of this double-strand break produces an intact vector that expresses Venus, a green fluorescent protein. Cells bearing the repaired NAV lack the ccdB gene which slows cell proliferation, the cultures are enriched in cells containing repaired double-strand breaks 2.7.7.31 DNA nucleotidylexotransferase analysis methodology for fluorescence turn-on detection of DNA methyltransferase (MTase) activity based on terminal deoxynucleotidyl transferase TdT using a thioflavin T probe. The method is highly selective and sensitive. The fluorescence intensity is directly proportional to Dam MTase concentration in the range from 0.1 to 8.0 U/ml with a detection limit of 0.1 U/ml. As no labeling with a fluorophore quencher pair is required, the method is simple and low cost 2.7.7.38 3-deoxy-manno-octulosonate cytidylyltransferase analysis adaptation of a simple colorimetric assay for diphosphate to the enzyme 3-deoxy-D-manno-octulosonate cytidylyltransferase. This assay can be combined with the malachite green assay for phosphate to form an assay system capable of determining phosphate and diphosphate in the same solution. The assay system has the potential for simultaneous screening of the 3-deoxy-D-manno-octulosonate biosynthesis pathway 2.7.7.47 streptomycin 3''-adenylyltransferase analysis the developed multiplex PCR assay is useful in verifying the GM status of a sample irrespective of the crop and GM trait 2.7.7.48 RNA-directed RNA polymerase analysis assembly of a productive isoform NS5B primer-template elongation complex stalled after formation of a 9-nucleotide primer. Under optimized conditions up to 65% of NS5B can be converted into active elongation complexes. The elongation complex is extremely stable, allowing purification away from excess nucleotide and abortive initiation products 2.7.7.49 RNA-directed DNA polymerase analysis the laser capture microdissection microscopycombined with multiplex quantitative real-time reverse transcriptase PCR, LCMM/qRTPCR, technique can be used to resolve and quantify GFP mRNA variability at high spatial resolution, the method is useful for quantification of any gene expressed by bacteria in their native environment, method development, overview 2.7.7.50 mRNA guanylyltransferase analysis valuable in a variety of analytical studies of RNA 2.7.7.50 mRNA guanylyltransferase analysis fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time and a secondary orthogonal assay that measures guanylation activity. This platform provides an avenue for identification of potent nsP1 inhibitors 2.7.7.60 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase analysis CTP:phosphocholine cytidylyltransferase enzyme assay that employs separation of non-radioactive 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol from CTP. The assay can be applied to glycerol 3-phosphate cytidylyltransferase and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively 2.7.7.72 CCA tRNA nucleotidyltransferase analysis a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. The selected gene region of the tRNA nucleotidyltransferase reveals a seven- to 30fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. Even in the presence of a 1000fold excess of human genomic DNA, no unspecific amplicons are produced 2.7.7.72 CCA tRNA nucleotidyltransferase analysis development of a tRNA sequencing method that is specifically tailored to assess the 3'-termini of Escherichia coli tRNA 2.7.7.84 2'-5' oligoadenylate synthase analysis modified coupled spectrophotometric assay to detect 2-5 oligoadenylate synthetase enzyme activity in prostate cell lines as a model system. The phosphates generated by the OAS enzymatic reaction are coupled with conversion of the substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside to a purine base product, 2-amino-6-mercapto-7-methylpurine and ribose1-phosphate via a purine nucleoside phosphorylase 2.7.7.89 [glutamine synthetase]-adenylyl-L-tyrosine phosphorylase analysis development of a continuous fluorometric assay of the deadenylylation reaction and for measurement of uridylated signal transduction protein PII 2.7.8.2 diacylglycerol cholinephosphotransferase analysis since CPT expression is associated with elaboration of new endoplasmic reticulum membrane, it may be useful as a marker protein for endoplasmic reticulum expansion 2.7.8.7 holo-[acyl-carrier-protein] synthase analysis enzyme can be utilized in an assay for apo-ACP in biological material 2.7.8.7 holo-[acyl-carrier-protein] synthase analysis method for the generation of crypto-AcpM loaded with a solvatochromic probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl, which is linked to the 4'-phosphopantetheine (Ppant) prosthetic group of AcpM by Sfp. The crypto-AcpM is employed to explore the elusive dynamics of Ppant arm in AcpM 2.7.8.13 phospho-N-acetylmuramoyl-pentapeptide-transferase analysis the enzyme is an attractive antibacterial drug target involved in peptidoglycan synthesis 2.7.8.B13 phosphatidylinositol phosphate synthase analysis synthesis of substrate 1l-myo-inositol 1-phosphate using a solution containing recombinant 1l-myo-inositol 1-phosphate synthase derived from the hyperthermophilic archaeon Aeropyrum pernix 2.7.8.24 phosphatidylcholine synthase analysis since choline is not a biosynthetic product of prokaryotes, the Pcs pathway is probably a direct sensor of environmental conditions, using choline availability as an indicator of the status of the location in which the bacterium is found 2.7.8.27 sphingomyelin synthase analysis development of a high-throughput screening assay 2.7.9.1 pyruvate, phosphate dikinase analysis PPDK is useful in DNA analysis, method development, overview 2.7.9.1 pyruvate, phosphate dikinase analysis pyruvate phosphate dikinase, expressed onto bacterial magnetic particles, PPDK-BacMPs, is utilized in pyrosequencing 2.7.10.1 receptor protein-tyrosine kinase analysis large-scale proteomic approach to identify over 200 substrates of the receptor tyrosine kinases kinase family in cancer cell lines driven by the c-Met, epidermal growth factor receptor, or platelet-derived growth factor receptor alpha receptor tyrosine kinases. For a subset of proteins with RxRxxS/T sites, phosphorylation is decreased by receptor tyrosine kinase inhibitors as well as by inhibitors of the PI3K, mTOR, and MAPK pathways. Phosphorylation of the protein chaperone small glutamine-rich tetratricopeptide repeat-containing protein alpha at Ser305 is essential for PDGFRalpha stabilization and cell survival in PDGFRalpha-dependent cancer cells 2.7.10.1 receptor protein-tyrosine kinase analysis proximity ligation assay-based methodology for in situ visualization and quantification of ligand-dependent EGFR receptor dimerization in intact cells. Using the approach combined with a universally applicable epitope tagging strategy, EGFR dimers can be detect in cells transiently co-expressing FLAG-tagged and MYC-tagged human EGFRs. Data strongly suggest that the method can be used to detect ligand-dependent EGFR dimerization, and the signal is generated in a protein interaction-based manner 2.7.10.2 non-specific protein-tyrosine kinase analysis development of a protein chip consisting of a silicone elastomer microwell array with recombinant enzyme covalently attached to the wells via a 3-glycidoxypropyltrimethoxysilane crosslinker for large scale activity assay, overview 2.7.10.2 non-specific protein-tyrosine kinase analysis peptide photoaffinity probe GAPEVIYA-4-azidophenylalanyl-PGAKKKGK(biotin) based on consensus substrate GAPEVIYATPGAKKK. Incubation of purified recombinant isoform Abl results in covalent incorporation of biotin. The label crosslinks to Abl specifically through binding to the active site cleft into the kinase. Abl activity is enhanced by phosphorylation at multiple sites, the extent of labeling increases with increasing catalytic activity 2.7.10.2 non-specific protein-tyrosine kinase analysis the fluorescence properties of bosutinib and related compounds allow inhibitor binding to be measured quantitatively, and the infrared absorption of the nitrile group reveals a different electrostatic environment in the conserved ATP-binding sites of Abl and Src kinases 2.7.10.2 non-specific protein-tyrosine kinase analysis SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex. An SH2 domain from lymphocyte-specific tyrosine kinase is genetically fused to a truncated biotin carboxyl carrier protein, and the resulting fusion protein is labeled through biotinylation with biotin protein ligase carrying multiple copies of a luminescent Tb3+ complex. The labeled SH2 fusion proteins are employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide is produced by phosphorylation to the substrate peptide by Src tyrosine kinase. The assay allows for a reliable determination of the activity of Src kinase lower than 10 ng/mL 2.7.11.10 IkappaB kinase analysis development of real-time imaging for continous enzyme detection and kinetics in intact cells and living mice utilizing a recombinant IkappaBalpha-firefly luciferase reporter construct, system can be used for determination of kinetics/pharmacodynamics of potential selective inhibitors, and for investigations of NF-kappaB signalling pathway activation 2.7.11.21 polo kinase analysis the development of an ELISA-based Plk1 assay is described that employs a principle to rapidly and accurately quantify the Plk1 activity with high sensitivity and specificity 2.7.11.22 cyclin-dependent kinase analysis development of a protein chip consisting of a silicone elastomer microwell array with recombinant enzyme covalently attached to the wells via a 3-glycidoxypropyltrimethoxysilane crosslinker for large scale activity assay, overview 2.7.11.22 cyclin-dependent kinase analysis the methodologies embodied in transitional dynamic analysis and positional impact vertex for entropy transfer provide a quick approach to identify local fluctuation change important for protein function and residue contacts of CDK2 that contributes to these changes. Further, these approaches can be used to check for possible errors in protein dynamic simulations of CDK2 and have the potential to facilitate a better understanding of the contribution of entropy to protein allostery and function 2.7.11.24 mitogen-activated protein kinase analysis development of a protein chip consisting of a silicone elastomer microwell array with recombinant enzyme covalently attached to the wells via a 3-glycidoxypropyltrimethoxysilane crosslinker for large scale activity assay, overview 2.7.11.24 mitogen-activated protein kinase analysis crystal structure of the MAP kinase binding domain of human MAP kinase phosphatase MKP5 2.7.11.24 mitogen-activated protein kinase analysis HitHunter EFC binding assay can be used to select compounds that specifically target inactive kinase 2.7.11.24 mitogen-activated protein kinase analysis a concept and implementation of a new simple inhibitor screening method applicable for a broad range of human proteins by using transformed bacteria is reported 2.7.11.24 mitogen-activated protein kinase analysis the use of 3'-UTR elements to regulate tissue-specific expression of pmk-2 provides a mechanism that endows context specificity to p38 paralogues 2.7.11.25 mitogen-activated protein kinase kinase kinase analysis development of a protein chip consisting of a silicone elastomer microwell array with recombinant enzyme covalently attached to the wells via a 3-glycidoxypropyltrimethoxysilane crosslinker for large scale activity assay, overview 2.7.11.25 mitogen-activated protein kinase kinase kinase analysis proline cis-trans isomerization of MEKK3 PB1 of the Gln38-Pro39 bond, first case of structural heterogeneity within PB1 domains, major difference between both isomers in the Pro39 region. Residues Gly37-Leu40 form a type VIb beta-turn in the cis conformation, whereas no obvious character of beta-turn is observed in the trans conformation. Internal motions in the beta3/beta4-turn on a microsecond-millisecond time scale 2.7.12.1 dual-specificity kinase analysis development of a protein chip consisting of a silicone elastomer microwell array with recombinant enzyme covalently attached to the wells via a 3-glycidoxypropyltrimethoxysilane crosslinker for large scale activity assay, overview 2.7.12.2 mitogen-activated protein kinase kinase analysis development of a protein chip consisting of a silicone elastomer microwell array with recombinant enzyme covalently attached to the wells via a 3-glycidoxypropyltrimethoxysilane crosslinker for large scale activity assay, overview 2.7.12.2 mitogen-activated protein kinase kinase analysis development of a continuous spectrophotometric assay for mitogen-activated protein kinase kinases 2.7.14.1 protein arginine kinase analysis analysis of the fragmentation behavior of peptides with arginine phosphorylation in mass spectrometry and its influence on phospho-site localization 2.7.14.1 protein arginine kinase analysis use of a Bacillus subtilis ywlE- strain lacking the phosphatase activity corresponding to protein arginine kinase MscB as a source for arginine-phosphorylated proteins and mass spectrometry protocols to analyze the arginine modification. A substrate-trapping mutant of the YwlE phosphatase retains binding affinity toward arginine-phosphorylated proteins but cannot hydrolyze the captured substrates 2.7.14.1 protein arginine kinase analysis detection of protein arginine kinase McsB via a sequential turn-off surface enhance Raman scattering (SERS) assay platform. The positive charged peptide initiates the aggregation of labelled Au nanoparticles to form hot spots, resulting to a higher SERS intensity. The SERS biosensor shows high sensitivity, selectivity and simplicity, detection limit for McsB is 46 pM 2.7.14.1 protein arginine kinase analysis reversible fluorescent activity probes for protein Arg kinases and phosphatases to allow real-time kinetic assays, probe is based on the chelation-enhanced fluorescence (CHEF) effect of the cysteine-sulfonamido-oxine fluorophore 2.8.1.1 thiosulfate sulfurtransferase analysis detection method for cyanide based on in-capillary enzymic reaction. Method shows a linear detection range from 0.015 to 0.5 mM with a detection limit of 0.003 mM and a limit of quantitation of 0.09 mM. The method is rapid, simple and can easily be automated 2.8.1.2 3-mercaptopyruvate sulfurtransferase analysis high-throughput screening of a large chemical library with a H2S-selective fluorescent probe, HSip-1, based on azamacrocyclic Cu2+ complex chemistry. HSip-1 can detect H2S in aqueous solution with high selectivity over biothiols, inorganic sulfur compounds, reactive oxygen species and reactive nitrogen species 2.8.2.1 aryl sulfotransferase analysis a modified variant of sulfotransferase assay employs permeabilized fission yeast cells (enzyme bags). A new and convenient SULT activity assay is based on the sulfation of a proluciferin compound, which is catalyzed by SULT1E1, SULT2A1, SULT4A1, and SULT6B1 2.8.2.1 aryl sulfotransferase analysis establishment of a reproducible method for simultaneous quantification of sulfotransferase SULT1A1 and SULT1A3/4 in S9 fractions and cell line samples 2.8.2.2 alcohol sulfotransferase analysis a modified variant of sulfotransferase assay employs permeabilized fission yeast cells (enzyme bags). A new and convenient SULT activity assay is based on the sulfation of a proluciferin compound, which is catalyzed by SULT1E1, SULT2A1, SULT4A1, and SULT6B1 2.8.2.4 estrone sulfotransferase analysis a modified variant of sulfotransferase assay employs permeabilized fission yeast cells (enzyme bags). A new and convenient SULT activity assay is based on the sulfation of a proluciferin compound, which is catalyzed by SULT1E1, SULT2A1, SULT4A1, and SULT6B1 2.8.2.4 estrone sulfotransferase analysis N-terminally fluorescein isothiocyanate (FITC)-labeled decapeptide (MIYKEGDVEK, FITC-hSULT1E1-P10) corresponding to residues 59-68 of hSULT1E1 can be used as an efficient fluorescent probe for the detection of Ox-LDL, LPC, and PAF, which could facilitate the mechanistic study, identification, diagnosis, prevention, and treatment of atherosclerosis 2.8.2.23 [heparan sulfate]-glucosamine 3-sulfotransferase 1 analysis usefulness of HS3ST-2 as a genetic marker for TrkC-positive mechanosensory neurons 2.8.3.1 propionate CoA-transferase analysis amperometric propionate sensor 2.8.4.1 coenzyme-B sulfoethylthiotransferase analysis identifying methanotrophic archaea with mcrA sequences 2.8.4.1 coenzyme-B sulfoethylthiotransferase analysis methanogen diversity evidenced by molecular characterization of methyl coenzyme M reductase A genes in hydrothermal sediments of the Guaymas Basin 3.1.1.1 carboxylesterase analysis use of fluorescence-activated cell sorting and methylumbelliferyl acetate substrate for identification of carboxylesterases and high-throughput screening 3.1.1.1 carboxylesterase analysis the enzyme with substrate (S)-1-(6-fluoro-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-(isoquinolin-5-yloxy)ethanone and inhibitor bis(p-nitrophenyl) phosphate are useful in a system to measure in plasma-free fraction for a plasma-labile compound 3.1.1.1 carboxylesterase analysis wild-type enzyme and mutant enzyme N44S/S48V have the potential to be a biosensor for pesticide detection 3.1.1.2 arylesterase analysis description of an easy and non-toxic method for large scale phenotyping and activity quantitation of arylesterase 3.1.1.2 arylesterase analysis the enzyme has the potential to be used as a catalytic bioscavenger of nerve agents. Insect production of the enzyme may provide a source for both in vitro enzymatic and crystallographic studies and in vivo stability and anti-nerve agent efficacy testing 3.1.1.2 arylesterase analysis quantification of arylesterase activity in routine clinical studies by monitoring the formation of acetic acid, upon the hydrolysis of phenyl acetate, using 10 microl of sample. The method accuracy is higher than 90% and intra-assay and inter-assay precisions are 96% and 95%, respectively. The procedure is suitable for use in human serum and heparinized plasma samples, while ethylenediaminetetra-acetic acid containing samples should be avoided 3.1.1.2 arylesterase analysis use of 9-(4-chlorophenyloxycarbonyl)-10-methylacridinium triflate as a substrate for serum PON arylesterase activity assay. The apparent Km value of a serum sample for the substrate is 85 nmol/l, close to the Km value of recombinant human isoform PON1. Recombinant human PON1 in presence of CaCl2 shows at least 7.8 times selectivity over acetylcholinesterase and lipases. The method allows reliable, cost-saving, and specific determination in a buffer of physiological pH 3.1.1.4 phospholipase A2 analysis simple and inexpensive protein expression, refolding and purification system may be useful for site-directed mutagenesis experiments of sPLA2-IID which will advance the understanding of the structure–function relationship and biological effects of the protein 3.1.1.4 phospholipase A2 analysis development of a spectrophotometric assay to measure, continuously and specifically, phospholipase A1 or phospholipase A2 activities using synthetic glycerophosphatidylcholines containing alpha-eleostearic acid. Substrates 1-alpha-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine or 1-octadecyl-2-alpha-eleostearoyl-rac-glycero-3-phosphocholine differentiate, with excellent accuracy, between PLA1 and PLA2 activity 3.1.1.6 acetylesterase analysis development of a heroin biosensor with high sensitivity 3.1.1.7 acetylcholinesterase analysis development of sensors for direct detection of organophosphates 3.1.1.7 acetylcholinesterase analysis detection of organophosphate and carbamate insecticides 3.1.1.7 acetylcholinesterase analysis pesticide and organophosphate analysis in different soil samples using the enzyme in a photometric assay, overview 3.1.1.7 acetylcholinesterase analysis the enzyme immobilized on porous silicon can be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds 3.1.1.8 cholinesterase analysis enzyme in immobilized forms has a potential application in environmental, water and air, pollution control as a functional part of monitoring system 3.1.1.8 cholinesterase analysis covalent coupling of the enzyme to a copolymerisate of maleinic anhydride and butanedioldivinylether provides the possibility to yield stable enzyme preparations 3.1.1.B10 p-coumaroyl esterase analysis assay for catalytic activity using reverse-phase HPLC analysis of 4-coumaric acid released. As little as 100 ng of acid can be determined 3.1.1.13 sterol esterase analysis immobilization of the enzyme alone or coimmobilization with cholesterol oxidase on zirkonia-coated acrylamine glass beads for analysis of total cholesterol in serum, long-term stability, precise measurements with the enzyme mixture at cholesterol concentration above 1 g/l 3.1.1.13 sterol esterase analysis co-immobilization of cholesterol oxidase and cholesterol esterase on oxygen electrode for use as cholesterol biosensor. Optimum pH value and temperature of immobilized enzymes are 6.0 and 25°C, resp. Sensor works linearily in the range of 2-59 mg cholesterol palmitate per dl, can be reused over 30 times and is stable up to 9 weeks. Use of sensor to determine cholesterol content in food samples was investigated 3.1.1.13 sterol esterase analysis covalent immobilization of cholesterol oxidase and cholesterol esterase on electrochemically prepared polyaniline films for use as cholesterol biosensor. Biosensing electrodes have a response time of about 40 s, linearity from 50 to 500 mg/dl of cholesterol oleate concentration, and are thermally stable up to 46°C. The optimum pH range is 6.5-7.5, sensitivity as 0.00075 nA/mg dl and a lifetime of 6 weeks 3.1.1.13 sterol esterase analysis covalent immobilization of cholesterol oxidase and cholesterol esterase onto tetraethylorthosilicate sol-gel films for use as cholesterol biosensor. Films show a thermal stability up to 55°C, response time as 180 s, linearity up to 780 mg/dl, shelf life of 1 month, and detection limit of 12 mg/dl cholesterol 3.1.1.17 gluconolactonase analysis response characteristics of a glucose-sensitive field-effect transistor. Coimmobilization of glucose oxidase and gluconolactonase in a membrane of a glucose sensor shows a sufficient response amplitude, whereas without gluconolactonase no detectable response is observed up to 3 mM glucose 3.1.1.17 gluconolactonase analysis auxiliary enzyme for assaying glucose-fructose oxidoreductase activity 3.1.1.32 phospholipase A1 analysis development of a spectrophotometric assay to measure, continuously and specifically, phospholipase A1 or phospholipase A2 activities using synthetic glycerophosphatidylcholines containing alpha-oleostearic acid. Substrates 1-alpha-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine or 1-octadecyl-2-alpha-eleostearoyl-rac-glycero-3-phosphocholine differentiate, with excellent accuracy, between PLA1 and PLA2 activity 3.1.1.53 sialate O-acetylesterase analysis detection of 9-O-acetylated sialic acids on sialoglycoconjugates immobilized on microtiter plates, nitrocellulose or separated on thin-layer chromatograms. The assay takes advantage of two different biological properties of influenza C virus, its high affinity-binding to 9-O-acetylated sialic acids and its sialate 9-O-acteylesterase that is used for detection of bound virus 3.1.1.72 acetylxylan esterase analysis beta-xylosidase-coupled assay of acetylxylan esterases using monoacetylated 4-nitrophenyl beta-D-xylopyranosides 3.1.1.72 acetylxylan esterase analysis colorimetric method based on the color changes produced in a pH indicator occurring during the deacetylating reaction of 7-aminocephalosporanic acid, cephalosporin C, or acetylated xylan caused by acetyl xylan esterase from Bacillus pumilus CECT 5072 3.1.1.75 poly(3-hydroxybutyrate) depolymerase analysis a method for poly(3-hydroxybutyrate) (PHB) depolymerase activity determination. The method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid and/or 3-hydroxybutyrate oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a softwarecontrolled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude 3.1.1.75 poly(3-hydroxybutyrate) depolymerase analysis a method to investigate quantitatively and qualitativly the hydrolysis of different types of poly(3-hydroxybutyrate) by selected PHB depolymerases. The method is based on the derivatization of 3-hydroxybutyrate oligomers into bromophenacyl derivates and separation by high performance liquid chromatography. The method allows the separation and quantification of 3-hydroxyutyrate and 3-hydroxybutyrate oligomers up to the octamer 3.1.1.77 acyloxyacyl hydrolase analysis potential use in studies of LPS structure and bioactivity 3.1.1.81 quorum-quenching N-acyl-homoserine lactonase analysis the enzyme can serve as biosensor for detection of quorum-sensing inhibitors or activators, overview 3.1.1.101 poly(ethylene terephthalate) hydrolase analysis determination of enzymatic hydrolysis by measuring the change of intensity of transmitted light due to the scattering effect of PET nanoparticles immobilized in an agarose gel 3.1.1.101 poly(ethylene terephthalate) hydrolase analysis fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples. The assay is robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins and can be used to quantify the amount of terephthalate obtained as the final degradation product of the enzymatic hydrolysis of PET in a microplate format 3.1.1.111 phosphatidylserine sn-1 acylhydrolase analysis immunoassay for determination of enzyme in blood serum samples. The mean standard deviation of the serum PS-PLA1 antigen concentration in 191 healthy subjects is 33.8 +- 16.6 microg/l. The concentration is significantly higher among men (13.8-80.6 microg/l) than among women (12.1-68.8 microg/l) 3.1.1.113 ethyl acetate hydrolase analysis method to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change 3.1.2.12 S-formylglutathione hydrolase analysis identification of a carboxylesterase expressed in protoplasts using fluorescence-activated cell sorting 3.1.3.1 alkaline phosphatase analysis CCP-2 cells are an important tool for investigation of chondrooclastogenesis/osteoclastogenesis and endochondrial ossification 3.1.3.1 alkaline phosphatase analysis enzyme is a potential marker for specific plasma membrane purification 3.1.3.1 alkaline phosphatase analysis ALP and fat storage are tightly linked during preadipocyte maturation. Measurement of ALP activity may be a sensitive and rapid technique for the quantification of intracellular lipid accumulation 3.1.3.1 alkaline phosphatase analysis application of intracellular alkaline phosphatase activity measurement in detection of neutrophil adherence in vitro 3.1.3.1 alkaline phosphatase analysis development of a simple in situ duodenal alkaline phosphatase activity assay based on the fluorogenic substrate ELF 3.1.3.1 alkaline phosphatase analysis simple, rapid, sensitive, and selective enzymatic procedures for determining zinc and magnesium based on their inhibiting and activating effects on the catalytic activity of alkaline phosphatases 3.1.3.1 alkaline phosphatase analysis construction o a fusion proein between the ZZ protein, two specific Fc-fragment-binding domains derived from the B-domain of SpA, and alkaline phosphatase. The ZZ-AP fusion retains full parental activities and exhibits an approximately tenfold higher sensitivity than that of ZZ-AP conjugate in enzyme-linked immunosorbent assay. The ZZ-AP fusion is a promising immunoreagent for IgG detection and a potential biolinker between antibodies and reporter enzymes. Compared with the parents, the equilibrium dissociation constant of ZZ-AP conjugate is decreased by 32% and catalytic activity is decreased by 24% 3.1.3.1 alkaline phosphatase analysis enzyme is capable of precipitating Cu2+ from 50 ppm CuCl2 solution with 64.3% precipitation. Phosphate generated from enzyme-mediated hydrolysis of 4-nitrophenyl phosphate binds with the copper ions 3.1.3.3 phosphoserine phosphatase analysis development of a malachite green-based high throughput assay system to identify isoform SerB2 inhibitors 3.1.3.4 phosphatidate phosphatase analysis radiolabeled and fluorescent lipid substrates for the detection, quantitation and analysis of the enzymatic activities of the LPPs measured using intact or broken cell preparations as the source of enzyme 3.1.3.8 3-phytase analysis comparison of assay methods and the impact of assay conditions on activity estimate using the molybdenum blue method, the molybdovanadate method, and the acetone phosphomolybdate method. Nearly identical activity of PhyA is determined from the molybdenum blue method and the acetone phosphomolybdate method. The molybdenum blue method and the molybdovanadate method give only 22% difference in PhyA activity 3.1.3.8 3-phytase analysis comparison of assay methods and the impact of assay conditions on activity estimate using the molybdenum blue method, the molybdovanadate method, and the acetone phosphomolybdate method. The activity values of AppA2 are more variable with the three assay methods than those of Aspergillus niger PhyA. The molybdenum blue method and the molybdovanadate method produce nearly a 3fold disparity for AppA2. Overall, the pH value, the type of buffers, and the inclusion of ancillary chemicals such as the detergents Triton X-100 and BSA each account for approximately one-third of the variations of AppA2 3.1.3.8 3-phytase analysis screening method to elucidate the ability of different yeast strains to utilize phytic acid as sole phosphorus source. The growth test in liquid culture in a microtiter plate with phytic acid as sole phosphorus source is a reliable, fast and easy-to-use screening method 3.1.3.8 3-phytase analysis gain of function mutants of the enzyme can be instrumental for the structure-function study of the enzyme and for industrial application 3.1.3.16 protein-serine/threonine phosphatase analysis assembly of a biosensor system involving protein phosphatase-2A and glycogen phosphorylase a activities, method optimization, overview 3.1.3.32 polynucleotide 3'-phosphatase analysis assay for the detection of T4 polynucleotide kinase/phosphatase activity based on a terminal transferase-aided photoinduced electron transfer strategy. The method is highly sensitive, the T4 PNKP detection limit is 0.01 U/ml 3.1.3.32 polynucleotide 3'-phosphatase analysis fluorescence polarization methods can detect specific binding of single-stranded DNAs with the phosphatase domain, but not specific interactions between the PNKP phosphatase and double-stranded substrates 3.1.3.32 polynucleotide 3'-phosphatase analysis fluorometric method for the determination of the activity of T4 polynucleotide kinase phosphatase PNKP. A partially doublestranded DNA substrate is dephosphorylated on addition of T4 PNKP to generate the long dsDNA, which acts as a template for synthesizing copper nanoclusters. The assay has an analytical range from 0.07 U/ml to 15 U/ml and a detection limit of 0.06 U/ml 3.1.3.32 polynucleotide 3'-phosphatase analysis label-free fluorescent assay for T4 polynucleotide kinase/phosphatase PNKP activity using poly(thymine)-templated fluorescent copper nanoparticles as a fluorescent indicator. A T-rich hairpin primer with a 3'-phosphoryl end, serves as both the substrate for T4 PNKP and DNA template for the formation of fluorescent copper nanoparticles. Upon hydrolysis by T4 PNKP, the resulting hairpin primer with a 3'-hydroxyl end is elongated to form a long double-strand product by DNA polymerase, which prohibits the formation of fluorescent copper nanoparticles 3.1.3.67 phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase analysis availability of the phosphorylated and unphosphorylated forms of recombinant PTEN permits future investigations into the three-dimensional structures of the phosphorylated and unphosphorylated forms of PTEN, and the role of phosphorylation in regulating PTEN activity, phospholipid- and protein-binding affinities 3.1.3.68 2-deoxyglucose-6-phosphatase analysis use of gene as a selectable marker gene to produce transgenic plants by Agrobacterium tumefaciens-mediated transfection of oil palm and regeneration of resistant tissues into whole plantlets on various regeneration media containing the same concentration of 2-deoxyglucose-6-phosphate. A transformation efficiency of about 1.0% is obtained 3.1.4.1 phosphodiesterase I analysis useful tool in nucleic acid research, characterization of oligonucleotides and as an aid in sequence analysis 3.1.4.1 phosphodiesterase I analysis enzyme system consisiting of phosphodiesterase and calf intestine alkaline phosphatese is used to examine the 3'-termini of strand break sites 3.1.4.3 phospholipase C analysis synthesis of fluorogenic analogue of phosphatidylcholine 1-O-(6-(p-methyl red)-amino-hexanoyl)-2-O-(12-(p-methyl red)-amino-dodecanoyl)-sn-glyceryl-N-(3-(5-BODIPY-pentanoyl)-amino-propyl)-N,Ndimethyl-phosphatidylethanolamine, and use as direct substrate for real-time measurement of enzyme activity. Substrate for both phospholipases C and D and lysophospholipase D, no substrate for Bacillus cereus PI-PLC or Clostridium perfringens PC-PLC 3.1.4.3 phospholipase C analysis enzymatic measurement of choline plasmalogen using PLD684 and phospholipase B 3.1.4.4 phospholipase D analysis synthesis of fluorogenic analogue of phosphatidylcholine 1-O-(6-(p-methyl red)-amino-hexanoyl)-2-O-(12-(p-methyl red)-amino-dodecanoyl)-sn-glyceryl-N-(3-(5-BODIPY-pentanoyl)-amino-propyl)-N,Ndimethyl-phosphatidylethanolamine, and use as direct substrate for real-time measurement of enzyme activity. 1-O-(6-(p-methyl red)-amino-hexanoyl)-2-O-(12-(p-methyl red)-amino-dodecanoyl)-sn-glyceryl-N-(3-(5-BODIPY-pentanoyl)-amino-propyl)-N,Ndimethyl-phosphatidylethanolamine is substrate for both phospholipases C and D 3.1.4.4 phospholipase D analysis chemical method for imaging phosphatidic acid synthesis by PLD enzymes in live cells. The method is specific to PLD-generated phosphatidic acid. PLD enzymes can accept azidoalcohols as reporters in a transphosphatidylation reaction. The resultant azidolipids are then fluorescently tagged enabling visualization of cellular membranes bearing active PLD enzymes. The method reveals a heterogeneity in PLD activities at both the cellular and subcellular levels 3.1.4.4 phospholipase D analysis enzymatic measurement of choline plasmalogen using PLD684 and phospholipase B 3.1.4.35 3',5'-cyclic-GMP phosphodiesterase analysis fusion protein of wild-type and mutant F163A GAFa domain of enzyme to Green Fluorescent Protein or Renilla luciferase for use in bioluminescence resonance energy transfer assay BRET as a biosensor of cGMP. BRET ratios of wild-type, but not mutant F163A, increase in presence of cGMP, but not cAMP 3.1.4.37 2',3'-cyclic-nucleotide 3'-phosphodiesterase analysis 2',3'-cyclic nucleotide 3'-phosphodiesterase is a stable marker for in situ detection of canine but not rat olfactory ensheathing cells 3.1.4.39 alkylglycerophosphoethanolamine phosphodiesterase analysis fluorogenic substrates, potential in screening for inhibitors and for monitoring spatiotemporal changes in activity in cells 3.1.4.39 alkylglycerophosphoethanolamine phosphodiesterase analysis the purified ATX protein is enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme 3.1.5.1 dGTPase analysis development of a continuous spectrophotometric enzyme-coupled assay. The deoxyguanosine released by Dgt is subjected to phosphorolysis by purine nucleoside phosphorylase, yielding deoxyribose-1-phosphate and guanine, which, in turn, can be oxidized to 8-oxoguanine by xanthine oxidase 3.1.5.B1 dNTPase analysis high-throughput colorimetric assay for dNTP hydrolase activity, using Escherichia coli inorganic pyrophosphatase to convert triphosphate to 3 phosphates 3.1.5.B1 dNTPase analysis high-throughput screen that directly measures SAMHD1 catalytic activity, utilizing bis(4-nitrophenyl) phosphate as the substrate and Mn2+ as the activating cation 3.1.6.1 arylsulfatase (type I) analysis cytochemical localization of enzyme in cytoplasmic vesicles as a specific detection method for lysosomes 3.1.6.1 arylsulfatase (type I) analysis distinction of lysosomes and reservosomes by cytochemical localization of enzyme in epimastigote and trypomastigote 3.1.6.1 arylsulfatase (type I) analysis development of a time-based mass spectrometric assay to analyze substrate conversion and substrate specificity 3.1.6.1 arylsulfatase (type I) analysis enzyme hydrolyzes dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate in a urine matrix. Purified arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates. The enzyme can be employed for short three-step chemoenzymatic synthesis of 5alpha-androstane-3beta,17beta-diol 17-glucuronide from epiandrosterone 3-sulfate 3.1.6.1 arylsulfatase (type I) analysis high-throughput screen for mutants expressed in Escherichia coli. The alkaline lysis buffer is 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature. The enzmye tolerates final 0.1 % Tween-20, and activities in lysates are steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing 3.1.6.2 steryl-sulfatase analysis method for the evaluation of aromatase and steryl-sulfatase activites in endometrial tumors using tritium-labeled steroids 3.1.6.2 steryl-sulfatase analysis enzyme hydrolyzes dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate in a urine matrix. Purified arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates. The enzyme can be employed for short three-step chemoenzymatic synthesis of 5alpha-androstane-3beta,17beta-diol 17-glucuronide from epiandrosterone 3-sulfate 3.1.6.B2 fucoidan-2-sulfatase analysis the enzyme hydrolyzes only the sulfate group at the 2-O position of the fucopyranoside. Therefore, this sulfoesterase is a helpful tool in the structure-activity study of the fucoidan, as the 2-O sulfation level plays a central role in the biological properties of the polysaccharide 3.1.6.9 chondro-4-sulfatase analysis the enzyme is a useful tool for structural studies of chondroitin sulfate and dermetan sulfate 3.1.6.13 iduronate-2-sulfatase analysis direct assay for enzyme activity using substrate 4-methylumbelliferyl alpha-L-idopyranosiduronic acid 2-sulfate and LC-MS/MS based detection 3.1.6.20 S-sulfosulfanyl-L-cysteine sulfohydrolase analysis the enzyme gene is used as a marker for detection of sulfur-oxidizing bacteria in coastal sediments 3.1.8.1 aryldialkylphosphatase analysis PON1 status can be used as a potential biomarker for insecticides metabolites in urine, e.g. dimethyl phosphate and diethyl phosphate, and of susceptibility to organophosphate toxicity, overview 3.1.8.1 aryldialkylphosphatase analysis development of a polymerized crystalline colloidal array photonic crystal sensing material which reversibly senses the organophosphate compound methyl paraoxon at micromolar concentrations in aqueous solutions using the enzyme, mechanism, overview 3.1.8.1 aryldialkylphosphatase analysis description of an easy and non-toxic method for large scale phenotyping and activity quantitation of arylesterase 3.1.8.1 aryldialkylphosphatase analysis PON1 has the potential to be used as a catalytic bioscavenger of nerve agents. Insect production of PON1 may provide a source for both in vitro enzymatic and crystallographic studies and in vivo stability and anti-nerve agent efficacy testing 3.1.8.2 diisopropyl-fluorophosphatase analysis biosensors using immobilized recombinant Escherichia coli cells expressing OPH are being employed for identifying OP nerve agents 3.1.11.1 exodeoxyribonuclease I analysis an exonuclease I hydrolysis assay for evaluating G-quadruplex stabilization by small molecules 3.1.11.2 exodeoxyribonuclease III analysis restriction analysis of the prototype strain of enteric adenovirus type 41 using exonulease 3.1.11.2 exodeoxyribonuclease III analysis analysis of 3'-terminal nucleotide sequences of duplex DNA. Under selected conditions the enzyme can be used to remove a small defined number of nucleotides from each 3'-terminus of duplex DNA 3.1.11.2 exodeoxyribonuclease III analysis use of the enzyme in a quantitative DNA probe assay based on exonuclease III digestion of probes hybridized on target DNA 3.1.11.2 exodeoxyribonuclease III analysis identification of DNA bonding sites of two pyrrolobenzodiazepine derivatives - tomamycin and anthramycin 3.1.11.2 exodeoxyribonuclease III analysis the enzyme is used for incremental truncation to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries have application in protein engineering as well as protein folding, enzyme evolution and the chemical synthesis of proteins 3.1.11.2 exodeoxyribonuclease III analysis use of exonuclease III to determine the site of stable lesions in defined sequences of DNA: the cyclobutane pyrimidine dimer and cis and trans dichlorodiamine platinum II examples 3.1.11.2 exodeoxyribonuclease III analysis enzymatically amplified surface plasmon resonance imaging detection of DNA by exonuclease III digestion of DNA microarrays. Through the use of ExoIII in conjunction with DNA microarrays, a 100-1000 improvement in the detection limit for the multiplexed surface plasmon resonance imaging detection of 16-mer oligonucleotides is achieved. This enhancement is not as great as RNase H amplification with RNA microarrays. The major advantage of ExoIII amplification compared to RNase H is that the additional difficulty in preparing and handling RNA microarrays is avoided. The ExoIII enzymatic amplification process can be used with the more robust and cost-effective DNA microarrays that are currently applied in research areas such as gene analysis and medical diagnostics. Most DNA sequences can be detected since the activity of ExoIII is not sequence dependent 3.1.11.2 exodeoxyribonuclease III analysis utility of a more versatile target assisted Exo III-catalyzed amplification fluorescence polarization, TAECA-FP, methodology for the highly sensitive and selective detection of DNA 3.1.11.2 exodeoxyribonuclease III analysis a combined treatment of nucleosomes with micrococcal nuclease and exonuclease III overcomes micrococcal nuclease sequence preference and produces nucleosomal DNA trimmed symmetrically and precisely at the core/linker junctions regardless of the underlying DNA sequence. Combined micrococcal nuclease/exonuclease III digestion can be applied to in situ chromatin for unbiased genome-wide mapping of nucleosome positions that is not ifluenced by DNA sequences at the core/linker junctions. The same approach can be also used for the precise mapping of the extent of linker DNA protection by H1 and other protein factors associated with nucleosome linkers 3.1.11.2 exodeoxyribonuclease III analysis development of a graphene oxide-based fluorescent aptasensor for adenosine detection by employing exonuclease III as a signal amplifying element. In the presence of adenosine, the adenosine aptamers associate with the targets, which leads to the formation of duplex DNAs between the cDNAs and the signal probes. Exonuclease III thereafter can digest the duplex DNAs from 3' blunt terminus of signal probes, liberating the fluorophore. The biosensor exhibits an ultrahigh sensitivity and holds a versatile platform for clinical diagnostics, molecular biology and drug developments 3.1.11.2 exodeoxyribonuclease III analysis cell cycle profiling by image and flow cytometry, optimised protocol for the detection of replicational activity using 5-bromo-2'-deoxyuridine, low concentration of hydrochloric acid and exonuclease III, method evaluation and optimization, detailed overview 3.1.11.3 exodeoxyribonuclease (lambda-induced) analysis use as reagent restriction mapping and substrate preparation 3.1.11.3 exodeoxyribonuclease (lambda-induced) analysis for studies of genetic recombination 3.1.11.3 exodeoxyribonuclease (lambda-induced) analysis use as reagent for determination of secondary structures at DNA termini 3.1.11.3 exodeoxyribonuclease (lambda-induced) analysis ultiplexed single-molecule assay for enzymatic activity on flow-stretched DNA can be applied to lambda exonuclease 3.1.11.3 exodeoxyribonuclease (lambda-induced) analysis a novel method for real-time monitoring of the activity and kinetics of T4 polynucleotide kinase (PNK) by use of a singly fluorophore-labeled DNA-hairpin smart probe (SP) coupled with lambda exonuclease (lambda exo) cleavage. 3.1.11.3 exodeoxyribonuclease (lambda-induced) analysis simple and homogeneous microRNA assay by integration of ligase chain reaction and lambda exonuclease-assisted cationic conjugated polymer biosensing. Ligase chain reaction is utilized for exponential amplification of microRNA, and lambda exonuclease is introduced to degrade excess fluorescein-labeled probes in ligase chain reaction for eliminating background signal. The method is sensitive enough to detect 0.1 fM target microRNA and specific to discriminate one-base difference of microRNAs 3.1.11.3 exodeoxyribonuclease (lambda-induced) analysis an ultrasensitive electrochemical biosensor for polynucleotide kinase assay based on gold nanoparticle-mediated lambda exonuclease cleavage-induced signal amplification is constructed. The presence of polynucleotide kinase (PNK) induces the phosphorylation of the strand 2-strand 1 hybrid and the subsequent cleavage of double-stranded DNA (dsDNA) by lambda exonuclease, resulting in the release of AuNP-strand 2 conjugates and [Ru(NH3)6]3+ from the gold electrode surface and consequently the decrease of electrochemical signal. The PNK activity can be simply monitored by the measurement of [Ru(NH3)6]3+ peak current signal. This assay is very sensitive with a very low detection limit and exhibits a large dynamic range from 0.001 to 10 U/ml. The method can be used to screen the PNK inhibitors, and it shows excellent performance in real sample analysis, thus holding great potential for further applications in biological researches and clinic diagnosis 3.1.11.4 exodeoxyribonuclease (phage SP3-induced) analysis determination of the direction of hydrolysis of processive exonucleases 3.1.11.5 exodeoxyribonuclease V analysis no recombination in recB2109CD mutants 3.1.11.5 exodeoxyribonuclease V analysis possible role in genetic recombination 3.1.11.5 exodeoxyribonuclease V analysis cloning of palyndromic structures in the Physarum actin gene in E. coli lacking the recBC gene 3.1.11.5 exodeoxyribonuclease V analysis lack of enzyme increases transfection frequencies with linear DNA 3.1.11.5 exodeoxyribonuclease V analysis recBCD inhibits recombination between closely related bacteria 3.1.11.5 exodeoxyribonuclease V analysis required for stability of plasmids 3.1.11.6 exodeoxyribonuclease VII analysis mapping of mRNA isoforms 3.1.11.6 exodeoxyribonuclease VII analysis xseA-mutants for measurement of single-stranded apurinic-apyrimidinic endonuclease 3.1.11.6 exodeoxyribonuclease VII analysis degradation of adenovirus-associated DNA 3.1.11.6 exodeoxyribonuclease VII analysis of nucleic acid structure 3.1.11.6 exodeoxyribonuclease VII analysis purification of DNA rings 3.1.11.6 exodeoxyribonuclease VII analysis removal of single-stranded DNA 3.1.11.6 exodeoxyribonuclease VII analysis of spliced RNA 3.1.11.6 exodeoxyribonuclease VII analysis excision of DNA segments from cloning vectors 3.1.13.1 exoribonuclease II analysis - 3.1.14.1 yeast ribonuclease analysis knowledge of amino acid sequences are useful for phylogenetic studies of several fungi 3.1.16.1 spleen exonuclease analysis kinetic studies on macromolecular substrates degradation 3.1.16.1 spleen exonuclease analysis end group and sequential analysis of polynucleotides 3.1.16.1 spleen exonuclease analysis detection and assay of RNA-linked nascent DNA pieces in E.coli strains 3.1.16.1 spleen exonuclease analysis broadly applicable, robust, and rapid method for complete sequence confirmation of highly modified oligonucleotides containing a mixture of 2'-deoxy, 2'-fluoro, 2'-O-methyl, abasic and ribonucleotides. The sense and antisense strands from synthetic short interfering RNA duplexes are digested individually using both 5'- and 3'-exonucleases and the resulting ladders are analyzed using MALDITOF mass spectrometry. Complete sequence confirmation for the antisense strands of four synthetic RNA duplexes is obtained, whereas a three-base sequence gap in the 5'-end is observed for all four sense strands. Outline of a general strategy for routine sequence confirmation of highly modified oligonucleotides 3.1.21.1 deoxyribonuclease I analysis study of chromatin structure, visualizing cellular microfilaments by DNase I-actin interaction 3.1.21.1 deoxyribonuclease I analysis preparation of substrates for the nick translation reaction, production of random DNA fragments, analysis of DNA-protein complexes 3.1.21.1 deoxyribonuclease I analysis enzymatic activity assay for determination of enzyme stability. Method bases on a colorimetric endpoint activity assay using degradation of a DNA/methyl green complex and is feasible on an automated analyzer system within a rather short time 3.1.21.1 deoxyribonuclease I analysis microtiter plate assay for quantification of enzyme activity in biological fluids. Assay is based on hydrolysis of 974 bp PCR product labeled with biotynilated forward and fluorescein-labeled reverse primers. Urine samples give an activity of 1.49 U/ml, blood plasma of 0.36 U/ml 3.1.21.1 deoxyribonuclease I analysis use of DNase I pre-treatment significantly increases the reliability and sensitivity of immunodetection of CIP/KIP cyclin-dependent kinase inhibitors. Applications in developmental neurobiology and cancer diagnosis 3.1.21.1 deoxyribonuclease I analysis construction of a sensor electrode carrying Fc-oligo-SH to achieve an electrochemical DNase I assay. Under the optimum conditions of DNase I digestion at 37°C for 30 min, a quantitative analysis can be achieved in the range of 0.0001-0.01 units/microl of DNase I 3.1.21.1 deoxyribonuclease I analysis graphene-based real-time fluorescent assay of deoxyribonuclease I activity and inhibition. The system is composed of graphene oxide and a fluorescent dye fluorescein amidite-labeled dsDNA substrate. At first, the fluorescence of the substrate is quenched upon addition of graphene oxide. When nuclease is added to the mixture of dsDNA and graphene oxide, hydrolysis of dsDNA is initiated and small DNA fragments are produced. The short fluorescein amidite-linked DNA fragments are released, and the fluorescence gets a restoration. DNase I activity can be quantitatively analyzed by the velocity of the enzymatic reaction, and 1.75 U/ml DNase I can be detected 3.1.21.1 deoxyribonuclease I analysis use of fluorescent DNA-templated gold/silver nanoclusters as a probe for sensitive detection of deoxyribonuclease I. The procedure is based on quenching fluorescence of DNA-templated gold/silver nanoclusters by DNase I digestion of the DNA 5'-CCCTTAATCCCC-3' template. This decrease in fluorescence intensity permits sensitive detection of DNase I in a linear range of 0.013-60 microg per ml, with a detection limit of 3 ng per ml at a signal-to-noise ratio of 3. The practicality of this probe for detection of DNase I in human serum and saliva samples has been validated 3.1.21.2 deoxyribonuclease IV analysis application for biosensing 3.1.21.4 type II site-specific deoxyribonuclease analysis the enzyme can be used in DNA-based diagnostic applications 3.1.21.4 type II site-specific deoxyribonuclease analysis method for following the digestion of DNA by restriction endonucleases in real time without the use of any extrinsic dyes or labels via linear dichroism spectroscopy 3.1.21.4 type II site-specific deoxyribonuclease analysis genetic analysis of 24 Hungarian canine parvovirus strains collected from 2004 to 2008 revealed that all of them are type 2a strains. Due to a seemingly constant point mutation present in most of the Hungarian canine parvovirus 2a strains, a previously described MboII-based rapid identification of CPV2c strains unfortunately cannot be reliably used any more 3.1.21.4 type II site-specific deoxyribonuclease analysis type II REases are widely used as tools for the dissection, analysis and reconstruction of DNA 3.1.21.5 type III site-specific deoxyribonuclease analysis transcriptome analysis 3.1.21.7 deoxyribonuclease V analysis determination of the minimum value of triplet energies for a photosensitizer to produce thymine cyclobutane dimer in DNA 3.1.22.1 deoxyribonuclease II analysis microtiter plate assay for quantification of enzyme activity in biological fluids. Assay is based on hydrolysis of 974 bp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers 3.1.25.1 deoxyribonuclease (pyrimidine dimer) analysis - 3.1.26.1 Physarum polycephalum ribonuclease analysis - 3.1.26.4 ribonuclease H analysis hybrid RNase H molecules with various oligodeoxyribonucleotides may facilitate structural studies of RNA and prove useful as tool for RNA manipulations 3.1.26.4 ribonuclease H analysis facile method that identifies single-stranded regions in RNA using short, randomized DNA oligonucleotides and RNase H cleavage. These regions are then used as constraints in secondary structure prediction. This method was used to improve the secondary structure prediction of Escherichia coli 5S rRNA. The addition of constraints from RNase H cleavage improves the prediction to 100% of base pairs. The same method was used to generate secondary structure constraints for yeast tRNAPhe, which is accurately predicted in the absence of constraints to 95%. The method is advantageous over other single-stranded nucleases since RNase H is functional in physiological conditions and can be used for any RNA to identify accessible binding sites for oligonucleotides or small molecules 3.1.26.4 ribonuclease H analysis quadruplex-based fluorescence assay for sensitive, facile, real-time, and label-free detection of RNase H activity and inhibition by using a G-quadruplex formation strategy. A RNA-DNA substrate is prepared, with the DNA strand designed as a quadruplex-forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G-rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G-quadruplex binder, N-methyl mesoporphyrin IX, giving a dramatic increase in fluorescence and serving as a reporter of the reaction. The assay is simple in design, fast in operation and convenient 3.1.26.5 ribonuclease P analysis potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure and a general approach for the engineering of RNase P ribozymes 3.1.26.5 ribonuclease P analysis coupling DNA micro-arrays with conditional mutants of RNase P in yeast is a powerful approach to conduct genomewide searches and identify new substrates (e.g., polycistronic mRNAs, snoRNAs) 3.1.26.5 ribonuclease P analysis fluorescein labeling has only a modest effect on the binding affinity of pre-tRNA and this fluorescent titration method may be useful for rapidly measuring the affinity of RNase P for a wide variety of substrates 3.1.26.11 tRNase Z analysis comparative studies both bis(p-nitrophenyl)phosphate hydrolysis and pre-tRNA processing of tRNase Z variants 3.1.26.12 ribonuclease E analysis the enzyme can be a model for investigation of the action of site specific nucleases 3.1.26.13 retroviral ribonuclease H analysis use of 6-phenylpyrrolocytidine as a sensitive fluorescent reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single 6-phenylpyrrolocytidine insert is an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reports cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The 6-phenylpyrrolocytidine-based assay for RNase H is superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease. The assay is amenable to high-throughput microplate assay format 3.1.26.13 retroviral ribonuclease H analysis use of a commercially available computed radiography system for dental radiography to produce images from radiolabeled polyacrylamide gel electrophoresis assays and its application for quantitative analyses of the human immunodeficiency virus type 1 reverse transcriptase polymerase-independent ribonuclease H activity monitored by PAGE analysis. The methodology allows quantifying effectively the RNase H catalyses and the obtained data are in good agreement with previous reference works 3.1.30.1 Aspergillus nuclease S1 analysis - 3.1.30.1 Aspergillus nuclease S1 analysis in DNA-RNA annaeling experiments as reagent for selective removal of nonhybridized polynucleotides 3.1.30.1 Aspergillus nuclease S1 analysis studies of nucleic acid secondary structure 3.1.30.1 Aspergillus nuclease S1 analysis analysis of heteroduplex and superhelical DNA, quantitation of nucleic acid hybridization 3.1.30.1 Aspergillus nuclease S1 analysis analysis of cap structures at the 5'-termini of eucaryotic mRNA 3.1.30.1 Aspergillus nuclease S1 analysis reagent for mapping splice points and ordered regions in RNA 3.1.30.1 Aspergillus nuclease S1 analysis analysis of tRNA for determination of amino acid specificity 3.1.30.1 Aspergillus nuclease S1 analysis gene isolation 3.1.30.1 Aspergillus nuclease S1 analysis used in S1 nuclease protection assays 3.1.30.1 Aspergillus nuclease S1 analysis development of a fluorescent method for simple detection of sequence-specific single-strand DNA in complex, contaminant-ridden samples directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio can be increased dramatically. It enables the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate from pork 3.1.31.1 micrococcal nuclease analysis peptide model LMYKGQPM from staphylococcal nuclease can serve as model for faster folding beta-hairpins pursuing fast folding events 3.1.31.1 micrococcal nuclease analysis purification of DNA from the cell-associated herpesvirus Marek’s disease virus 3.1.31.1 micrococcal nuclease analysis staphylococcal nuclease fused at its N-terminus to signal peptide of the lactococcal Usp45 protein (SP Usp45-NucB), as reporter for expression and secretion in Lactobacillus bulgaricus 3.1.31.1 micrococcal nuclease analysis the enzyme is a useful tool for mapping chromatin structure in eukaryotes, usage of the enzyme to determine the positions of nucleosomes within a region of DNA to identify dynamic changes induced during gene regulation 3.1.31.1 micrococcal nuclease analysis combined MNase/exoIII digestion can be applied to in situ chromatin for unbiased genome-wide mapping of nucleosome positions that is not influenced by DNA sequences at the core/linker junctions. The same approach can be also used for the precise mapping of the extent of linker DNA protection by H1 and other protein factors associated with nucleosome linkers 3.1.31.1 micrococcal nuclease analysis the enzyme is very widely used to map nucleosomes 3.1.31.1 micrococcal nuclease analysis the existence of MNase can be the standard to identify Staphylococcus aureus, and the content of MNase can be used to evaluate the pathogenicity of Staphylococcus aureus, usage of a graphene oxide-based biosensor, method development, overview 3.2.1.1 alpha-amylase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.1 alpha-amylase analysis the alpha-amylase from Anoxybacillus amylolyticus could be used in a sensitive method to detect very low concentrations of heavy metals 3.2.1.3 glucan 1,4-alpha-glucosidase analysis method for anchoring native acarbose to gold chip surfaces for surface plasmon resonance studies employing glucoamylase as analyte. The key method is the chemoselective and protecting group-free oxime functionalization of the pseudo-tetrasaccharide-based inhibitor acarbose. At pH 7.0 the association and dissociation rate constants for the acarbose-glucoamylase interaction are 10000 per M and s and 103 per s, respectively, and the conformational change to a tight enzyme-inhibitor complex affects the dissociation rate constant by a factor of 100 per s. The acarbose-presenting surface plason resonance surfaces can be used as a glucoamylase sensor that allows rapid, label-free affinity screening of small carbohydrate-based inhibitors in solution 3.2.1.4 cellulase analysis endo-beta-1,4-D-glucanase can be used as a marker to study root development in Arabidopsis 3.2.1.4 cellulase analysis labeled Trichoderma reesei cellulase is useful as a marker for Acanthamoeba cyst wall cellulose in infected tissues 3.2.1.4 cellulase analysis specific and sensitive assay of endo-1,4-beta-glucanase (cellulase). The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-beta-cellotrioside in the presence of thermostable beta-glucosidase. Hydrolysis by exo-acting enzymes such as beta-glucosidase and exo-beta-glucanase is prevented by the presence of the benzylidene group on the non-reducing end D-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-beta-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free D-glucose by the beta-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation 3.2.1.4 cellulase analysis method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation. Cellulases are printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms are detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose, while exocellulase isoforms are detected in electrophoresis gel with 4-methylumbelliferyl-beta-D-cellobioside 3.2.1.4 cellulase analysis polymerization-based assay for determining the potency of cellulolytic enzyme formulations on pretreated biomass substrates by monitoring the autofluorescence of cellulose. The one-pot method is label-free, rapid, highly sensitive, and requires only a single pipetting step. Using model enzyme formulations derived from Trichoderma reesei, Trichoderma longibrachiatum, Talaromyces emersonii and recombinant bacterial minicellulosomes from Clostridium thermocellum, enzyme performance based on differences in thermostability, cellulose-binding domain targeting, and endo/exoglucanase synergy can be differentiated 3.2.1.4 cellulase analysis screening for thermostable cellulase using 2% carboxymethyl cellulose and congo red as an indicator at temperatures 0°C, 37°C, 45°C and 50°C,respectively. Eight isolates were selected for further screening and show the abilities to secrete cellulases by forming distinct halo zones on selective agar plate. The maximum halo zones ranging from 32 mm to 35 mm are obtained after 72 hours incubation at 50°C 3.2.1.4 cellulase analysis construction of a pipeline based on Leishmania tarentolae cell-free system to characterize 30 putative thermostable endo-1,4-beta-glucanases and xylanases identified in public genomic databases. The system uses high-throughput assays for glucanase and xylanase activities that rely on solubilisation of labelled particulate substrates performed in multiwell plates 3.2.1.4 cellulase analysis rapid, selective, quantitative assay based on substrate 44-nitrophenyl 4,6-O-(3-oxobutylidene)-beta-D-cellopentaoside and reaction of product 4,6-O-(3-oxobutylidene)-beta-D-cellotriose with beta-glucosidase 3.2.1.6 endo-1,3(4)-beta-glucanase analysis use in structural analysis of polysaccharides 3.2.1.6 endo-1,3(4)-beta-glucanase analysis assay procedure for the measurement of beta-glucanase and lichenase in crude enzyme extracts. Substrate 4,6-O-benzylidene-2-chloro-4-nitrophenyl-beta-3'-cellotriosyl-beta-glucopyranoside is a more generally applicable reagent. Standard curves allow the assay absorbance response to be directly converted to beta-glucanase/lichenase activity on barley beta-glucan. Manual and automated assay formats aloow the analysis of beta-glucanase in malt flour and lichenase enzyme extracts and the assays are repeatable 3.2.1.8 endo-1,4-beta-xylanase analysis construction of a pipeline based on Leishmania tarentolae cell-free system to characterize 30 putative thermostable endo-1,4-beta-glucanases and xylanases identified in public genomic databases. The system uses high-throughput assays for glucanase and xylanase activities that rely on solubilisation of labelled particulate substrates performed in multiwell plates 3.2.1.8 endo-1,4-beta-xylanase analysis enzyme coupled assay procedure, using substrate 4-nitrophenyl 4,6-O-(3-oxobutylidene)-beta-D-glucosyl-(1->4)-[(1->4)-beta-D-xylopentaoside] and release of 4-nitrophenol by beta-xylosidase 3.2.1.14 chitinase analysis the polymorphic gene encoding the enzyme is a marker for species determination in Entamoeba 3.2.1.14 chitinase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.14 chitinase analysis transglycosylation reaction takes place under the conditions used for HPLC analysis, but not under the ESI-MS conditions 3.2.1.14 chitinase analysis isozyme YlCts1p can be used as a good model for studying O-glycosylation in the dimorphic yeast Yersinia lipolytica 3.2.1.14 chitinase analysis strain C61 displays antifungal activities in vitro and is used successfully for the biocontrol of plant diseases under field conditions 3.2.1.15 endo-polygalacturonase analysis kinetic approach in a commercial enzyme preparation under pulsed electric fields explains satisfactorily evolution of PG activity and permits obtaining valuable information about the conditions for which either inactivation or enhancement of PG activity may happen. The mechanism offers simplicity and flexibility and can explain occurrences such as flat and activation shoulders at the beginning of inactivation curves for enzymes under pulsed electric field treatments 3.2.1.17 lysozyme analysis cross-linking method for lysozyme and catalytic activity assay. Catalytic activity of lysozyme dimer is the same as monomer 3.2.1.17 lysozyme analysis establishment of a precise fluorimetric assay for determination and characterisation of lysozyme activity immobilised in the initial in situ formed pellicle. For in situ pellicle formation, bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times on buccal and palatal sites. The mean immobilised activity over all samples amounted to 68.67 U/cm2. The enzyme activity exposed at the pellicles' surfaces increases in a time-dependant manner and shows a Michaelis-Menten kinetic. Chlorhexidine and black tea reduce lysozyme activity of the in situ pellicle significantly 3.2.1.17 lysozyme analysis kinetics of tertiary conformation of lysozyme adsorbed on 90 nm silica nanoparticles. A rapid initial unfolding, followed by a much slower refolding and subsequent unfolding is observed, with the extent of unfolding being higher at lower surface concentration, higher ionic strengths, higher 2,2,2-trifluoroethanol and dithiothreitol concentrations and at pH 9. alpha-Helix content is lower for adsorbed lysozyme compared to bulk with a corresponding increase in beta-sheet and random coil 3.2.1.18 exo-alpha-sialidase analysis neuraminidase assay based on PNA hemagglutanation is highly sensitive, reproducible and can be used as a tool to detect sialidase activity in anaerobic bacteria, particularly, in species of the bacteroides fragilis group 3.2.1.18 exo-alpha-sialidase analysis ensemble-based virtual screening and relaxed complex scheme approach for structure-based drug discovery 3.2.1.18 exo-alpha-sialidase analysis subcloning of carbohydrate-binding module CBM40 of sialidase, showing high affinity for sialic acid and specificity to alpha(2,3), alpha(2,6), and alpha(2,8)-linked sialosides. Creation of polypeptides containing up to four CBM40 modules in tandem show increased affinities compared with the single module molecule. Variation in linker length has little effect on affinity 3.2.1.18 exo-alpha-sialidase analysis virtual screening method for inhibitors based on consensus scoring and ligand efficiency indices, which allows the combination of pharmacodynamic and pharmacokinetic properties into unique measures 3.2.1.21 beta-glucosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.22 alpha-galactosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.22 alpha-galactosidase analysis determinantion of alpha-galactosidase A activity in samples from patients with Fabry disease and healthy controls. Average enzyme activity in dried blood spot samples prepared using EDTA tubes is higher compared to those spotted directly irrespective of disease status 3.2.1.23 beta-galactosidase analysis enzyme can be used as a tool in the structural analysis of D-galactose-containing oligosaccharide chains, as well as for the synthesis of glycoconjugates 3.2.1.23 beta-galactosidase analysis the enzyme is a reliable marker for the course of replicative cell senescence 3.2.1.23 beta-galactosidase analysis comparison of commercially available kits to assess water quality and evaluation of their ability to detect Escherichia coli. Chromocult, MI agar, Readycult, and Colilert detect beta-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 Escherichia coli strains tested. These four methods detect beta-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The high level of false-negative results for Escherichia coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive Escherichia coli strains 3.2.1.23 beta-galactosidase analysis electrophoretic mobility and catalytic activity of individual molecules of enzyme are reproducible for each molecule but different for individual molecules. The observed ranges in electrophoretic motility are similar for different experimental protocols. There is no relationship between the observed activities and electrophoretic motilities 3.2.1.23 beta-galactosidase analysis precise and reliable detection of Escherichia coli strains for differentiation from biochemically and phylogenetically related bacteria. Method is based on polymerase chain reaction, in which four genes coding for lactose permease, cytochrome bd complex, beta-D-glucuronidase, and beta-D-galactosidase, serve as target DNA sequences 3.2.1.23 beta-galactosidase analysis enzyme functions both as a marker, promoting colony color development in the presence of a lactose analogue S-gal, and as a reporter enabling quantitative measurement by a simple colorimetric assay. The combination of BgaB expressed from promoters of varying strength with S-gal produces distinct black colonies in aerobic and anaerobic conditions at temperatures ranging from 37 to 60°C, being an advantage over the conventional beta-galactosidase (LacZ) and substrate X-gal. The system allows for construction and measurement of expression levels of a library in just 4 days 3.2.1.24 alpha-mannosidase analysis characterization of asparagine-linked oligosaccharides 3.2.1.24 alpha-mannosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.25 beta-mannosidase analysis the enzyme together with alpha-galactosidase and endo-beta-D-mannanase, can be used to characterize the structures of more complex oligosacharides 3.2.1.25 beta-mannosidase analysis application in carbohydrate research 3.2.1.25 beta-mannosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.26 beta-fructofuranosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.B26 Sulfolobus solfataricus beta-glycosidase analysis reporter genes in molecular biology 3.2.1.28 alpha,alpha-trehalase analysis highly specific tool for the assay of trehalose 3.2.1.31 beta-glucuronidase analysis assay for beta-glucuronidase is able to distinguish Escherichia coli from other Escherichia species 3.2.1.31 beta-glucuronidase analysis enzymatic assay adapted to study the fate of fecal coliforms in survival experiments, and appears to be rapid and efficient way to estimate the microbiological quality of surface waters. The major advantage of the enzymtic assay is the very short time response, and thus this method offers a powerful, rapid, and efficient way to estimate the microbiological quality of bathing and fishing areas, and to monitor disinfection efficiencies 3.2.1.31 beta-glucuronidase analysis reporter enzyme which is used for studies in higher plants because endogenous activities are low and sensitive assays are available. A version of GUS with phenylalanine at the mature N-terminus accumulates a minimum of 3fold lower than GUS with methionine at its mature N-terminus. This altered protein can be useful for promoter studies which require more rapid changes in the accumulation of the reporter protein 3.2.1.31 beta-glucuronidase analysis the enzyme is useful in analysis of phytoestrogens and related compounds in human biofluids, e.g. urine 3.2.1.31 beta-glucuronidase analysis comparison of commercially available kits to assess water quality and evaluation of their ability to detect Escherichia coli. Chromocult, MI agar, Readycult, and Colilert detect beta-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 Escherichia coli strains tested. These four methods detect beta-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The high level of false-negative results for Escherichia coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive Escherichia coli strains 3.2.1.31 beta-glucuronidase analysis comparison of commercially available kits used for the simultaneous detection of coliforms and Escherichia coli from water. Membrane lactose glucuronide agar, Colilert-18, MI agar, Colitag and Chromocult agar to detect beta-D-glucuronidase activity are tested with over 1000 isolates of Escherichia coli recovered from naturally contaminated water samples. Four of the media give very similar results but membrane lactose glucuronide agar fails to detect glucuronidase activity in 15.6% of the cultures tested 3.2.1.31 beta-glucuronidase analysis in environmental toxicology, techniques used to visualise lysosomes in order to determine their responses to pollutants are the lysosomal structural changes test and the lysosomal membrane stability test are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase and hexosaminidase. beta-Glucuronidase, but not hexosaminindase, histochemistry provides an appropriate marker for the lysosomal structural changes test and that, although both lysosomal marker enzymes can be employed in the lysosomal membrane stability test, different values would be obtained depending on the marker enzyme employed 3.2.1.31 beta-glucuronidase analysis precise and reliable detection of Escherichia coli strains for differentiation from biochemically and ohylogenetically related bacteria. Method is based on polymerase chain reaction, in which four genes coding for lactose permease, cytochrome bd complex, beta-D-glucuronidase, and beta-D-galactosidase, serve as target DNA sequences 3.2.1.31 beta-glucuronidase analysis Sulfolobus gene can be used as reporter in any thermophilic microorganism lacking an endogenous beta-glucuronidase activity 3.2.1.31 beta-glucuronidase analysis construction of a noncompetitive homogeneous biosensor system for immunodetection of small molecules based on beta-glucuronidase complementation 3.2.1.33 amylo-alpha-1,6-glucosidase analysis useful in structural determination of glycogen and amylopectin 3.2.1.35 hyaluronoglucosaminidase analysis cancer cell lines often secrete hyaluronidase, the enzyme might therefore be a marker for growing tumours 3.2.1.37 xylan 1,4-beta-xylosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.38 beta-D-fucosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.39 glucan endo-1,3-beta-D-glucosidase analysis structural analysis of laminarins 3.2.1.B47 exo-fucoidanase analysis the enzyme is useful for structural analysis of sulfated fucan 3.2.1.51 alpha-L-fucosidase analysis studies of structures and functions of glycoconjugates and oligosaccharides due to unique substrate specificity 3.2.1.51 alpha-L-fucosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.51 alpha-L-fucosidase analysis low cost and accurate method for the detection and analytical determination of the activity of enzyme alpha-L-fucosidase. The method is based upon measuring the fluorescence intensity of the complex ion associate of the ion associate of rhodamine-B and the compound 2-chloro-4-nitrophenol at 580 nm in phosphate buffer, pH 5 against the reagent blank. The correlation between the fluorescence activity of the enzyme by the developed procedures and the standard method is positive and highly significant in patients and controls. The developed method is simple and proceeds without practical artifacts compared to the standard method 3.2.1.51 alpha-L-fucosidase analysis the facile recombinant expression procedure and the peculiar substrate specificities make the enzyme an attractive candidates for bioanalytical applications 3.2.1.51 alpha-L-fucosidase analysis the facile recombinant expression procedure and the peculiar substrate specificities make these enzymes attractive candidates for bioanalytical applications 3.2.1.51 alpha-L-fucosidase analysis tool for the analysis and modification of human and animal milk oligosaccharides 3.2.1.52 beta-N-acetylhexosaminidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.53 beta-N-acetylgalactosaminidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.54 cyclomaltodextrinase analysis synthesis of radiolabeled linear and cyclic maltodextrins as tools for metabolism investigation 3.2.1.55 non-reducing end alpha-L-arabinofuranosidase analysis - 3.2.1.55 non-reducing end alpha-L-arabinofuranosidase analysis commercially available enzyme, potential for use in the structural analysis of complex carbohydrates 3.2.1.55 non-reducing end alpha-L-arabinofuranosidase analysis enzymatic tool for structural analysis of oligo- and polysaccharides 3.2.1.58 glucan 1,3-beta-glucosidase analysis the small enzyme is an ideal computational model for its family of enzymes, GH5, and can be used to create several enzyme-substrate models starting from a crystallographic glucanase inhibitor structure 3.2.1.60 glucan 1,4-alpha-maltotetraohydrolase analysis bioconversion of starch into maltotetraose in a membrane recycle bioreactor 3.2.1.65 levanase analysis the enzymatic activity can be used to characterize the aquatic ecosystems impurity degree, the evolution prognosis and water quality maintenance, the Gilau dam reservoir sediment has a decreased and quite unstable enzymatic potential 3.2.1.67 galacturonan 1,4-alpha-galacturonidase analysis studying fine structure of xylogalacturonan in pectin 3.2.1.67 galacturonan 1,4-alpha-galacturonidase analysis development of an Escherichia coli-based metabolic selection system for the uncovering of new oligogalacturonate-active enzymes. Based on the expression of the specific permease TogMNAB, this system enables the entry of oligogalacturonates into the cytoplasm of Escherichia coli thus providing a modified strain usable for this purpose. This tool is used for the metabolic selection of Thermotoga maritima exopolygalacturonase (TmGalU) mutants enabling the uptake of sodium trigalacturonate as the sole carbon source by the bacterium. In one round of error-prone PCR and selection, mutants of TmGalU with a 4fold increased turnover at pH 7.0 and 2fold more active at 37°C than wild-type enzyme are isolated 3.2.1.68 isoamylase analysis - 3.2.1.68 isoamylase analysis elucidating of the finestructures of various polysaccharides 3.2.1.68 isoamylase analysis structural determination of glycogen and starch components 3.2.1.68 isoamylase analysis bioinformatics, microarray and reporter gene analyses reveal that AtISA1 and AtISA2, AtISA2 and AtISA3 or AtISA1, AtISA2 and AtISA3 are coexpressed under certain conditions 3.2.1.68 isoamylase analysis composition and starch molecular structure of eight rice varieties are studied, rice starches were debranched using isoamylase, different amount and size of longbranch chain of amylopectin influences the compactness of starch structure 3.2.1.68 isoamylase analysis Di-O-a-maltosyl-beta-cyclodextrin is fromed via a transglycosylation reaction using TreX, a Sulfolobus solfataricus P2 debranching enzyme, isoamylase is used to examine the structure of the product and to produce branched cyclodextrins via reverse reaction 3.2.1.68 isoamylase analysis isoamylase-type debranching enzymes (ISAs) play an important role in determining starch structure 3.2.1.73 licheninase analysis the enzyme is used for oligosaccharide profiling of mixed-linkage glucan, beta-1,3-1,4-glucan, a characteristic hemicellulose in primary cell walls of grasses, that adsorps onto microcrystalline cellulose in a slow, irreversible, and temperature-dependent manner. The binding of the oligomer is reduced if the cellulose samples are first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan 3.2.1.73 licheninase analysis assay procedure for the measurement of beta-glucanase and lichenase in crude enzyme extracts. Substrate 4,6-O-benzylidene-2-chloro-4-nitrophenyl-beta-3'-cellotriosyl-beta-glucopyranoside is a more generally applicable reagent. Standard curves allow the assay absorbance response to be directly converted to beta-glucanase/lichenase activity on barley beta-glucan. Manual and automated assay formats aloow the analysis of beta-glucanase in malt flour and lichenase enzyme extracts and the assays are repeatable 3.2.1.74 glucan 1,4-beta-glucosidase analysis tandem mass spectrometry to compare gas-phase and solution binding of small-molecule inhibitors to the wild type and three mutant forms of Cex 3.2.1.75 glucan endo-1,6-beta-glucosidase analysis the enzyme is used for analysis of yeast polysaccharides cell wall composition in a method combines acid and enzymatic hydrolysis, and involves a chitinase from Streptomyces griseus, an endo/exo-beta-(1,3)-glucanase from Trichoderma species, recombinant endo-beta-(1,6)-glucanase from Trichoderma harzianum, and a beta-glucosidase from Aspergillus niger, method development and evaluation, overview 3.2.1.81 beta-agarase analysis use of enzyme in agarolysis for recovery of DNA from agarose gels, and use of enzyme as a reporter in the construction of a secretion signal trap, a simple and efficient molecular tool for the selction of genes encoding secretion proteins from both gram-positive and gram-negative bacteria 3.2.1.83 kappa-carrageenase analysis studies of carrageenan structure 3.2.1.88 non-reducing end beta-L-arabinopyranosidase analysis comparison of glycolytic and chitinolytic enzyme activities between desert and oasis flies of Phlebotomus papatasi to evaluate potential differences in susceptibility to infection with Leishmania major 3.2.1.91 cellulose 1,4-beta-cellobiosidase (non-reducing end) analysis polymerization-based assay for determining the potency of cellulolytic enzyme formulations on pretreated biomass substrates by monitoring the autofluorescence of cellulose. The one-pot method is label-free, rapid, highly sensitive, and requires only a single pipetting step. Using model enzyme formulations derived from Trichoderma reesei, Trichoderma longibrachiatum, Talaromyces emersonii and recombinant bacterial minicellulosomes from Clostridium thermocellum, enzyme performance based on differences in thermostability, cellulose-binding domain targeting, and endo/exoglucanase synergy can be differentiated 3.2.1.96 mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase analysis study of glycans and protein backbones as discrete entities and for defining the nature of the glycan-protein interface 3.2.1.96 mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase analysis essential reagent for the investigation of the structure and functions of glycoproteins 3.2.1.96 mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase analysis the enzyme will be an excellent tool for clarification of the structures and functions of complex oligosaccharides in glycoproteins 3.2.1.96 mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase analysis useful in metabolic studies dealing with oligosaccharide-lipid assembly and their involvement in the N-glycosylation of proteins 3.2.1.96 mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase analysis commonly used reagent in glycobiology research, including the characterization of oligosaccharides in glycoproteins 3.2.1.96 mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase analysis the enzyme is a useful tool for analyzing oligosaccharide contents of glycoproteins. Endo-CC1 would potentially be a valuable tool for analyzing oligosaccharides on glycoproteins, as large quantities of it can be made available easily and less economically 3.2.1.97 endo-alpha-N-acetylgalactosaminidase analysis the enzyme can release T-antigen, Galbeta(1-3)GalNAc, a carcinoma-associated marker from human cells, enzyme is useful for detection and determination of T-antigen from human erythrocytes and native human gastric carcinoma cells 3.2.1.97 endo-alpha-N-acetylgalactosaminidase analysis the enzyme is useful in the study of the structures of the carbohydrate moieties of various complex carbohydrates 3.2.1.97 endo-alpha-N-acetylgalactosaminidase analysis the enzyme is ideally suited to explore the distribution and function of mucin-type glycoproteins on normal and cancer cell surfaces 3.2.1.99 arabinan endo-1,5-alpha-L-arabinanase analysis - 3.2.1.99 arabinan endo-1,5-alpha-L-arabinanase analysis structural analysis of plant cell pectic polysaccharides and arabinans 3.2.1.99 arabinan endo-1,5-alpha-L-arabinanase analysis useful for studies on structure of protopectin 3.2.1.99 arabinan endo-1,5-alpha-L-arabinanase analysis useful in structural analysis of primary cell wall polysaccharides 3.2.1.101 mannan endo-1,6-alpha-mannosidase analysis used to determine the structure of mannan-protein linkage region 3.2.1.101 mannan endo-1,6-alpha-mannosidase analysis used to define kinetics of mannosylation of pro-alpha-factor, pro-alphaf, to distinguish alpha-1,6-mannosylated and alpha-1,2-mannosylated forms of glycoproteins 3.2.1.102 blood-group-substance endo-1,4-beta-galactosidase analysis endo-beta-galactosidase-Griffonia-simplicifolia-agglutinin-II-staining procedure into carbohydrate histochemistry of human tissues. The procedure may provide a powerful tool for analysing the backbone structures carrying the blood-group-related antigens in tissue sections 3.2.1.103 keratan-sulfate endo-1,4-beta-galactosidase analysis useful for studying the structure of glycoproteins and oligosaccharides 3.2.1.103 keratan-sulfate endo-1,4-beta-galactosidase analysis the enzyme can be used to distinguish between GlcNAcbeta1-6Gal and GlcNAcbeta1-3Gal units within linear backbone sequences of all known types of oligo-(N-acetyllactosamino)glycans 3.2.1.103 keratan-sulfate endo-1,4-beta-galactosidase analysis enzyme can be useful in studying the precise structures of keratan sulfates, related glycoproteins and oligosaccharides 3.2.1.106 mannosyl-oligosaccharide glucosidase analysis structural analysis of lipid-linked oligosaccharide precursor of asparaginyl oligosaccharides 3.2.1.106 mannosyl-oligosaccharide glucosidase analysis using the processing glucosidase activity as latency marker for topological studies with microsomal vesicles from nongluconeogenic tissues in addition to brain and cultured mammalian cells lacking glucose 6-phosphate phosphatase, to assess the intactness of microsomal vesicles 3.2.1.106 mannosyl-oligosaccharide glucosidase analysis contributes to knowledge of N-glycan biosynthesis 3.2.1.123 endoglycosylceramidase analysis the enzyme can be used for comprehensive profiling of glycosphingolipid glycans in a high-throughput workflow. The existing robotized N-glycan analysis platform is adapted for the quantitative high-throughput profiling of 2AB-fluorescent-labeled mammalian glycosphingolipid head groups using ultraperformance hydrophilic interaction liquid chromatography with fluorescence detection (UPLC-HILIC-FLD). An enabling component of the glycosphingolipid glycan workflow is the identification and characterization of the recombinant EGCase I enzyme from Rhodocococcus triatomea that exhibits a broad glycosphingolipid specificity, including the release of globo-series glycosphingolipids and Gal(beta1<->1)Cer, important classes of glycosphingolipids that are not efficiently released by known EGCases. The ability is demonstrated to characterize glycosphingolipid head groups from both mammalian cell surfaces and small volumes of blood serum. This analytical workflow will permit further exploration of the glycosphingolipid headgroup repertoire of glycosphingolipids from a broad range of biological sources and will enable studies aiming to identify cellular or serum glycosphingolipid-glycan biomarkers of disease 3.2.1.127 1,6-alpha-L-fucosidase analysis the enzyme is potentially useful for glycan sequencing and analysis of more complex glycoconjugates 3.2.1.132 chitosanase analysis development of a simple plate assay based on hydrolysis of soluble chitosan, for high-throughput screening for enzyme activity 3.2.1.132 chitosanase analysis fluorimetric determination of transition temperature is a reliable method for rapid assessment of the thermal behaviour of chitosanases and applicable to structure-function studies 3.2.1.139 alpha-glucuronidase analysis development of a precise alpha-glucuronidase assay by coupling the alpha-glucuronidase catalyzed formation of 4-nitrophenyl beta-D-xylopyranoside with its efficient hydrolysis by xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, is used as source of beta-xylosidase 3.2.1.140 lacto-N-biosidase analysis the enzyme may be useful in identifying type 1 structure in glycoconjugates and to distinguish type 1 oligosaccharides from type 2 oligosaccharides 3.2.1.142 limit dextrinase analysis methods for a rapid and cost efficient assay of both beta-amylase and limit dextrinase thermostability 3.2.1.142 limit dextrinase analysis assay for limit dextrinase activity using a chromogenic assay. The assay utilizes a small soluble chromogenic substrate which is hydrolyzed selectively by limit dextrinase in a coupled assay to release the chromophore 2-chloro-4-nitrophenol. The results obtained are comparable with the results obtained from a commercially available assay. The improved assay uses a soluble substrate, being suited for high-throughput screening 3.2.1.142 limit dextrinase analysis substrate 4,6-O-benzylidene-2-chloro-4-nitrophenyl-beta-63-alpha-D-maltotriosyl-maltotrioside (BzCNPG3G3, Hexachrom) is not susceptible to transglycosylation and serves amiably as a routine quantitative assay tool with the potential to run kinetic assays due to the low pKa (about 5.5) of the chromogenic moiety. Substrate 4,6-O-benzylidene-4-methylumbelliferyl-beta-63-alpha-D-maltotriosyl-maltotrioside (BzMUG3G3, Hexafluor) is susceptible to transglycosylation. Both substrates are applicable to industrial standard assays 3.2.1.143 poly(ADP-ribose) glycohydrolase analysis non-radioactive and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. The maximum velocity Vmax and the Michaelis constant Km of PARG reaction according to this method are 4.46 microM and 128.33 micromol/min/mg, respectively 3.2.1.145 galactan 1,3-beta-galactosidase analysis the activity of the enzyme is well suited for the study of type II galactan structures and provides an important tool for the investigation of the biological role of arabinogalactan proteins in plants 3.2.1.147 thioglucosidase analysis micellar electrokinetic capillary chromatography method for monitoring the myrosinase catalyzed hydrolysis of 2-hydroxy substituted glucosinolates and formation of the corresponding oxazolidine-2-thiones and nitriles. Method has a detection limit of 0.04 mM and a limit of quantification of 0.2 mM and 0.3 mM for the glucosibarin-derived oxazolidine-2-thione and nitrile, respectivityl 3.2.1.157 iota-carrageenase analysis usage of the enzyme for the analysis of the distribution of the carrabiose moieties in hybrid carrageenan chains, hybrid iota-/alpha-carrageenan, by Pseudoalteromonas atlantica a 4S-iota carrageenan sulfatase that converts iota-carrabiose into alapha-carrabiose units 3.2.1.166 heparanase analysis a simple, accurate, and robust biochemical assay for heparanase activity that uses a commercially available homogeneous substrate (fondaparinux) with a single enzymatic cleavage point and, thus, does not have the problems associated with using heparan sulfate-based assays. The assay is suitable for testing heparanase inhibitors and could easily be adapted for use in high-throughput screening applications 3.2.1.176 cellulose 1,4-beta-cellobiosidase (reducing end) analysis development of an optical tweezers-based single-molecule motility assay for precision tracking of Cel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Cel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. The fundamental stepping cycle likely includes energy from glycosidic bonds and other sources. The catalytic domain alone is sufficient for processive motion 3.2.1.176 cellulose 1,4-beta-cellobiosidase (reducing end) analysis polymerization-based assay for determining the potency of cellulolytic enzyme formulations on pretreated biomass substrates by monitoring the autofluorescence of cellulose. The one-pot method is label-free, rapid, highly sensitive, and requires only a single pipetting step. Using model enzyme formulations derived from Trichoderma reesei, Trichoderma longibrachiatum, Talaromyces emersonii and recombinant bacterial minicellulosomes from Clostridium thermocellum, enzyme performance based on differences in thermostability, cellulose-binding domain targeting, and endo/exoglucanase synergy can be differentiated 3.2.1.207 mannosyl-oligosaccharide alpha-1,3-glucosidase analysis synthetic route for fluorescent and biotin-modified activity-based probes for in vitro and in situ monitoring of alpha-glucosidases. alpha-Glucopyranose configured cyclophellitol aziridines label distinct retaining alpha-glucosidases including lysosomal acid alpha-glucosidase and ER alpha-glucosidase II, and this labeling can be tuned by pH 3.2.2.1 purine nucleosidase analysis construction of a Saccharomyces cerevisiae strain that requires expression of the Leishmania major enzyme for growth, and application of this strain for screening of enzyme inhibitors 3.2.2.5 NAD+ glycohydrolase analysis simple, fast and reliable fluorescence-based assay method to evaluate the NAD+ glycohydrolase activity of cell-permeable C3 proteins 3.2.2.5 NAD+ glycohydrolase analysis NADase fused to an amino-terminal fragment of anthrax toxin lethal factor is translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to the fused NADase in the presence of protective antigen results in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death 3.2.2.9 adenosylhomocysteine nucleosidase analysis adaptation of an enzyme-coupled colorimetric assay based on the quantification of homocysteine produced from S-adenosyl-L-homocysteine in presence of enzyme and S-ribosylhomocysteinase to cyclopropane fatty acid synthase for high-throughput screening of compound libraries 3.2.2.9 adenosylhomocysteine nucleosidase analysis enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine, the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase. Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase. Application of the assay to other enzymes that produce S-adenosylhomocysteine 3.2.2.17 deoxyribodipyrimidine endonucleosidase analysis analysis of cellular repair mechanisms 3.2.2.17 deoxyribodipyrimidine endonucleosidase analysis quantification of cyclobutane pyrimidine dimers formation. The ability of T4 endonuclease V to make single-stranded cuts at cyclobutane pyrimidine dimers sites is used to calculate the occurrence of cyclobutane pyrimidine dimers in the irradiated pAJ and Beauveria bassiana DNA 3.2.2.17 deoxyribodipyrimidine endonucleosidase analysis the comet assay, modified for plant protoplast preparations and T4 endonuclease V treatment, can detect UV-B-induced DNA damage in Spirodela polyrhiza sensitively and specifically 3.2.2.22 rRNA N-glycosylase analysis detection method based on N-glycosidase activity of ricin. Ricin is captured by a monoclonal antibody directed against the B chain and immobilized. Detection is realized via the release of adenine by the ricin A chain. Limit of detection is around 0.1 ng/ml after concentration of the toxin 3.2.2.22 rRNA N-glycosylase analysis detection method for ricin based on its inhibitory effects on protein synthesis. Development of an array of protein expression units to accomodate controls and multiple samples. Detection limit is around 0.01 nM ricin 3.2.2.22 rRNA N-glycosylase analysis electrochemiluminescence-based detection of RNA N-glycosidase activities with a limit of detection of 0.1 ng/ml of ricin 3.2.2.22 rRNA N-glycosylase analysis evaluation of rapid measurement platform using fluorogenic hand-held immunoassays for ricin A chain. Detection limit is 14 ng/ml or 140 pg/test. The assays are inclusive for ricin A60, ricin A120, ricin A chain, and ricin B chain and exclusive in discrimination of ricin A60 from other toxins 3.2.2.22 rRNA N-glycosylase analysis highly selective and sensitive aptamer-based abrin assay using a molecular light switching reagent [Ru(phen)2(dppz)]2+ with a limit of detection of 1 nM and a wide linear range from 1 to 400 nM with the correlation coefficient of 0.993. Assay can be performed in physiological buffers as well as diluted serum 3.2.2.22 rRNA N-glycosylase analysis identification of single domain antibodies bound to ricin immobilized on the surface of microspheres. Use as effective capture molecules in sandwich immunoassays 3.2.2.22 rRNA N-glycosylase analysis isolation of aptamers that specifically recognize ricin by affinity chromatography and by capillary electrophoresis/systematic evolution of ligands by exponential enrichment, i.e. CE-SELEX. Identification of three aptamers with Kd values in the nanomolar range that do not recognize abrin toxin 3.2.2.22 rRNA N-glycosylase analysis rapid and selective detection of ricin using fluorescently tagged RNA aptamers. Dissociation constant is 134 nM for RNA aptamer and ricin A chain, detection limit for ricin is around 1 nM 3.2.2.22 rRNA N-glycosylase analysis sensitive immuno-polymerase chain reaction assay for detection of ribosome-inactivating proteins, limit of detection is about 10 fg/ml of dianthin or ricin, and the test can be applied to human serum 3.2.2.22 rRNA N-glycosylase analysis validation of a cell-free translation assay for determining ricin biological activity. Assay is specific for determining ricin in food-based matrixes and discriminates ricin from other ribosome-inactivating proteins 3.2.2.22 rRNA N-glycosylase analysis facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy 3.2.2.22 rRNA N-glycosylase analysis simple and cost effective N-glycosidase assay based on the direct determination of the released adenine by thin-layer chromatography and TLC-densitometry. An adenine based single stranded oligonucleotide is used as substrate. The released adenine is quantified on silica glass plates by UV absorbance at 260 nm 3.2.2.23 DNA-formamidopyrimidine glycosylase analysis comparison of human Ogg1, Escherichia coli Fpg and endonuclease III for the ability to modify the sensitivity of the comet assay. All three endonucleases recognize oxidative DNA damage and, in addition, Fpg and endonuclease III also recognize alkylation damage. Use of human Ogg1 in the modified comet assay offers a useful alternative to Fpg and is more specific for 8-oxoguanine and methyl-fapy-guanine 3.2.2.23 DNA-formamidopyrimidine glycosylase analysis comparison of human Ogg1, Escherichia coli Fpg and endonuclease III for the ability to modify the sensitivity of the comet assay. Use of human Ogg1 in the modified comet assay offers a useful alternative to Fpg and is more specific for 8-oxoguanine and methyl-fapy-guanine 3.2.2.23 DNA-formamidopyrimidine glycosylase analysis DNA repair kinetics can be investigated with the comet assay and differences between cell types can be observed 3.2.2.23 DNA-formamidopyrimidine glycosylase analysis simple and sensitive FPG activity assay using target-induced self-primed rolling circle amplification and magnetic nanoprobes. A 8-oxoguanine is positioned in duplex DNA containing a P-circle and P1, which together serve as a FPG substrate, rolling circle amplification template, and rolling circle amplification primer probe. The presence of FPG specifically binds and cleaves 8-oxoG containing duplex DNA, resulting in 5'-phosphoryl termini. The rolling circle amplification reaction produces an amount of long tandem-repeat DNA tiles with multiple recognizing regions for the modified DNA probes and biotin-modified DNA probes: A detection limit of 1.033 U/ml for FPG is obtained. FPG assays in human blood serum are also obtained using fluorescence and confocal laser scanning microscopy 3.2.2.27 uracil-DNA glycosylase analysis fluorescence method for sensitive detection of uracil-DNA glycosylase using TdT-activated Endonuclease IV-assisted hyperbranched signal amplification. The method exhibits high sensitivity with a limit of detection of 0.090 U/ml for pure uracil-DNA glycosylase and shows a dynamic range from 0.1 to 50 U/ml, and can be applied for accurate detection of uracil-DNA glycosylase in HeLa nuclear extract. The method can be used for discrimination of uracil-DNA glycosylase from other DNA glycosylases 3.3.2.2 lysoplasmalogenase analysis use in an assay system for phospholipase A2 3.3.2.9 microsomal epoxide hydrolase analysis development of a fluorescence-based assay using substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate and application as a useful tool for the discovery of structure-activity relationships among mEH inhibitors and for the screening chemical library with high accuracy and with a Z' value of approximately 0.7 3.3.2.10 soluble epoxide hydrolase analysis development of a high-throughput screen assay for soluble epoxide hydrolase inhibition using (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester as a substrate. Assay is accurate and precise 3.4.11.2 membrane alanyl aminopeptidase analysis detection of Pseudomonas aeruginosa based on chromogenic aminopeptidase N substrate 7-N-(beta-alanyl)amino-1-pentylphenoxazin-3-one with low degree of diffusion in nutrient media and a bright red colour of the product 3.4.11.2 membrane alanyl aminopeptidase analysis synthesis of chromogenic selective beta-alanyl aminopeptidase substrates, producing colored bacterial colonies with Pseudomonas aeruginosa and Serratia marcescens, which is enhanced after 48 h of incubation. The compounds display sensitivity to a wider range of Pseudomonas aeruginosa strains tested 3.4.11.5 prolyl aminopeptidase analysis biochemical tests for Neisseria gonorrhoeae often rely on the activity of prolyliminopeptidase. The overall prevalence of enzyme-negative isolates is about 4.3% with significant geographical variation and higher frequency of these isolates among men who have sex with men 3.4.11.5 prolyl aminopeptidase analysis chromogenic substrate tests for Neisseria gonorrhoeae based on the activity of prolyliminopeptidase should be combined with other tests. A study of a prolyliminopeptidase-negative subtype among Neisseria gonorrhoeae isolates in Australia reveals that up to 22% of isolates may be negative for enzyme activity 3.4.11.5 prolyl aminopeptidase analysis widespread dissemination, mainly among men who have sex with men, of indistinguishable and highly related genotypes that have evolved from a single N gonorrhoeae PIP-negative serovar IB-4 strain among several countries worldwide. An increased awareness of PIP-negative N gonorrhoeae strains is crucial 3.4.11.10 bacterial leucyl aminopeptidase analysis evaluation of a single-stage MS-based technique for amino acid sequencing involving partial, heterogenous digestion of a peptide by a processive, non-specific, thermotropic Bacillus subtilis-derived aminopeptidase (BsuAP), which allows single-shot sequencing to be carried out through simultaneous accumulation, and detection of subpopulations of peptides of progressively reducing length 3.4.11.13 Clostridial aminopeptidase analysis immobilized enzyme can be used for peptide sequence determination, together with other peptidases, reactive in solutions containing up to 10% DMSO 3.4.11.22 aminopeptidase I analysis two-photon fluorescent probe SNCL exhibits high sensitivity toward ERAP1, with about a 95-fold fluorescence enhancement at 550 nm. SNCL can be applied for monitoring ERAP1 in tumor tissue with an imaging depth of 50-120 microm 3.4.11.24 aminopeptidase S analysis the enzyme is clinically important as a model for undestanding the structure and mechanism of action of other metallopeptidases 3.4.11.26 intermediate cleaving peptidase 55 analysis identification of 36 substrates utilizing charge-based fractional diagonal chromatography, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two Saccharomyces cerevisiae samples within 10 h of LC-MS, starting from only 50 microg of protein per condition and analyzing only 40% of the obtained fractions 3.4.13.9 Xaa-Pro dipeptidase analysis applications using prolidase to detoxify organophosphorous compounds nerve agents include its incorporation into fire-fighting foams and as biosensors for organophosphorous compound detection 3.4.13.17 non-stereospecific dipeptidase analysis improved method for hydrolyzing proteins and peptides without inducing racemization and for determining their true D-amino acid content 3.4.13.20 beta-Ala-His dipeptidase analysis a validated method for measuring serum carnosinase activity in serum and heparin plasma 3.4.13.22 D-Ala-D-Ala dipeptidase analysis although catalytically ineffecient, the VanX substrate D-leucine-p-nitroanilide is an alternative substrate for VanX because it can be monitored directly and assayed spectrophotometrically which facilitates the routine analysis of enzyme catalysis and the screening discovery of potential VanX inhibitors. In addition, it is with leucine in its D form that possible activities from other contaminated species (other than VanX) in Escherichia coli JM109 are greatly reduced. Moreover, D-leucine-p-nitroanilide needs essentially no sophisticated synthetic chemistry for preparation 3.4.13.22 D-Ala-D-Ala dipeptidase analysis continuous assay of VanX in vitro and in vivo from hydrolysis of D-Ala-D-Ala, based on the heat-rate changes measured with isothermal titration calorimetry 3.4.14.1 dipeptidyl-peptidase I analysis use in peptide sequence studies 3.4.14.1 dipeptidyl-peptidase I analysis application of substrate beta-(2-thienyl)Ala-beta-(7-methoxycoumarin-4-yl)Ala-Ser-Gly-Tyr(3-NO2) for detection of cathepsin C activity in various cell lysates, urine and bronchoalveolar lavage fluid samples 3.4.14.2 dipeptidyl-peptidase II analysis the enzyme is a potential urinary marker for the biochemical detection of kidney damage 3.4.14.5 dipeptidyl-peptidase IV analysis the enzyme can be used for peptide sequence analysis 3.4.14.5 dipeptidyl-peptidase IV analysis surface marker for a subpopulation of human T-lymphocytes 3.4.14.5 dipeptidyl-peptidase IV analysis the enzyme is a marker for T lymphocytes 3.4.14.5 dipeptidyl-peptidase IV analysis significant levels of enzyme activity are present in commercial human serum albumin. This activity is abolished using a specific DPP-IV inhibitor. Fully 70 to 80% DPP-IV activity remains at 60°C compared with the 37°C incubate. No DPP-IV activity is present in recombinant human serum albumin, suggesting that DPP-IV activity is present only in serum albumin produced using the Cohn fractionation process. DPP-IV activity contributes to the formation of aspartate-alanine diketopiperazine, a known immunomodulatory molecule from the N terminus of human albumin, could account for some of the clinical effects of commercial human serum albumin 3.4.14.5 dipeptidyl-peptidase IV analysis method for specific recognition and imaging for DPP IV in living cells by by coupling the inhibitor alogliptin to a carboxyl-modified fluorophore. The probe shows photostability and good biocompatibility. Alogliptin interacts with residues Glu206, Arg125 and Tyr631 3.4.14.9 tripeptidyl-peptidase I analysis development of fluorescence resonance energy transfer peptides using tryptophan as the fluorophore to study TPP-I hydrolytic properties. Assay can be applied to spleen and kidney homogenate 3.4.14.12 Xaa-Xaa-Pro tripeptidyl-peptidase analysis continuous assay for endoprotease activity of meprin alpha and beta using prolyl tripeptidyl aminopeptidase and the chromogenic substrate KKGYVADAP-p-nitroanilide 3.4.16.2 lysosomal Pro-Xaa carboxypeptidase analysis enzymatic sequencing by MALDI-MS combined with carboxypeptidase cleavage 3.4.16.4 serine-type D-Ala-D-Ala carboxypeptidase analysis Forster resonance energy transfer assay that uses the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry to detect periplasmic protein interactions in fixed and in living bacteria, in single samples or in plate reader 96-well format 3.4.16.5 carboxypeptidase C analysis application of CPY as sensing element in a biosensor for the direct detection of ochratoxin A and comparison with thermolysin. Thermolysin and CPY both exhibit an optimal activity under the conditions 35 min cross-linking time, working pH of 7 and temperature of 25°C and exhibit comparable analytical performances in the biosensor. Ochratoxin A concentrations can be determined without pretreatment of the sample and no matrix effect is observed 3.4.17.1 carboxypeptidase A analysis the enzyme can serve as specific molecular maker for mast cells amongst normal hematopoietic cell populations 3.4.17.1 carboxypeptidase A analysis the enzyme serves as model enzyme for design of specific inhibiors for zinc proteases 3.4.17.2 carboxypeptidase B analysis the immobilized carboxypeptidase B is used for stepwise digestion of cytochrome c 3.4.17.2 carboxypeptidase B analysis carboxypeptidase B treatment of recombinant monoclonal antibodies indicates that C-terminal lysine variants are present mainly among the basic species but also to some extent among the acidic species 3.4.17.21 Glutamate carboxypeptidase II analysis synthesis of a series of PSMA-targeted 99mTc-chelate complexes, with core ligand 2-[3-(3-benzyloxycarbonyl-1-tert-butoxycarbonyl-propyl)-ureido]pentanedioic acid di-tert-butyl ester, for imaging PSMA-expressing human prostate cancer cells, in silico docking studies using the crystal structure of PSMA, overview. The method offers a potential for use in localizing prostate cancer masses, monitoring response to therapy, detecting prostate cancer recurrence following surgery, and selecting patients for subsequent PSMAtargeted chemotherapy 3.4.17.23 angiotensin-converting enzyme 2 analysis mass spectrometric assay for angiotensin-converting enzyme 2 using angiotensin II as substrate will have applications in drug screening, antagonist development, and clinical investigations 3.4.17.23 angiotensin-converting enzyme 2 analysis easy-to-use method for determining ACE2 activity in brain tissue and cerebrospinal fluid, based on a quenched fluorescent substrate and presence and absence of inhibitor DX600. The method can be adapted for other tissues, plasma, cell extracts, and cell culture supernatants 3.4.17.23 angiotensin-converting enzyme 2 analysis method for measurement of ACE2 activity in biological fluids, using hydrolysis of an intramolecularly quenched fluorogenic ACE2 substrate, in the absence or presence of the ACE2 inhibitors MLN-4760 or DX600. ACE2 detection ranges from 1.56 to 50 ng/ml. MLN-4760 potently inhibits the activity of both human and mouse ACE2, DX600 (linear form) only effectively blocks human ACE2 activity in this assay. In biological samples of human and mouse urine, cell culture medium from mouse proximal tubular cells, and mouse plasma, the mean intra- and interassay coefficients of variation of the assay range from 1.43 to 4.39 %, and from 7.01 to 13.17 %, respectively 3.4.17.23 angiotensin-converting enzyme 2 analysis method for measurement of ACE2 activity in biological fluids, using hydrolysis of an intramolecularly quenched fluorogenic ACE2 substrate, in the absence or presence of the ACE2 inhibitors MLN-4760 or DX600. ACE2 detection ranges from 1.56 to 50 ng/ml. MLN-4760 potently inhibits the activity of both human and mouse ACE2, DX600 (linear form) only effectively blocks human ACE2 activity in this assay. In biological samples of human and mouse urine, cell culture medium from mouse proximal tubular cells, and mouse plasma, the mean intra- and interassay coefficients of variation of the assay range from 1.43 to 4.39%, and from 7.01 to 13.17%, respectively 3.4.17.23 angiotensin-converting enzyme 2 analysis ACE2-based biosensor designed to detect both SARS-CoV-2 S1 mutations and neutralizing antibodies. In binding mode, the biosensor works by detecting binding of the spike protein to an immobilized ACE2 receptor and is able to detect S1 proteins of the alpha (500 pg/ml) and beta variants (10 ng/ml), as well as wild-type S1 (10 ng/ml), of SARSCoV-2 and it distinguishes wild-type SARS-CoV-2 S1 from the S1 alpha and beta variants via color differences. A modification to the protocol enables the ACE2-based biosensor to operate in blocking mode to detect neutralizing antibodies in serum samples from COVID-19 patients 3.4.17.23 angiotensin-converting enzyme 2 analysis analysis of the interaction between ACE2 and SARS-CoV-2 spike protein using a surface plasmon resonance-based assay that reduces the heterogeneity introduced from multivalent binding interactions to enable the determination of the kinetic rate constants for multivalent binding interactions. Controlling the sensor surface heterogeneity enables the deconvolution of the avidity-induced affinity enhancement for the SARS-CoV-2 spike protein and ACE2 interaction 3.4.17.23 angiotensin-converting enzyme 2 analysis application of CEBIT, i.e. Condensate-aided Enrichment of Biomolecular Interactions in Test tubes for high-throughput screening of drugs to inhibit the interaction between the receptor-binding domain of SARS-CoV-2 spike protein and its obligate receptor ACE2 3.4.19.1 acylaminoacyl-peptidase analysis the enzyme is a tool in sequencing blocked peptides and proteins. Useful for unblocking acetylated proteins prior to protein sequence analysis 3.4.19.1 acylaminoacyl-peptidase analysis enzyme is useful for deblocking of peptides 3.4.19.1 acylaminoacyl-peptidase analysis the enzyme may be useful for the removal of the N-terminal acylamino acid from some N-terminal blocked peptides and proteins in amino acid sequence analysis 3.4.19.1 acylaminoacyl-peptidase analysis the enzyme is expected to be useful for the removal of Nalpha-acylated residues in short peptide sequence analysis at high temperatures 3.4.19.3 pyroglutamyl-peptidase I analysis application to N-terminal pyroglutamyl unblocking prior to Edman sequential degradation in peptide and protein sequencing 3.4.19.3 pyroglutamyl-peptidase I analysis pyroglutamyl aminopeptidase is an auxiliary enzyme in a spectrometric assay for glutaminyl cyclase activity. It is a fast, continuous, and reliable determination of glutaminyl cyclase activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis 3.4.19.3 pyroglutamyl-peptidase I analysis the enzyme may be a useful single-test adjunct for distinguishing Staphylococcus aureus from Staphylococcus delphini and other members of the Staphylococcus intermedius group 3.4.19.9 folate gamma-glutamyl hydrolase analysis trienzyme extraction is a combined enzymatic digestion by Pronase, alpha-amylase, and conjugase, i.e. gamma-glutamyl hydrolase, to liberate the carbohydrate and protein-bound folates from food matrices prior to total folate analysis by microbiological assay using Lactobacillus casei ssp. rhamnosus, ATCC 7469, determine the contribution of the enzymes to the growth response, the extraction method is used in AOAC Official Method 2004.05 for the folate analysis of cereal foods 3.4.19.9 folate gamma-glutamyl hydrolase analysis folate analysis. Isoform GGH2 is able to completely deconjugate folates using 50-100 microg/g of sample for 1 h at 37°C in all matrices tested. It removes the tails of folylpolyglutamates from tomato fruit, black bean and peanut seeds, and alfalfa sprouts extracts 3.4.19.12 ubiquitinyl hydrolase 1 analysis the proportion of inclusion-bearing cells showing high levels of ubiquitin C-terminal hydrolase may provide a marker of the activity of a degenerative process 3.4.19.12 ubiquitinyl hydrolase 1 analysis UCH-L1 is a neuronal marker 3.4.21.B1 hyaluronan-binding serine protease analysis substrate Ala-Lys-Nle-Arg-7-amido-4-methylcoumarin can be used to measure FSAP activity in plasma 3.4.21.B3 duodenase analysis comparative evaluation of specificity of different proteases can give rise to methods employing new proteases in analytical protein chemistry 3.4.21.4 trypsin analysis application of biotinylated trypsin as a sensitive versatile probe for the detection and characterization of an ovine chondrocyte serine proteinase inhibitor using Western blotting 3.4.21.4 trypsin analysis experimental system for the structural and physico-chemical analysis of factor Xa inhibitors in trypsin variants with factor Xa phenotype: E97N/Y99L, S190A, E97N/Y99L/S190A 3.4.21.4 trypsin analysis the double mutant trypsinogen D189S/DELTA223 lacks trypsin-like activity but aquires a rather unique selectivity, it preferentially hydrolyses peptide bonds C-terminal to tyrosyl residues. This narrow specificity should be useful in peptide-analytical applications such as sequence-specific fragmentation of large proteins prior to sequencing 3.4.21.4 trypsin analysis detection of enzyme hydrolytic activity by use of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate coupled to a chymotryptic petide obtained from hemoglobin on silica gel plate. Detection of 45 fmol of enzyme at 25°C within 1 min 3.4.21.4 trypsin analysis comparison of five crystals of bovine trypsin obtained under analogous conditions. The Calpha and backbone atoms of the structures superpose very well. The occupancy of ligands in regions of low thermal motion is reproducible, whereas solvent molecules containing heavier atoms (such as sulfur) or those located on the surface can differ significantly. The coordination lengths of the calcium ion are conserved. A large proportion of the multiple conformations refine to similar occupancies and the residues adopt similar orientations. The protonation states of histidine residues and carboxylate moieties is consistent for all of the models. However, several features of residues or ligands located in flexible parts of the macromolecule may vary significantly, such as side-chain orientations and the occupancies of certain fragments 3.4.21.5 thrombin analysis detection of enzyme complexed with polyclonal anti-prothrombin antiserum. Enzyme retains enzymatic activity, and binding to antibody may enhance the efficiency of proenzyme activation 3.4.21.5 thrombin analysis affinity probe capillary electrophoresis/laser-induced fluorescence polarization assay for detection of human thrombin using a specific aptamer as probe. Monovalent and bivalent cations promoting the formation of a stable G quadruplex conformation in the aptamer may enhance the binding of the aptamer and thrombin, while cations like K+ and Mg2+ cannot stabilize the affinity complex. Without the use of typical cations, a highly sensitive assay of human thrombin was developed with the corresponding detection limits of 4.38×10?19 and 2.94×10?19 mol in mass for standard solution and human serum, respectively 3.4.21.5 thrombin analysis conjugation of angiomax to a 5'-amino oligonucleotide and assembly into a two-dimensional DNA lattice for observation of the binding of thrombins to the DNA lattice. Use of the functionalized DNA lattices as a platform for investigation of biomolecular interactions such as drug-protein, protein-protein, DNA-RNA, and DNA-protein interactions in the nano- and subnanoscales 3.4.21.5 thrombin analysis development of a biosensor for thrombin detection using surface-enhanced Raman spectroscopy. The method utilizes the electrostatic interaction between capture thrombin aptamer and probe crystal violet molecules. Procedure shows a highly specific selectivity and a linear detection of thrombin in the range from 0.1 nM to 10 nM with a detection limit of about 20 pM and realizes the thrombin detection in human blood serum solution directly 3.4.21.5 thrombin analysis development of a direct and an indirect competitive assay for thrombin by an electrochemical aptamer-based assay coupled to magnetic beads. With the direct competitive assay, when the aptamer is immobilised onto the magnetic beads, a detection limit of 430 nM for thrombin is achieved. A detection limit of 175 nM is obtained by detecting the product of the enzymatic reaction catalysed by thrombin. A sandwich assay reaches a detection limit of 0.45 nM of thrombin 3.4.21.5 thrombin analysis development of a modified electrochemical sandwich model for target protein detection using differential pulse voltammetry. With model target analyte thrombin, the sensor shows a linear response for thrombin in the range 1-60 nM with a detection limit of 0.5 nM 3.4.21.5 thrombin analysis development of an aptamer-based surface enhanced resonance Raman scattering sensor with high sensitivity, specificity, and stability for the detection of human alpha-thrombin. The sensor displays a limit of detection of 100 pM by monitoring the signal change upon the single-step of thrombin binding to immobilized thrombin binding aptamer and specifically discriminates thrombin from other proteins. The sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, and is sufficiently robust for clinical diagnostic applications 3.4.21.5 thrombin analysis study on the effects of dilution on the thrombin generation process in assessing the clotting function. Anticoagulant pathways are far more affected by dilution than the procoagulant pathways. Plasma dilution causes a loss of sensitivity towards thrombomodulin and activated protein C. At dilutions above 1:12 a second wave of prothrombinase-activity is observed 3.4.21.5 thrombin analysis design of an aptamer-based suspension array detection platform for the sensitive, specific and rapid detection of human alpha-thrombin as a model. Thrombin is first recognized by a 29-mer biotinylated thrombin-binding aptamer in solution. Then 15-mer TBA modified magnetic beads capture the former aptamer-thrombin to form an aptamer-thrombin-aptamer sandwich complex. The median fluorescence intensity obtained via suspension array technology is positively correlated with the thrombin concentration. The dynamic quantitative working range of the aptamer-based suspension array is 18.37-554.31 nM, and the coefficients of determination R2 are greater than 0.9975. The lowest detection limit of thrombin is 5.4 nM. The method is highly specific for thrombin without being affected by other analogs and interfering proteins. The recoveries of thrombin spiked in diluted human serum are in the range 82.6-114.2% 3.4.21.6 coagulation factor Xa analysis development of an assay for the monitoring of anticoagulants inhibiting factor Xa and/or factor IIa. Assay is based on the addition of factor Xa and snake venom RVV-V, i.e. Russell viper venom factor V activator specifically activating factor V and phospholipids to platelet-poor plasma. Assay shows an almost linear dose-response and high sensitivity for unfractionated heparin, low molecular weight heparins, r-hirudin, and argatroban 3.4.21.7 plasmin analysis electrochemical assay of plasmin activity based on a ferrocenyl peptide substrate having a plasmin-specific substrate sequence, Lys-thr-Phe-Lys, and immobilized on a gold electrode. Detection limit for plasmin is around 50 ng/ml or 0.15 mU/ml. Ratio of kcat/Km values is 0.063 microM/s 3.4.21.9 enteropeptidase analysis cellular libraries of peptide substrates, CLiPS, are used to study substrate specificities, fluorescent reporter substrates on the surface of Escherichia coli as N-terminal conjugates are used as whole-cell protease activity assays 3.4.21.9 enteropeptidase analysis enteropeptidase activity is influenced by accessibility of the target site and by downstream sequences 3.4.21.10 acrosin analysis the enzyme activity can be used as a reliable predictor of boar sperm freezability 3.4.21.B12 prostase analysis enzyme-specific sandwich-type immunoassay using a monoclonal antibody, detection limit is 0.02 microgram per liter, and less than 0.1% cross-reactivity with other human kallikreins 3.4.21.19 glutamyl endopeptidase analysis study of structure-function organization of anti-HIV and antitumor proteins MAP30 and GAP31 by limited proteolysis with V8-GSE, enzym may be useful for studying other proteins as well 3.4.21.19 glutamyl endopeptidase analysis increase in selectivity of immobilized metal affinity chromatography for phosphopeptides by use of enzyme for protein digestion. Enzyme cleaves proteins at acidic residues and reduces the number of acidic residues in peptides. Application of method to a selction of model proteins and to bovine milk 3.4.21.25 cucumisin analysis subcellular localization of protease C1 and pH conditions in protein storage vacuoles in parenchyma cells of seedling cotyledons and maturing seeds examined 3.4.21.36 pancreatic elastase analysis elastase digests yield a large proportion of transmembrane peptides facilitating membrane protein identification 3.4.21.50 lysyl endopeptidase analysis the enzyme is used for rapid lysis of gram-positive cocci for pulsed-field gel electrophoresis 3.4.21.50 lysyl endopeptidase analysis the enzyme is useful in the determination of primary structure of proteins 3.4.21.50 lysyl endopeptidase analysis middle-down approach, where the antibody is subjected to limited digestion using the endoproteinase Lys-C, is potentially useful for the accurate, sensitive and routine characterization of recombinant antibodies 3.4.21.50 lysyl endopeptidase analysis development of an achromopeptidase lysis procedure for high-volume testing and preparation of samples for PCR detection of methicillin-resistant Staphylococcus aureus. The BD GeneOhm MRSA kit method shows a sensitivity of 92.0%, specificity of 94.6%, positive predictive value of 75.4%, and negative predictive value of 98.5%. The assay is an accurate and rapid test for detection of MRSA colonization 3.4.21.50 lysyl endopeptidase analysis method for identification of both the amino and the carboxyl termini of proteins. The method independently uses two proteases, Lys-C and peptidyl-Lys metalloendopeptidase Lys-N, to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified as the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest, the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest, and all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different. Acetylation on N-terminus and protein isoforms, which have different termini, is also determined 3.4.21.50 lysyl endopeptidase analysis the enzyme is attractive for the proteolytic generation of peptides to be analyzed by mass spectrometry, usage in quantitative mass spectrometry, overview. It can be used in the presence of protein denaturants which allow better access to cleavage sites and hence better proteolysis. The enzyme has advantages over trypsin, namely, reduced missed cleavage (because KR and RK are cleaved specifically), tolerance to denaturants and requirement for only a single labelled amino acid in experiments using isotopic labelling 3.4.21.50 lysyl endopeptidase analysis endoproteinase lysine-C (Lys-C)/trypsin sequential digestion can be used in human 293T cell proteomics sample preparation, evaluation of the advantages of Lys-C/trypsin sequential digestion over trypsin digestion in solution conditions in different aspects, method optimzation, overview 3.4.21.62 Subtilisin analysis model for understanding the enormous rate enhancements affected by enzymes 3.4.21.62 Subtilisin analysis popular system for protein engineering studies 3.4.21.62 Subtilisin analysis analytical on-line liquid chromatography/bioassay with which both chemical and biological information on inhibitory effect of proteins in mixtures can be obtained simultaneously. Assay consists of protein separation using gel filtrationand subsequent homogeneous assay to distinct between active enzyme inhibitors and non-active compounds 3.4.21.62 Subtilisin analysis due to high pI-value of enzyme, it does not migrate in the electrophoretic field in the Laemmli buffer system. Fast and simple identification of enzyme by over-running electrophoretic technique with a miniscale culture supernatant 3.4.21.64 peptidase K analysis protein engineering approach for increasing activity and heat stability of proteinase K. Protein design algorithms that only require the testing of a small number of variants represent a significant step towards a generic, resource-optimized protein engineering process 3.4.21.64 peptidase K analysis nonspecific protease proteinase K can be used as an alternative to trypsin for cross-linking studies, digestion by proteinase K results in a family of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results. Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links are found for all of the possible cross-linking sites of native and oligomeric forms of prion protein substrates, overview. After digestion with proteinase K, the mass distribution of the crosslinked peptides is very suitable for MALDI-MS analysis 3.4.21.64 peptidase K analysis improvement of RNA extraction from papillary cancer-derived K1 cells and thyroid fine-needle aspiration biopsy (FNAB) specimens suspended in liquid-based cytology (LBC) solutions during fine-needle aspiration biopsy diagnostics of thyroid diseases. Commercial proteinase K treatment is essential for efficient RNA extraction from the fixed cells. Proteinase K treatment facilitates RNA recovery from rigid cells after dehydration. RNA molecules are largely released from the fixed cells even after 1-h treatment. U6 small nuclear RNA was detected in these RNA samples by reverse transcription-PCR. Method overview 3.4.21.66 Thermitase analysis use of enzyme for generation of peptides for mass spectrometric characterization 3.4.21.72 IgA-specific serine endopeptidase analysis the ability of IgA1 protease to cleave exclusively IgA1 without affecting IgA2 molecules allows a more economical and reliable method for determination of these antibody subclasses in human sera based on recombinant Neisseria meningitidis IgA1 protease. Estimation of IgA1 and IgA2 content in human serum and of IgA subclass levels and IgA1/IgA2 ratio using recombinant active and inactive forms of IgA1 protease from Neisseria meningitidis, method evaluation, overview 3.4.21.73 u-Plasminogen activator analysis assay of cellular internalization and localization of enzyme:PAI-2 inhibitor complex based on the use of inhibitor labelled with Alexa488 fluorochrome and a polyclonal antibody 3.4.21.73 u-Plasminogen activator analysis optical zymography technique that specifically detects enzyme activity in biological samples via fluorescence emission at 695 nm. Method can efficiently distinguish the active two-chain enzyme from its proenzyme and directly measure enzyme activities in different cancer cell lines 3.4.21.73 u-Plasminogen activator analysis quantification of uPA and inhibitor PAI-1 mRNA expression in breast cancer cell lines as well as in tumor tissue of breast cancer patients by sensitive quantitative real-time PCR assays, based on the LightCycler technology. In breast cancer cell lines, mRNA and antigen values are highly correlated for both uPA and PAI-1 I. Correlations between uPA/PAI-1 mRNA and protein in the breast cancer samples were found to be distinctly weaker or not significant. Quantitative determination of mRNA expression for both factors does not mirror antigen levels in breast cancer tissue 3.4.21.74 venombin A analysis the enzyme may be useful for developing potential diagnostic agents for detecting coagulation disorders 3.4.21.75 Furin analysis bioluminogenic probes can specifically image furin activity in xenografted breast cancer tumors in mice 3.4.21.75 Furin analysis sensitive and specific assay for furin activity using an antibody capture step to immobilise furin from whole cell lysates. The assay has a minimum detection limit of 0.006 nM and is sensitive enough to determine the furin activity of many of the cell lines tested 3.4.21.76 Myeloblastin analysis knowledge of the protonation states of the ionizable residues in an enzyme like PR3 is a prerequisite to an accurate description of its structure and mechanism 3.4.21.77 semenogelase analysis development and employment of counter-SELEX (systematic evolution of ligands by exponential enrichment) procedures to identify specific RNA aptamers against the purified active PSA, wich is not only a specific marker but also a target molecule for diagnosis and therapy of prostate cancer. The aptamers have a specific binding activity against the active PSA, but not for GST or proPSA 3.4.21.77 semenogelase analysis development of a simultaneous electrochemical biosensor, using the goldmodified screen-printed carbon dual sensor, for free and total PSA for monitoring PSA production from three different cultures of human androgen-sensitive prostate tumor cells 3.4.21.77 semenogelase analysis development of an electrical immunosensor for the detection of PSA using a microgapped electrode array based on enzymatic silver deposition. This electrical immunosensor exhibits a linear response with PSA concentrations over a 6-decade range from 1.0 pg/l to 1.0 microg/l, with detection limit of 0.9 pg/l. PSA concentrations using this immunosensor agree within 10% of those obtained using a commercial chemiluminescent immunoassay 3.4.21.77 semenogelase analysis mass spectrometry annotation can identify more molecular forms of PSA compared with Western and zymographic analyses. Observation of various isoforms of PSA in patients may contribute to the further identification of disease-relevant heterogeneity of PSA, including transcriptional and post-translational modifications present due to various stages and causes of prostate disease 3.4.21.77 semenogelase analysis development of PSA and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization LAMI, aiming at the detection and quantification of PSA, as a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulfide bridges, conserved in both PSA and Fab molecules. The thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. LAMI technology is successful in immobilizing biomedically relevant molecules while preserving their activity 3.4.21.77 semenogelase analysis real-time detection of prostate-specific antigen PSA in diluted human serum without labeling by use of an amplitude-sensitive paired surface plasma wave biosensor PSPWB. The detection limit of PSPWB is 8.4 × 10-9 refractive index units, and the PSPWB can measure PSA in a phosphate buffered saline solution from 10 fg/ml to 100 pg/ml, i.e. about 3 pM, successfully, with a linear relationship between PSA concentrations and surface plasmon resonance signals. The PSPWB successfully detects PSA in diluted human serum as well 3.4.21.79 granzyme B analysis caution in the design and interpretation of experiments using GrBs from different species due to distinct tetrapeptide specificities and abilities to recruit the BID pathway 3.4.21.84 limulus clotting factor C analysis recombinant factor C assay for measuring endotoxin in house dust: comparison with LAL, and (1->3)-beta-D-glucans. In the Limulus amebocyte lysate assay endotoxin is detected through a reaction cascade, initiated by the binding of endotoxins to Factor C. The advantage of an assay using the genetically engineered factor Cbar in measuring endotoxins is that recombinant factor Cbar does not contain factor G that can produce interference from (1->3)-beta-D-glucans 3.4.21.86 Limulus clotting enzyme analysis screen-printed endotoxin sensor based on amperometric detection of protease activity. The amperometric measurement with a screen-printed electrode provides compact, low-cost, and easy-to-use sensors for on-site monitoring of endotoxin 3.4.21.89 Signal peptidase I analysis development of a fluorescence resonance energy transfer-based assay method as a rapid and reliable tool in future research for the identification and validation of potential SPase I inhibitors 3.4.21.91 Flavivirin analysis NS2B-NS3pro K48A mutant construct is well suited for follow-up purification and structural and drug design studies 3.4.21.91 Flavivirin analysis two sets of prediction approaches. Secondary structure prediction performed using available structure prediction servers. A second approach makes use of the information on the secondary structures extracted from structure prediction servers, threading techniques and DSSP database of some of the templates used in the threading techniques. Consensus on the one-dimensional secondary structure of Dengue virus type 2 protease obtained from each approach and evaluated against data from the recently crystallised Dengue virus type 2 NS2B/NS3 obtained from the Protein Data Bank. The second approach shows higher accuracy compared to the use of prediction servers only and thus is applicable to the initial stage of structural studies of proteins with low amino acid sequence homology against other available proteins in the Protein Data Bank 3.4.21.92 Endopeptidase Clp analysis development of a ClpXP protein degradation systemusing purified ClpXP in a cell-free transcription-translation system 3.4.21.93 Proprotein convertase 1 analysis highly specific and potent PC1 inhibitors proSAAS-(235-246) and proSAAS-(235-244) may be useful in development of an effective affinity procedure for the purification of PC1 3.4.21.97 assemblin analysis cell growth selection system in the yeast Saccharomyces cerevisiae that can detect and characterize HCMV protease activity and is applicable to screen for HCMV protease inhibitors in a high-throughput format 3.4.21.98 hepacivirin analysis generation of NS3/4A/Lap/LC-1 triple transgenic mouse liver-specifically and conditionally expressing reporter luciferase Fluc, Cre recombinase and reverse tetracycline-controlled transcriptional activator. NS3/4A protein is strictly and conditionally expressed in the liver of doxycycline-induced triple transgenic mice 3.4.21.105 rhomboid protease analysis screening assay using fluorescence polarization activity-based protein profiling 3.4.21.118 kallikrein 8 analysis binding assessment of ligands to kallikrein-8 using a residue-wise decomposition of the binding energy. Use of chemical energy-wise decomposition or CHEWD for residue-wise decomposition of binding energies 3.4.21.119 kallikrein 13 analysis substrate substrate ABZ-Val-Arg-Phe-Arg-Ser-Thr-Gln-Tyr(3-NO2)-NH2 is suitable for determination of KLK13 activity and is successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The modified subvsrtae is applicable in complex biological samples 3.4.21.122 transmembrane protease serine 2 analysis development of a cell-based fusion assay for the S protein in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase-based split reporter proteins. S-protein is stably expressed in the effector cells, and the corresponding receptor for S, CD26, is stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between the effector and target cells is quantitatively measured by determining the Renilla luciferase activity. The assay is optimized for a 384-well format 3.4.21.122 transmembrane protease serine 2 analysis use of TMPRSS2 to produce high titre influenza hemagglutixadnin lentiviral pseudotypes from Group 2 influenza viruses. These lentiviral pseudotypes transduce HEK293T/17 cells with high efficiency. The Group 2 influenza pseudotype particles can be used as surrogate antigens in neutralization assays and are efficiently neutralized by corresponding influenza virus reference sera 3.4.22.2 papain analysis use of the enzyme in sequence determination 3.4.22.2 papain analysis study on the cross-sectional distribution of methylene blue and papain in porous silicon layers by TOF-SIMS. The larger Papain molecule distributes itself in a similar manner to methylene blue demonstrating larger molecules can be effectively incorporated into such pore structures 3.4.22.2 papain analysis study on the mixed micellar behavior of anionic surfactant, sodium dodecylsulfate, and cationic surfactant, dodecylethyldimethylammonium bromide, in aqueous solution of papain. The effect of concentration of papain on mixed micellar behavior indicates that with increase in the concentration of protein, the critical aggregation concentration and critical micellizationconcentration values increase. The unfolding of the polypeptide chain in the presence of mixed surfactant has been observed 3.4.22.3 ficain analysis use for antibody screening of blood donor samples with the microplate system autoanalyzer 3.4.22.3 ficain analysis the enzyme should prove generally useful in peptide sequencing 3.4.22.56 caspase-3 analysis cell-permeable substrate N-acetyl-L-Asp-L-Glu-L-Val-L-Asp-N’-morpholinecarbonyl-rhodamine 110, high turnover rate and sensitivity both in enzyme solution and in living cells 3.4.22.56 caspase-3 analysis the highly cell-permeable caspase-3 substrate is obtained by linking a fluorogenic DNA-binding dye to the caspase-3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD-NucView488 detects caspase-3 activation within a live-cell population much earlier and with higher sensitivity compared with other apoptosis reagents. Cells incubated with DEVD-NucView488 exhibit no toxicity and normal apoptotic progression. DEVD-NucView488 is an ideal substrate for kinetic studies of caspase-3 activation because it detects caspase-3 activity in real-time and also efficiently labels DNA in nuclei of caspase-3-activated cells for real-time fluorescent visualization of apoptotic morphology 3.4.22.69 SARS coronavirus main proteinase analysis developing a novel red-shifted fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms 3.4.22.69 SARS coronavirus main proteinase analysis development of a dual-color probe for the simultaneous detection of Mpro and PLpro by FRET in vitro and in cells. The probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro 3.4.22.69 SARS coronavirus main proteinase analysis FRET-based method to assess the proteolytic activity of SARS-CoV-2 3CLpro using intramolecularly quenched fluorogenic peptide substrates corresponding to the cleavage sequence of SARS-CoV-2 3CLpro 3.4.22.69 SARS coronavirus main proteinase analysis quantitative structure-activity relationship model, molecular docking studies and molecular dynamics simulation to identify inhibitors, the model is applied to three large databases and reports top 25 compounds from each database 3.4.22.69 SARS coronavirus main proteinase analysis SARS-CoV-2-infected Vero E6 cell viability assay for detection of antiviral activity 3.4.22.70 sortase A analysis development of a reverse-phase HPLC assay to identify and characterize sortase reaction intermediates 3.4.22.70 sortase A analysis semisynthetic active site mutant enzymes containing selenocysteine and homocysteine might represent useful tools for further biochemical investigations and engineering approaches of sortases A 3.4.22.71 sortase B analysis method for sequence specificity determination of sortases or other bond-forming enzymes that recognize an amino acid sequence. Using mixtures of recognition peptides of limited complexity, which are reacted with biotinylated substrates, the biotinylated transpeptidation products are isolated with magnetic streptavidin beads and analyzed via liquid chromatography and mass spectrometry 3.4.22.B79 nsP2 protease analysis assay for quantitative measurement of Nsp2 protease activity based on a substrate fusion protein consisting of eGFP and Gaussia luciferase linked together by a small peptide containing a Nsp2 protease cleavage sequence. The expression of the substrate protein in cells along with recombinant Nsp2 protease results in cleavage of the substrate protein resulting in extracellular release of free Gaussia luciferase 3.4.22.B79 nsP2 protease analysis nsP2 protease-based cell free high throughput screening assay 3.4.22.B80 SARS-CoV papain-like protease analysis development of a dual-color probe for the simultaneous detection of Mpro and PLpro by FRET in vitro and in cells. The probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro 3.4.23.1 pepsin A analysis combined use of a theoretical model that relates electrophoretic behaviour of peptides to their sequence together with capillary electrophoresis-mass spectrometry to characterize the cleavage specificity of recombinant enzymes. Characterization of a protein lysate using recombinant and natural pepsin 3.4.23.B1 napsin analysis enzyme is a specific marker in diagnosis of primary lung adenocarcinoma and to distinguish it from metastatic adenocarcinoma 3.4.23.B1 napsin analysis identification of suitable fluorogenic protease substrates for the pharmaceutically important napsin A by an easy, solid-phase combinatorial assay technology 3.4.23.B5 murine leukemia virus protease analysis development of an in vitro enzymatic assay system to characterize XMRV protease-mediated cleavage of host-cell proteins 3.4.23.B10 Rous sarcoma virus retropepsin analysis coexpression of human prorenin receptor hPRR and RSV protease domain-deleted mutant in silkworm larvae results in display of human prorenin receptor on the surface of RSV virus-like particles in an active form. This transmembrane protein display system using RSV Gag in silkworm larvae is applicable to expression of intact transmembrane proteins and binding assays of transmembrane proteins to its ligands 3.4.23.B10 Rous sarcoma virus retropepsin analysis development of a rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus. The test strip can detect 600 pg purified recombinant p27 protein but also quantifies avian leukosis virus as low as 70 TCID50, which is comparable to the commercial enzyme-linked immunosorbent assay 3.4.23.B14 plasmepsin IV analysis PSM4 is unusable for structural studies by hydrogen/deuterium exchange coupled to mass spectrometry, DXMS, since the method requires the use of proteases working at acidic pH and low temperatures, in contrast to PSM2, EC 3.4.23.39 3.4.23.16 HIV-1 retropepsin analysis method for picomolar electrochemical detection of human immunodeficiency virus type-1 protease using ferrocene-pepstatin-modified surfaces studied by cyclic voltammetry and electrochemical impedance spectroscopy. Both gold nanoparticles and thiolated single walled carbon nanotubes/gold nanoparticles electrode materials show enhanced electrochemical responses to increasing concentrations of HIV-1 protease with shifting to higher potentials as well as decrease in the overall signal intensity. The sensing electrode modified with thiolated ingle walled carbon nanotubes/gold nanoparticles shows an estimated detection limit of 0.8 pM 3.4.23.B24 signal peptide peptidase analysis application of the photophore walking technique for probing the active sites of SPP. Nontransition state gamma-secretase inhibitors inhibit labeling of gamma-secretase by activity-based probes but enhance labeling of SPP. The opposite is true of gamma secretase modulators, which have little effect on the labeling of gamma-secretase but diminish labeling of SPP 3.4.23.39 plasmepsin II analysis cheap and high-throughput heterogeneous enzymatic assay for measuring plasmepsin II activity in order to use it as a tool in the discovery of new inhibitors of this enzyme 3.4.23.46 memapsin 2 analysis development of robust assay for cerebrospinal fluid beta-secretase-1 activity with sensitivity down to 1 pM of recombinant enzyme 3.4.23.46 memapsin 2 analysis identification of potential inhibitors of beta-secretase using in silico multi-filter techniques, substructure screening, computer-aided ligand docking, binding free energy calculations, and partial interaction energy analyses. Method retrieves all known inhibitors from the compound database investigated 3.4.23.46 memapsin 2 analysis development and validation of novel plasma BACE activity, soluble amyloid precursor proteins alpha and beta assays. Plasma BACE activity assay is sensitive and specific 3.4.23.48 plasminogen activator Pla analysis multiplex real-time polymerase chain reaction assay for the detection of Yersinia pestis and Yersinia pseudotuberculosis using the Yersinia pestis pla gene coding for plasminogen activator and the ypo2088 gene. Assays are both sensitive and specific, the lower detection limit is 10-100 fg of extracted total DNA 3.4.23.48 plasminogen activator Pla analysis development of a highly sensitive one-step PLA-enzyme immunoassay and an immunostrip, usable as a rapid test under field conditions. The PLA-immunometric tests allow a rapid and easy detection of Yersinia pestis in environmental and flea samples 3.4.24.B4 matrix metalloproteinase-13 analysis MMP-13 expression might be an indicator for increased extracellular matrix remodeling and early signs of vertebral compression in farmed Atlantic salmon 3.4.24.B4 matrix metalloproteinase-13 analysis generation of a neutralizing antibody specifc for the active form of MMP-13, but not for the latent form, or other MMPs. antibody can be used to measure active MMP-13 selectively by an enzyme-linked immunosorbet assay. The antibody suppresses the cleavage of type II collagen in human articular chondrocyte cultures, and is thought to inhibit MMP-13 activity effectively 3.4.24.7 interstitial collagenase analysis detection of localized extracellular sites of protease activity by use of fluorescent biosensor rhodamine 6G-labeled KDP-6-aminohexanoic acid-GPLGIAGIG-6-aminohexanoic acid-PKGY. Protease activity is localized at the polarized leading edge of migrating tumor cells rather than further back on the cell body. The path of proteolytic cleavage by a migrating cell can be visualized in 2- and 3-dimensional matrices. Probe can be used to determine inhibitor concentrations needed to suppress cell-surface protease activity 3.4.24.7 interstitial collagenase analysis generation of specific recombinant human monoclonal antibody SP1, which may serve as building block for the development of antibody-based therapy strategies in mouse models of pathology 3.4.24.11 neprilysin analysis the synthetic fluorogenic peptide substrate qf-Abeta(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM can be detected at peptide concentration of 0.002 mM. It can be used for high-throughput screening of compounds that upregulate NEP 3.4.24.17 stromelysin 1 analysis generation of specific recombinant human monoclonal antibody SP3, which may serve as building block for the development of antibody-based therapy strategies in mouse models of pathology 3.4.24.17 stromelysin 1 analysis unspecific staining in tissue sections of both animal strains with commercial anti-MMP-3 antibody JM3523 and positive but enzyme unspecific staining with the anti-MMP-3 antibody MAB 548 in both MMP-3 wild-type and MMP-3-deficient mouse skin wounds 3.4.24.23 matrilysin analysis method to localize enzyme activity within tissues by selective degradation of crosslinked carboxymethylated transferrin on polyethylene films 3.4.24.23 matrilysin analysis use of substrate PB-M7vis as a proteolytic beacon and optical molecular imaging contrast reagent for in vivo detection of enzyme activity 3.4.24.27 thermolysin analysis thermolysin degrades cellular prion protein while preserving both proteinase K-sensitive and proteinase K-resistant isoforms of disease-related prion protein in both rodent and human prion strains. In variant Creutzfeldt-Jakob disease, up to 90% of total prion protein present in the brain resists degradation with thermolysin, whereas only about 15% of this material resists digestion by proteinase K 3.4.24.27 thermolysin analysis selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes. Development and evaluation of a method providing information at the surface of the outer envelope membrane, based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. Envelope, thylakoid, and stroma proteins are separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry, overview 3.4.24.27 thermolysin analysis thermolysin offers a tool for complete solubilization of cartilage prior to comprehensive glycosaminoglycans(GAG)omic analysis, and is likely applicable to other collagen-rich tissues such as ligaments, skin, and blood vessels 3.4.24.33 peptidyl-Asp metalloendopeptidase analysis analysis of isoaspartic acid by selective proteolysis with Asp-N and electron transfer dissociation mass spectrometry, overview. IsoAsp formation and repair is central to the survival and germination of plant seeds. Also once administered into patients and thus exposed to physiological conditions of pH 7 and 37 °C, protein pharmaceuticals, particularly those with long circulation time, may generate significant amount of isoAsp 3.4.24.56 insulysin analysis immunocapture-based assay that uses the fluorogenic peptide substrate (7-methoxycoumarin-4-yl)acetyl-RPPGFSAFK-2,4-dinitrophenyl and allows the specific measurement of insulin-degrading enzyme activity in brain tissue homogenates. The fluorogenic substrate can be cleaved by a number of enzymes including neprilysin endothelin-converting enzyme-1 and angiotensin-converting enzyme, as well as IDE. Discrimination between these individual enzymes is not readily achieved in tissue homogenates, even in the presence of selective inhibitors and pH conditions. Immunocapture with antibody to the inactive domain of IDE prior to the addition of fluorogenic substrate allows sensitive, linear at 156-2500 ng/ml, and specific measurement of IDE activity and negligible cross-reactivity with neprilysin, endothelin-converting enzyme-1 or angiotensin-converting enzyme 3.4.24.65 macrophage elastase analysis the enzyme can be utilized for pharmacological evaluation of anti-inflammatory mechanisms of action 3.4.24.67 choriolysin H analysis developmental expression patterns of the Chrysiptera parasema HCE gene are highly similar to that of the medaka HCE gene, hatching enzyme system is highly conserved between marine and freshwater fish species 3.4.24.67 choriolysin H analysis the eel enzymes resemble the high choriolytic enzyme of medaka Oryzias latipes, and the hatching enzymes of the zebra fish Danio rerio and masu salomon Oncorhynchus masou, early differentation profile of eel hatching gland cells is similar to that of medaka, masu salomon and zebra fish, whereas the final location of the gland cells was different among fishes 3.4.24.69 bontoxilysin analysis development and evaluation of molecular detection tool for tracking and tracing Clostridium botulinum types A, B, E, F and other botulinum neurotoxin producing Clostridia using the real-time PCR-based GeneDisc cycler and BoNTs, overview. No cross reactivity with non human-toxigenic bacteria, Clostridium botulinum types C, D and G 3.4.24.69 bontoxilysin analysis the recombinant VAMP2 peptide substrate can be used as in vitro bioassay for the detection of BONT/B in food samples and provide a relevant replacement assay for the estimation of the toxin potency in contaminated therapeutic preparations 3.4.24.75 lysostaphin analysis improvement of lipid extraction of staphylococcal cells 3.4.24.80 membrane-type matrix metalloproteinase-1 analysis screening, identification, and characterization of affinity peptides, AF7p and Cy5.5-HS7, specific to MT1-MMP and application in tumor imaging, overview. Feasibility of using the subtraction biopanning strategy to screen the affinity peptide targeting MT-loop regions and HS7 is a superior probe for noninvasively imaging MT1-MMP expression in MT1-MMP-positive tumor models. Usage of HS7 in early diagnosis of tumors and in peptide-mediated drugs 3.4.24.86 ADAM 17 endopeptidase analysis development of a fluorescence resonance energy transfer-based biosensor that quantitatively reports the kinetics of TACE activity in live cells 3.4.24.87 ADAMTS13 endopeptidase analysis development of a 77-amino acid flow cytometry substrate to measure ADAMTS13 activity. Substrate is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. It contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594-1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. Substrate is bound to streptavidin-bearing microspheres with varyable polyethylene glycol spacer lengths. Recombinant ADAMTS13 activity can be quantified using substrates with all varyable polyethylene glycol repeat-lengths, but only a construct with the longer 77 PEG-unit can quantify proteolysis in blood plasma. Plasma ADAMTS13 down to 5% of normal levels can be detected within 30 min. Enzyme catalytic activity is tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage 3.4.25.1 proteasome endopeptidase complex analysis simultaneous labeling and detection of constitutive 20S proteasome subunits using an affinity-based probe cocktail and application to biological samples 3.4.99.B2 D-aspartyl endopeptidase analysis a system for screening D-Asp-containing proteins is developed by using D-aspartyl endopeptidase and a two-dimensional gel electrophoresis 3.5.1.1 asparaginase analysis direct measurement of L-asparagine in human plasma samples through the use of Escherichia coli Lasparaginase in the soluble form is a major clinical application of this system 3.5.1.1 asparaginase analysis biosensor for asparagine using a thermostable recombinant asparaginase from Archaeoglobus fulgidus immobilized in front of an ammonium-selective electrode. The biosensor has a detection limit of 0.06 mM for L-asparagine. It shows high stability 3.5.1.1 asparaginase analysis assembly a microplate-based biosensor for the determination of L-asparagine in biological samples. The enzyme is immobilized by crosslinking with glutaraldehyde in a microplate in 96-well format. The sensing is based on the colorimetric measurement of ammonia formation using the Nessler's reagent. The sensor enables monitoring of L-asparagine levels in serum and foods samples in the concentration range 10-200 microM, with a detection limit of 10 microM for L-asparagine 3.5.1.1 asparaginase analysis method based on IR spectroscopy to determine PEG-chitosan copolymer composition as well as composition of copolymer-enzyme conjugates. The method is reagentx02free and allows fast and reliable determination of parameters 3.5.1.2 glutaminase analysis construction of a L-glutaminase enzyme reactor based on immobilization of enzyme onto bamboo sticks through a glutaraldehyde modification to achieve covalent bonding. L-glutaminase-bamboo exhibits improved enzymatic hydrolysis performances, including high hydrolysis efficiency, prolonged stability (14 days) and good reusability and can efficiently be uesd for inhibitor screening 3.5.1.3 omega-amidase analysis use of soluble omega-amidase-glutamate dehydrogenase to determine alpha-ketoglutaramate in biological samples 3.5.1.5 urease analysis urea is estimated in different blood samples with the help of agar-immobilized urease and the results are consistent with those from clinical pathology laboratory through an autoanalyzer 3.5.1.5 urease analysis urease is used to calculate the amount of urea in biological solutions in order to remove urea from blood, waste water, fruit juice and foods. Enhancement of stability of immobilised urease by biocompatible polymer-conjugated magnetic beads for industrial application based on removal of urea 3.5.1.12 biotinidase analysis identification of the site of carboxylation of the biotinyl prosthetic group of several biotin enzymes 3.5.1.14 N-acyl-aliphatic-L-amino acid amidohydrolase analysis enzyme widely used as reagent to resolve amino acid racemates 3.5.1.19 nicotinamidase analysis determination of nicotinamide 3.5.1.19 nicotinamidase analysis nicotinamidases are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases 3.5.1.23 ceramidase analysis development of a method for the specific visualization of catalytically active acid ceramidase in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered enzyme activity 3.5.1.32 hippurate hydrolase analysis enzymatic determination of Bacillus subtilis siderophore itoic acid and 2,3-dihydroxybenzoic acid 3.5.1.41 chitin deacetylase analysis the enzyme can be used for production of chitin particles with only 1% deacetylated chitin which is still rigid and insoluble in acidic environment, but shows highly increased ovalbumin binding, chitosan as well is a good material for column chromatography showing no hydrogel formation 3.5.1.52 peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase analysis valuable tool to characterize the peptide portion of glycoproteins and glycopeptides to release asparagine-linked oligosaccarides for structural analysis, recombinant PNGase F contains no contaminant Endo F, proteases or exoglycosidases 3.5.1.52 peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase analysis a procedure to map N-glycosylation sites is presented, it can be applied to purified proteins as well as to highly complex mixtures. The method exploits deglycosylation by PNGase F in a diagonal, reverse-phase chromatographic setup 3.5.1.52 peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase analysis the facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of the enzyme makes recombinant PNGase H+ a versatile tool in N-glycan analysis 3.5.1.69 glycosphingolipid deacylase analysis the enzyme is immobilised on magnetic macroporous cellulose and is used to semisynthesise C17:0 glucosylceramide and C17:0 sulfatide, which are required as internal standards for quantification of the corresponding glycosphingolipids by tandem mass spectrometry 3.5.1.75 urethanase analysis the enzyme is used in spectrophotometric determination of ethyl carbamate through bi-enzymatic cascade reactions, method, overview. Detection of ethyl carbamate (urethane) in Chinese rice wine. Urethane is known as a genotoxic carcinogen1 that widely exists in fermented foods and alcoholic beverages, such as bread, yogurt, cheese, brandy, Chinese rice wine, sake, and wine, due to the natural biochemical processes in the fermentation process 3.5.1.88 peptide deformylase analysis novel class of N-formylated peptides for routine kinetic characterization and for screening PDF inhibitors 3.5.1.103 N-acetyl-1-D-myo-inositol-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase analysis fluorescence-based assay for measuring N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase activity that does not require HPLC analysis and can be carried out in multiwell plates. The fluorescamine-based assay can be used to determine the steady-state parameters for the deacetylation of N-acetyl-glucosamine by MshB 3.5.1.119 Pup amidohydrolase analysis development of an in vitro activity assay for Dop, based on fluorescence anisotropy measurements. The assay is simple, sensitive, and compatible with a high-throughput format for screening purposes. It can be reliably and conveniently used for detailed kinetic measurements of Dop activity and can be employed with other Dop enzymes 3.5.2.10 creatininase analysis analysis of serum creatinine concentration 3.5.2.10 creatininase analysis construction of a biosensor by co-immobilization of creatininase, creatinase, and sarcosine oxidase onto iron oxide nanoparticles/chitosangraft-polyaniline, Fe3O4-NPs/CHIT-g-PANI, composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. The creatinine biosensor uses enzymes/Fe3O4-NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The biosensor exhibits an optimum response within 2 s at pH 7.5 and 30°C, when polarized at 0.4 V versus Ag/AgCl. The electrocatalytic response shows a linear dependence on creatinine concentration ranging from 1 to 800 microM. The sensitivity of the biosensor is 3.9 microA per microM and cm2, with a detection limit of 1 microM. The biosensor shows only 10% loss in its initial response after 120 uses over 200 days, when stored at 4°C. The biosensor measures creatinine in the serum of apparently healthy persons 3.5.2.14 N-methylhydantoinase (ATP-hydrolysing) analysis measurement of serum and urine creatinine 3.5.2.20 isatin hydrolase analysis a fluorescence-based enzymatic assay is developed that can quantify isatin, a putative stress biomarkerin blood samples. A phase extraction of isatin followed by a second phase extraction combined with an enzymatic reaction performed by an isatin hydrolase is used to extract and quantify isatin in whole blood samples 3.5.3.1 arginase analysis specific detection of NO production in intact mouse tissue, inhibition of enzyme by N-hydroxy-nor-L-arginine to avoid disturbances 3.5.3.1 arginase analysis arginase activity may interfere in nitric oxide activity assay. A nitric oxide synthase-independent radioactive signal in mitochondrial samples analyzed for nitric oxide synthase-catalyzed [14C]-L-arginine to [14C]-L-citrulline conversion is due to the arginase-catalyzed conversion of [14C]-L-arginine to [14C]-urea. The results, in addition to reconfirming the absence of nitric oxide synthase activity in rat liver MT, show the need to include arginase inhibitors in studies using mitochondrial samples in order to avoid confounding results when using nitric oxide synthase activity assays 3.5.3.1 arginase analysis assay method based on a combination of moderately selective host-guest binding with the specificity of enzymatic transformations which allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting supramolecular tandem assays exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene is used as the macrocycle, which displays binding constants of 6400 per M with arginine, 550 per M with ornithine, and 60 000 per M with the selected fluorescent dye 1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene, the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. Assays can be successfully used to probe the inhibition of enzymes 3.5.3.1 arginase analysis development of a high-throughput semiquantitative assay system using a colorimetric 96-well plate assay to monitor the formation of urea. The assay has a dynamic range of about 5-300 microM for the ureido product 3.5.3.3 creatinase analysis biomatrix fabricated to investigate immobilization of creatine amidinohydrolase 3.5.3.3 creatinase analysis construction of a biosensor by co-immobilization of creatininase, creatinase, and sarcosine oxidase onto iron oxide nanoparticles/chitosangraft-polyaniline, Fe3O4-NPs/CHIT-g-PANI, composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. The creatinine biosensor uses enzymes/Fe3O4-NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The biosensor exhibits an optimum response within 2 s at pH 7.5 and 30°C, when polarized at 0.4 V versus Ag/AgCl. The electrocatalytic response shows a linear dependence on creatinine concentration ranging from 1 to 800 M. The sensitivity of the biosensor is 3.9 microA per microM and cm2, with a detection limit of 1 microM. The biosensor shows only 10% loss in its initial response after 120 uses over 200 days, when stored at 4°C. The biosensor measures creatinine in the serum of apparently healthy persons 3.5.3.9 allantoate deiminase analysis development of an enzyme cycling method for measuring allantoin concentrations in human serum 3.5.3.12 agmatine deiminase analysis development of a multiplex PCR method for the simultaneous detection of four genes involved in the production of histamine, i.e. histidine decarboxylase hdc, tyramine, i.e.tyrosine decarboxylase tyrdc, and putrescine, via either ornithine decarboxylase odc, or agmatine deiminase agdi. A collection of 810 lactic acid bacteria strains isolated from wine and cider was screened. The most frequent gene corresponds to the agdi gene detected in 112 strains, 14% of all lactic acid bacteria strains, of 10 different lactic acid bacteria species 3.5.3.15 protein-arginine deiminase analysis adding bicarbonate to commercial peptidyl arginine deiminase activity kits could increase assay performance and biological relevance 3.5.3.15 protein-arginine deiminase analysis development of a fluorescent probe to detect citrulline with high sensitivity and broad dynamic range and establishment of multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with the probe 3.5.3.18 dimethylargininase analysis use of inhibitor N-(but-3-yn-1-yl)-2-chloroethanimidamide as a broad-specificity probe for labeling endogenous DDAH isoforms and enzymes with similar pharmacophores. Inhibitor labels the active fraction of DDAH-1 in intact mammalian cells and can be blocked by the presence of competitive reversible and irreversible inhibitors. Incorporation of the alkyne tag allows to derivatize with a variety of reagents after in vivo tagging 3.5.4.1 cytosine deaminase analysis crystallization strategy named microseed matrix screening, differential chelation pattern of cations by acidic surfaces of proteins within crystal lattice as a critical parameter of crystal nucleation and growth 3.5.4.1 cytosine deaminase analysis in a selection-rooting assay CodA-expressing aerial tissues or shoot cuttings of Glycine max are inhibited for root formation in media containing 5-fluorouracil 3.5.4.3 guanine deaminase analysis colorimetric and HPLC assay method using guanosine as a prosubstrate. Method is suitable for routine assays for measuring plasma enzyme over a wide range of activites 3.5.4.4 adenosine deaminase analysis a capillary electrophoresis method for simultaneous analysis of adenosine deaminase in red blood cells is developed. The method is also successfully applied in the inhibitor screening from traditional Chinese medicines 3.5.4.4 adenosine deaminase analysis analysis of adenosine deaminase in saliva could be used as a simple, rapid, economic and non-invasive diagnostic tool in porcine production in field conditions 3.5.4.6 AMP deaminase analysis assessment of synthetic nitro- and polycyclic musks, imidazolium ionic liquids and N-glucopyranosyl ammonium salts 3.5.4.21 creatinine deaminase analysis analysis of creatinine in serum and urine 3.5.4.21 creatinine deaminase analysis the use of an ammonium ion-selective composite material, based on polyaniline and copper is reported for use as a transducer in electrochemical hydrolase-based urea and creatinine biosensors. The experimental designs and analytical performance of the biosensors are detailed together with their application for the determination of creatinine and urea in serum samples from patients with chronic kidney disease. A combination of creatinine deaminase and urease is chosen as a model system to demonstrate the construction of urea and creatinine biosensors to illustrate the principle 3.5.5.1 nitrilase analysis tools in the biodegradation and biodetection of cyanide 3.6.1.2 trimetaphosphatase analysis histochemical marker for lysosomes in light and electron microscopy 3.6.1.5 apyrase analysis immune sera that recognize specifically the B domain of NTPDase 1 are produced against synthetic peptides (LbB1LJ and LbB2LJ) derived from this domain of NTPDase from Leishmania brasiliensis. The polyclonal antibodies have effective anti-leishmanial effect, reducing significantly in vitro promastigotes growth (21-25%), an antiproliferative effect is also demonstrated by immune sera produced against recombinant r-pot B domain, and two other synthetic peptides (potB1LJ and potB2LJ). In addition, using these biomolecules in ELISA technique, IgG1 and IgG2 subclasses reactivities of either healthy dogs or infected by Leishmania infantum and classified clinically as asymptomatic, oligosymptomatic and symptomatic are tested. Analysis of distinct IgG1 and IgG2 seropositivities patterns suggest antibody subclasses binding epitopes along B domain for protection against infection, indicating this domain as a tool for prophylactic and immunotherapeutic investigations 3.6.1.5 apyrase analysis usage of the enzyme in a two-dimensional array ATP/ADP sensitive image sensor, with a uniform distribution of chemically immobilized apyrase (via two different methods (3-APTES and CEST)). The enzyme sensor changes ATP into AMP, and the concentration of ATP reduces with time. The time-dependent profile of the potential response is different from the common ion sensor on which the concentration of the analyte ion is constant at an equilibrium state. Two ways of the measurement are used: 1. the addition measurement and 2. the steady measurement, overview 3.6.1.6 nucleoside diphosphate phosphatase analysis because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic parameter to estimate this effect of lipid peroxidation on the microsomal membrane 3.6.1.10 endopolyphosphatase analysis the overexpression of the processed form of the enzyme should provide a unique and powerful reagent to analyze inorganic polyphosphate when the chain termini are unavailable to the actions of polyPase and polyP kinase 3.6.4.B1 kinesin K16 analysis photoreactiveATP derivative, 2'(3')-O-(4-benzoylbenzoyl)-1,N6-etheno-ATP acts as a cross-linker to detect conformational changes in the kinesin motor domain 3.6.4.B7 RadA recombinase analysis the highly thermostable RadA protein from the archaeon Pyrococcus woesei enhances the specificity of simplex and multiplex PCR assays 3.6.4.10 non-chaperonin molecular chaperone ATPase analysis high-throughput screening method in 96-well plates for ATPase activity of DnaK. Screening of potential inhibitors 3.6.5.2 small monomeric GTPase analysis nucleotide exchange kinetics with small GTPases can be used to obtain important information about the binding kinetics of GTP molecules under different activation states and with different activator molecules 3.8.1.2 (S)-2-haloacid dehalogenase analysis the enzyme is covalently linked to an N-hydroxysuccinimidyl Sepharose resin to construct a highly specific sensor with long shelf life for the detection of L-2-haloacids 3.8.1.3 haloacetate dehalogenase analysis selection marker on cloning vectors 3.8.1.8 atrazine chlorohydrolase analysis fiber-optic biosensors for detection of atrazine at low concentrations in soil using the atrazine chlorohydrolase and quantification of hydrochloric acid release, optimization, overview 3.9.1.2 protein arginine phosphatase analysis development of a substrate-trapping mutant that retains binding affinity toward arginine-phosphorylated proteins but cannot hydrolyze the captured substrates. Mutant C9A stably binds to arginine-phosphorylated proteins. The substrate-trapping efficiency is improved by impeding the oligomerization of the phosphatase mutant. The engineered YwlE trap efficiently captures arginine-phosphorylated proteins from complex Bacillus subtilis ywlE- cell extracts, thus facilitating identification of phosphoarginine sites in the large pool of cellular protein modifications 3.11.1.2 phosphonoacetate hydrolase analysis activity stain for detection of carbon-phosphorus cleavage by phosphonoacetate hydrolase in PAGE gels 3.12.1.B1 tetrathionate hydrolase analysis measurement of enzyme activity is based on a continuous spectrophotometric method that detects soluble intermediates that absorb in the UV region 3.12.1.B1 tetrathionate hydrolase analysis measurement of the enzyme activity during purification is based on the absorbance of the initial intermediates formed from tetrathionate in the ultraviolet region. Enzyme activity can also be measured by the scattering of insoluble sulfur in the visible region 3.13.2.1 adenosylhomocysteinase analysis study of methylation reactions 3.13.2.1 adenosylhomocysteinase analysis simple colorimetric method for the sensitive detection of S-adenosylhomocysteine hydrolase activity and inhibition using fluorosurfactant-capped gold nanoparticles. After hydrolysis of S-adenosylhomocysteine, the produced homocysteine molecules are bound to the surface of Au nanoparticles through the formation of Au-S bonds. The approach has a minimum detectable concentration of 100 units/l, or about 6 nM. The addition of adenosine analogs to a solution containing S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase results in the suppression of hydrolyzed S-adenosylhomocysteine-induced nanoparticle aggregation 4.1.1.4 acetoacetate decarboxylase analysis the enzyme is a valuable tool for clinical analysis of ketone bodies in human plasma 4.1.1.15 glutamate decarboxylase analysis most important application of glutamate decarboxylase antibodies is to identify those neurons and neuronal projections that use 4-aminobutanoate as their neurotransmitter by immunocytochemical visualization 4.1.1.15 glutamate decarboxylase analysis the diabetes autoantibody standardization program, DASP, aims to improve and standardize measurement of autoantibodies, e.g. glutamic acid decarboxylase autoantibodies and islet antigen-2, against associated with type 1 diabetes, overview 4.1.1.21 phosphoribosylaminoimidazole carboxylase analysis sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Method is applied to accumulation of 5-aminoimidazole ribotide in AdeD cells 4.1.1.28 aromatic-L-amino-acid decarboxylase analysis development o a high-throughput assay for testing of inhibitors 4.1.1.28 aromatic-L-amino-acid decarboxylase analysis loss of dopa decarboxylase appears to result in similar phenotypes as those of aromatic L-amino acid decarboxylase deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of aromatic L-amino acid decarboxylase deficiency in children 4.1.1.39 ribulose-bisphosphate carboxylase analysis 1 pg of plant mRNA, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, is used to determine the efficiency of RT-PCR in general, and as a external reference gene for normalization of gene expression 4.1.1.39 ribulose-bisphosphate carboxylase analysis a method is established to remove ribulose bisphosphate carboxylase/oxygenase from plant samples to obtain high quality and high resolution 2D gels in proteome analysis 4.1.1.39 ribulose-bisphosphate carboxylase analysis a sensitive and robust mixed-mode high performance liquid chromatography-tandem mass spectrometry method is developed for the qualitative and quantitative determination of sugar phosphates 4.1.1.49 phosphoenolpyruvate carboxykinase (ATP) analysis bicarbonate assay 4.1.1.50 adenosylmethionine decarboxylase analysis development of a simple, economic, and non-radioactive spectrometric enzymatic assay, which can be adapted for experimental high-throughput screening of AdoMetDC inhibitors 4.1.1.53 phenylalanine decarboxylase analysis use of enzyme for decarboxylation of phenylalanine to increase its volatility for continuous-flow isotopic analysis without introducing extraneous C or significant isotopic fractionation 4.1.1.65 phosphatidylserine decarboxylase analysis development of a fluorescence-based assay for selectively monitoring production of phosphatidylethanolamine in reactions using a maltose-binding protein fusion with Plasmodium knowlesi PSD as the enzyme. The product detection by fluorescence occurs after the lipid reacts with a water-soluble distyrylbenzene-bis-aldehyde and provides strong discrimination against the phosphatidylserine substrate. The assay is amenable to application in 96- and 384-well microtiter plates 4.1.1.65 phosphatidylserine decarboxylase analysis use of the complementation of a Saccharomyces cerevisiae mutant for screening inhibitors 4.1.2.20 2-dehydro-3-deoxyglucarate aldolase analysis enzymatic determination of D-glucaric acid 4.1.2.20 2-dehydro-3-deoxyglucarate aldolase analysis specific enzymatic assay for D-glucarate in human serum 4.1.2.22 fructose-6-phosphate phosphoketolase analysis the gene encoding xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp) is conserved among Bifidobacterium species within a more variable region of the genome, useful for strain identification 4.1.2.27 sphinganine-1-phosphate aldolase analysis use of BODIPY-labeled sphinganine substrate, allows fluorescent product detection by HPLC. The reaction is linear over a 30 min time period 4.1.2.27 sphinganine-1-phosphate aldolase analysis easy and sensitive procedure to determine sphingosine-1-phosphate lyase activity. The assay uses C17-sphinganine-1-phosphate as substrate and the aldehyde product, pentadecanal, is quantified as its pentafluorobenzyloxime derivative by GC/MS. Derivatization of pentadecanal is performed as a one-step reaction, and the oxime product is directly injected for GC/MS analysis without any further purification. Limit of detection is 281 fmol. The assay is linear with both protein concentration and incubation time up to 20 microg and 40 min, respectively 4.1.2.27 sphinganine-1-phosphate aldolase analysis the addition of sphingosine to cultures of cell lines or primary cells provides a source of intracellular sphingosine 1-phosphate that is susceptible to degradation by the lyase and, hence, increases on inhibition or silencing of the enzyme. Development of a biochemical assay optimized with respect to sphingosine concentration, incubation time, and cell density and establishment for routine use with HEK293 cells. The assay is suitable for the testing of novel active site-directed sphongosine-1-phosphate lyase inhibitors 4.1.2.40 tagatose-bisphosphate aldolase analysis use in measuring intracellular tagatose 1,6-diphosphate 4.1.2.47 (S)-hydroxynitrile lyase analysis cyanide-based high-throughput screening assay is developed. The assay is useful to detect activity and enantioselectivity of hydroxynitrile lyases theoretically towards any cyanohydrin substrate 4.1.3.3 N-acetylneuraminate lyase analysis enzymatic determination of sialic acid 4.1.3.3 N-acetylneuraminate lyase analysis generation of a dynamic combinatorial library of sialic acid analogues using sialic acid aldolase 4.1.3.6 citrate (pro-3S)-lyase analysis analytical method for detection of adulteration of citrus products by a flow-injection method based on the use of enzyme reactors 4.1.3.6 citrate (pro-3S)-lyase analysis determination of citrate in urine with a new citrate lyase method 4.1.3.6 citrate (pro-3S)-lyase analysis quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. lactis biovar diacetilactis 4.1.99.1 tryptophanase analysis the metabolic enzyme tryptophanase (TPase) is the key component in an odor-based sensor system. The enzyme is able to convert an odorless substrate like S-methyl-L-cysteine or L-tryptophan into the odorous products methyl mercaptan or indole. For biosensor construction, TPase is biotinylated so that it can be coupled with a molecular recognition element, such as an antibody, to develop an ELISA-like assay. This method is used for the detection of an antibody present in nM concentrations by the human nose. TPase can also be combined with the enzyme pyridoxal kinase (PKase) for use in a coupled assay to detect adenosine 5'-triphosphate (ATP). When ATP is present in the low mM concentration range, the coupled enzymatic system generates an odor that is easily detectable by the human nose. Biotinylated TPase can be combined with various biotin-labeled molecular recognition elements, thereby enabling a broad range of applications for this odor-based reporting system 4.1.99.3 deoxyribodipyrimidine photo-lyase analysis the enzyme is of primary biological interest as an analytical tool 4.1.99.3 deoxyribodipyrimidine photo-lyase analysis the novel substrate (a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine) is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase 4.1.99.3 deoxyribodipyrimidine photo-lyase analysis the novel substrate (a modified thymidine 10-mer with a central cyclobutane pyrimidine dimer and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine) is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase 4.1.99.14 spore photoproduct lyase analysis a rapid separation technique for detecting and quantitating SP by chromatography : tritiated thymine-containing photoproducts from trifluoroacetic acid-hydrolyzed DNA purified from UV-irradiated cells or spores of Bacillus subtilis are identified and isolated from paper chromatograms, subjected to HPLC on a Microsorb phenyl 5-micrometer column using 100% water as the mobile phase, and detected by scintillation counting of collected fractions 4.2.1.1 carbonic anhydrase analysis carbonic anhydrase assay based on membrane inlet mass spectrometry. A quantitative assessment of the carbonic anhydrase activity in photosystem II 4.2.1.1 carbonic anhydrase analysis carbonic anhydrase may serve as a biochemical marker for photosynthetic capacity in teak genotypes 4.2.1.2 fumarate hydratase analysis fumarate hydratase activity can be useful in the diagnosis of hereditary leiomyomatosis and renal cell cancer in cases with atypical presentation and undetectable fumarate hydratase mutations. Furthermore, fumarate hydratase activity testing is of value in laboratory investigations to elucidate the mechanism of hereditary leiomyomatosis and renal cell cancer 4.2.1.9 dihydroxy-acid dehydratase analysis the enzyme is used to quantify and investigate the biological oxidant stress activity of various redox-cycling chemicals. High sensitivity to inactivation by oxidants makes the enzyme useful for identification of compounds which increase oxyradical flux in the cell and for probing their mechanism of action 4.2.1.10 3-dehydroquinate dehydratase analysis development of a high-throughput method of DHQase activity assay 4.2.1.20 tryptophan synthase analysis the enzyme complex is a model enzyme for understanding allosteric regulation 4.2.1.22 cystathionine beta-synthase analysis usefulness of the CBS domains as predictors of osmoregulatory activity 4.2.1.22 cystathionine beta-synthase analysis development of 7-azido-4-carbamoylmethylcoumarin as a modified fluorogenic probe for H2S detection with improved solubility in aqueous solutions 4.2.1.22 cystathionine beta-synthase analysis development of a LC-MS/MS and HPLC methods to simultaneously measure formation of thioethers and hydrogen sulfide from mixtures of cystathionine beta-synthase substrates 4.2.1.24 porphobilinogen synthase analysis measurement of ethanol consumption in alcoholics 4.2.1.24 porphobilinogen synthase analysis indirect measurement of blood lead in human subjects 4.2.1.24 porphobilinogen synthase analysis the enzyme can be used as a biomarker for Pb2+ contamination 4.2.1.30 glycerol dehydratase analysis determination of cobamide derivates in picomole quantities 4.2.1.30 glycerol dehydratase analysis determination of vitamin B12 in the range 0.057 to 1.0 pmol using glycerol dehydrase. The sensitivity and accuracy of this method are lower than those of other methods. But because the procedure for the method is simple, it can be performed in any laboratory in a short time. The method can be useful as a complement to other methods 4.2.1.40 glucarate dehydratase analysis assay of D-glucarate in human serum 4.2.1.41 5-dehydro-4-deoxyglucarate dehydratase analysis by establishing a pH-shift assay, an easy measurement method for 5-dehydro-4-deoxy-D-glucarate dehydratases/decarboxylase is developed. It can be used for measurements of the purified enzymes or using crude extracts. It is especially suitable as the method of choice within an engineering approach for further optimization of the enzyme 4.2.1.41 5-dehydro-4-deoxyglucarate dehydratase analysis by establishing a pH-shift assay, an easy measurement method for 5-keto-4-deoxy-D-glucarate dehydratases/decarboxylase is developed. It can be used for measurements of the purified enzymes or using crude extracts. It is especially suitable as the method of choice within an engineering approach for further optimization of the enzyme 4.2.1.42 galactarate dehydratase analysis a continuous assay for L-talarate/galactarate dehydratase is develped using circular dichroism. The advantages that this assay method offers are that initial rate measurements may be obtained much more rapidly than is possible using fixed-time assays (e.g., semicarbazide derivatization or NMR methods) and it is much less complex than a coupled assay, which is often limited by the availability and specific properties of the coupling enzymes 4.2.1.42 galactarate dehydratase analysis a convenient, direct continuous circular dichroism-based assay is developed for following the L-talarate/galactarate dehydratase-catalyzed conversion of meso-galactarate to 5-dehydro-4-deoxy-D-glucarate. The advantages that this assay method offers are that initial rate measurements may be obtained much more rapidly than is possible using fixed-time assays (e.g., semicarbazide derivatization or NMR methods) and it is much less complex than a coupled assay, which is often limited by the availability and specific properties of the coupling enzymes 4.2.1.46 dTDP-glucose 4,6-dehydratase analysis the highly conserved sequence of the dTDP-glucose-4,6-dehydratase gene is used to explore the Streptomyces tenebrarius H6 tabromycin/kanamycin biosynthetic gene cluster 4.2.1.46 dTDP-glucose 4,6-dehydratase analysis an absorbance-based microtiter plate assay is developed for RmlB activity. It can be used for high-throughput screening of RmlB inhibitors 4.2.1.66 cyanide hydratase analysis optical biotest for determination of cyanide traces in marine fish using microbial cyanide hydratase and formate dehydrogenase 4.2.1.66 cyanide hydratase analysis tools in the biodegradation and biodetection of cyanide 4.2.1.77 trans-L-3-hydroxyproline dehydratase analysis estimation of the content of L-hydroxyprolines using coupling systems with metabolic enzymes of the trans-4-hydroxy-L-proline pathway (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and the trans-3-hydroxy-L-proline pathway (trans-3-hydroxy-L-proline dehydratase (T3LHypD) and DELTA1-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. A functional expression system of recombinant HypDH with a heterooligomeric structure is constructed in Escherichia coli cells. Enzymological characterization reveals that the beta-subunit acts as a catalytic subunit, and also that assembly with other subunit(s) improves the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline are successfully estimated within the ranges of 0.004-1 mM and 0.05-1 mM, respectively, and are consistent with the contents determined by HPLC. This enzymatic method is used to measure the content of trans-4-hydroxy-L-proline in the acid-hydrolysate of collagen, and blood plasma 4.2.1.84 nitrile hydratase analysis the enzyme is used in production of acrylamide from acrylonitrile. For maximum production of Co2+ containing nitrile hydratase, is cultured in the medium containing lactose (18.0 g/l), peptone (1.0 g/l), yeast extract (2.0 g/l), MgSO4 (0.5 g/l), K2HPO4 (0.6 g/l), urea (9.0 g/l), and CoCl2 (0.01 g/l), pH 7.0, and incubated at 35°C for 24 h in an incubator shaker (160 rpm). Nitrile hydratase exhibits relatively high specificity for aliphatic nitriles. Free cells are immobilized using 2% (w/v) agar solution to enhance enzyme stability and reusability in repetitive cycles of acrylamide production. Under optimized conditions, nearly complete bioconversion of acrylonitrile is achieved with a fair recovery of 85% using free and immobilized cells equivalent to 500 mg/dry cell weight/l 4.2.2.1 hyaluronate lyase analysis application for the specific determination of hyaluronan concentration 4.2.2.3 mannuronate-specific alginate lyase analysis the enzyme, wild-type and mutant variants, can be used for alginate fine structure elucidation 4.2.2.5 chondroitin AC lyase analysis useful tool for the identification and structural characterization of chondriotin and dermatan sulfates 4.2.2.7 heparin lyase analysis development of a simple and rapid colorimetric method, which can detect heparinase activity and thus help to identify likely candidates for future anti-metastatic and anti-inflammatory drugs 4.2.2.7 heparin lyase analysis the GSTfused enzyme with high purity and specific activity may be useful in solid phase clinical applications and sequence characterizations of structurally specific heparin and heparan sulfate motifs 4.2.2.7 heparin lyase analysis detection of heparin and heparinase using a ratiometric fluorescence method based on benzoperylene derivative, 6-(benzo [ghi]perylene-1,2-dicarboxylic imide-yl)hexanoic acid excimer in an aequeous buffer solution. The assay is simple, rapid, inexpensive, sensitive and selective and can be used for assay of heparin and heparinase in complex sample mixtures 4.2.2.7 heparin lyase analysis in heparinized blood plasma samples of cancer patients, heparinase addition removes the heparin inhibition. Treatment significantly improves PCR efficacy, enabling quantification of circulating tumor DNA 4.2.2.8 heparin-sulfate lyase analysis analysis of heparitinase induced changes in Xenopus laevis embryos 4.2.2.19 chondroitin B lyase analysis method for quantitative detection of dermatan sulfate in heparin. Selective detection of deramtan sulfate in heparin is possible based on the increased value of UV absorption at 232 nm resulting from exhaustive depolymerization of dermatan sulfate. The first method facilitates analysis in heparin with dermatan sulfate concentrations greater than 0.1 mg/ml at 232 nm, with good linearity, precision and recovery. The second method allows sensitive and accurate detection of dermatan sulfate at concentrations below 0.1 mg/ml, with good linearity, precision and recovery. The linear range of dermatan sulfate detection is between 0.01 and 0.5 mg/ml 4.2.2.21 chondroitin-sulfate-ABC exolyase analysis micromethods for the measurement of as little as 3 pg of chondroitin sulfate A, B, or C in mixtures with other mucopolysaccharides or in urine 4.2.2.21 chondroitin-sulfate-ABC exolyase analysis enzyme combined with chodroitin sulfatase ABC, EC 4.2.2.4, can be applied for the sequencing of chondroitin sulfate hexa- and octasaccharides with complex structures 4.2.3.1 threonine synthase analysis continuous, coupled spectrophotometric threonine synthase assay. The sequential actions of threonine deaminase and hydroxyisocaproate dehydrogenase convert the L-Thr product of TS to alpha-ketobutyrate and then to 2-hydroxybutyrate, respectively, and are monitored as the decrease in absorbance at 340 nm resulting from the concomitant oxidation of beta-nicotinamide adenine dinucleotide to NAD+ by hydroxyisocaproate dehydrogenase 4.2.3.8 casbene synthase analysis optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis. Expression of casbene synthases leads to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products aere exported onto the leaf surface for structure elucidation of unknown diterpenes 4.2.3.23 germacrene-A synthase analysis in GC-MS analysis of reaction products, beta-elemene is identified as product of farnesyl diphosphate reaction. beta-Elemene is the Cope rearrangement product of germacrene A that is formedupon injection into the hot injection port of the GC-MS 4.2.3.27 isoprene synthase analysis method for measuring isoprene emission based on postillumination isoprene release after rapid temperature transient, and application to determine the rate constant of isoprene synthase, the pool size of its substrate dimethylallyldiphosphate, and to separate the component processes of the temperature dependence of isoprene emission 4.2.3.27 isoprene synthase analysis development of a method to measure the isopentenyl diphosphate levels and the ratio of dimethyallyl diphosphate/isopentenyl diphosphate in bacteria and plant tissues. The amount of dimethyallyl diphosphate is measured by conversion to isoprene using isoprene synthase IspS, and the total amount of dimethyallyl diphosphate plus isopentenyl diphosphate is measured by including isopentenyldiphosphate isomerase IDI in the assay mixture. The in vitro equilibrium ratio of dimethylallyl diphosphate/isopentenyl diphosphate is 2.11:1. Isopentenyl diphosphate and dimethylallayl diphosphate pools are significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio dimethyallyl diphosphate/isopentenyl diphosphate is 3.85. An Escherichia coli strain transformed with IspS but no additional IDI has a lower dimethylallyl diphosphate level and a dimethyallyl diphosphate/isopentenyl diphosphate ratio of 1.05 4.2.3.61 5-epiaristolochene synthase analysis method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product 4.2.3.69 (+)-alpha-barbatene synthase analysis method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product 4.2.3.77 (+)-germacrene D synthase analysis use of NMR spectroscopy as a tool to analyze the kinetics of enzyme reactions using progress curves. The protocol presented is also a simple and direct approach for the measurement of enzyme kinetics in the presence of synthetic inhibitors. The conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase involves an amphiphilic substrate forming micelles and a water insoluble product. Using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function 4.2.3.107 (+)-car-3-ene synthase analysis PCR-based approach to identify BAC clones for two target genes, 3-carene synthase and a cytochrome P450 from a non-arrayed genomic BAC library. Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp long for 3CAR. Inspection of the genomic sequences reveals the intron-exon structures, the putative promoterregions and putative cis-regulatory elements. Sequences related to transposable elements, high complexity repeats and simple repeats are prevalent and comprise approximately 40% of the sequenced genomic DNA 4.2.3.163 (+)-corvol ether B synthase analysis PMAs and EIMS fragmentation mechanisms for the fragment ions of reaction product 4.2.3.171 (+)-corvol ether A synthase analysis PMAs and EIMS fragmentation mechanisms for the fragment ions of reaction product 4.2.99.18 DNA-(apurinic or apyrimidinic site) lyase analysis chemical footprinting-mass spectrometric assay using N-ethylmaleimide, an irreversible Cys modifier, to characterize the interaction of the redox inhibitor, E3330, with APE1. When APE1 is incubated with E3330, two N-ethylmaleimide-modified products are observed, one with two and a second with seven added N-ethylmaleimide molecules 4.2.99.18 DNA-(apurinic or apyrimidinic site) lyase analysis development an effective in vitro system for monitoring G quadruplex structure formation in damage-containing DNA, a tool to investigate the mechanistic details of repair occurring at this noncanonical DNA structure 4.2.99.18 DNA-(apurinic or apyrimidinic site) lyase analysis development of assay method using a methylation-sensitive restriction endonuclease 4.3.1.1 aspartate ammonia-lyase analysis scaled-down method for determination of aspartase activity in a 96-well microtitre plate 4.3.1.2 methylaspartate ammonia-lyase analysis a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae 4.3.1.2 methylaspartate ammonia-lyase analysis procedure for the synthesis of mesaconic acids that are specifically mono- or di-deuterated in the methyl group in order to investigate the mechanisms of enzyme-catalyzed reactions 4.3.1.3 histidine ammonia-lyase analysis a potentiometric sensor is made by immobilizing histidine ammonia-lyase on an ammonia gas-sensing electrode 4.3.1.4 formimidoyltetrahydrofolate cyclodeaminase analysis formiminotransferase cyclodeaminase enzyme complex represents a marker with which to explore ER-Golgi dynamics 4.3.1.15 Diaminopropionate ammonia-lyase analysis a specific enzymatic procedure for the determination of neurotoxic components, derivatives of L-2,3-diaminopropanoate with diaminopropanonate ammonia-lyase 4.3.1.17 L-serine ammonia-lyase analysis online-determination of L-serine concentation in bioreactor 4.3.1.17 L-serine ammonia-lyase analysis development of a simple, direct, and continuous assay based on the absorbance of pyruvate, the product of the dehydratase reaction 4.3.1.18 D-Serine ammonia-lyase analysis development of a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free D-serine in tissue samples. The method uses chicken D-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. The presence of Zn2+ or EDTA dioes not have any effect on pyruvate formation under the present assay conditions. The method is not affected by the presence of a large excess of L-serine, nor by the presence of tissue extracts, and accurately determines concentrations of 2-30 microM of D-serine. The entire assay requires only 60 min 4.3.1.18 D-Serine ammonia-lyase analysis enzymatic assay for D-serine. D-serine dehydratase converts D-serine to pyruvate, which is in turn oxidized by pyruvate oxidase. Then, in the presence of horseradish peroxidase, hydrogen peroxide formed during the oxidation converts 10-acetyl-3,7-dihydroxy-phenoxazine, i.e. Amplex Red, to resorufin, which exhibits a strong fluorescence. This improved assay can be used to determine the concentration of D-serine in calf serum 4.3.1.24 phenylalanine ammonia-lyase analysis useful for determining Phe or removing Phe from mammalian systems 4.3.1.24 phenylalanine ammonia-lyase analysis immobilization of phenylalanine ammonia-lyase into gelatin on polyester films to determine phenylalanine in urine for the prediagnosis of phenylketonuria. Immobilized enzyme retaines 100% apparent activity after 30 days and as much as 75% of activity is retained after 2 months. The method is sufficiently sensitive to determine the phenylalanine concentration in phenylketonuric infants' urine 4.3.1.24 phenylalanine ammonia-lyase analysis an HPLC method for the determination of phenylalanine ammonia-lyase, flavanone 3-hydroxylase and flavonol synthase enzyme activity is proposed. This method is based on the determination of the compounds produced and consumed on the enzymatic reaction in just one chromatographic analysis. Optimisation of the method consideres kinetic studies to establish the incubation time to perform the assay. The method is an approach to measure the activities of the three enzymes simultaneously increasing the rapidity, selectivity and sensitivity over other methods 4.3.1.25 phenylalanine/tyrosine ammonia-lyase analysis rapid quantization of Phe and Tyr in plasma and serum from subjects with phenylketonuria 4.3.2.1 argininosuccinate lyase analysis the rapid and sensitive assay method is used to detect increases in the activity in serum from patients with liver diseases 4.3.2.7 glutathione-specific gamma-glutamylcyclotransferase analysis development of a 5-oxoprolinase-coupled, sensitive, 5-oxoproline detection-based gamma-GCT assay. Development of an alternative assay that exploits the Cys-Gly generation from glutathione. Cys-Gly is converted into free cysteine and glycine with the help of Dug1p (a peptidase from Saccharomyces cerevisiae), followed by estimation of cysteine 4.3.2.7 glutathione-specific gamma-glutamylcyclotransferase analysis development of a a 5-oxoprolinase-coupled, sensitive, 5-oxoproline detection-based gamma-GCT assay. Development of an alternative assay that exploits the Cys-Gly generation from glutathione. Cys-Gly is converted into free cysteine and glycine with the help of Dug1p (a peptidase from Saccharomyces cerevisiae), followed by estimation of cysteine 4.3.2.8 gamma-glutamylamine cyclotransferase analysis endpoint-assay, L-lysine is converted to alpha-keto epsilon-aminocaproate semicarbazone in the presence of semicarbazide, excess L-lysine alpha-oxidase, and catalase 4.4.1.1 cystathionine gamma-lyase analysis use in biosensor for measurement of L-cysteine 4.4.1.4 alliin lyase analysis microwave inactivation of the enzyme alliinase, to eliminate the interference from alliinase sulfo products, is used for a rapid detection method for determination of herbicides in onions, overview 4.4.1.4 alliin lyase analysis Flow-injection enzymatic analytical system for determination of alliin based on the immobilized alliinase and an ammonia gas electrode provids linearity in the 1 × 10-5 to 1 × 10-3 mol/l alliin concentration range and exhibited good repeatability and operational stability 4.4.1.4 alliin lyase analysis development of a qRT-PCR assay that enables the quantification and normalisation of alliinase and cysteine synthase transcripts 4.4.1.5 lactoylglutathione lyase analysis practical experiments for students to determine the effect of exposure to oxidative stress conditions on yeast glyoxalase I, investigation of kinetic parameters 4.4.1.5 lactoylglutathione lyase analysis flow cytometry method for GLO-1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO-1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients. Expression index of GLO-1-positive cells is slightly increased in mononuclear leukocytes from diabetic patients. This result correlates with the increase in GLO-1 activity in the whole blood samples of type 2 diabetes patients 4.4.1.11 methionine gamma-lyase analysis the enzyme can be used for detection of S-adenosyl-L-homocysteine hydrolase activity and inhibition in a simple and robust fluorogenic enzymatic assay, method development, overview 4.4.1.11 methionine gamma-lyase analysis assay for methionine gamma-lyase-catalyzed gamma-elimination reactions of L-methionine and its analogues with 2-oxobutanoate as product. The assay employs UV-Vis spectrophotometry to continuously monitor the rate of formation of 2-oxobutanoate by its absorbance at 315 nm 4.4.1.20 leukotriene-C4 synthase analysis although structural differences near the active site and along the C-terminal alpha-helix V suggest that the mouse and human enzymes may function differently in vivo, the mouse enzyme is a useful tool in pharmacological research and drug development 4.4.1.20 leukotriene-C4 synthase analysis cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying LTC4S activity and investigating LTC4S inhibitors 4.4.1.21 S-ribosylhomocysteine lyase analysis rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of the metabolites and precursors of the activated methyl cycle. Analytes are extracted from Escherichia coli MG1655 and chemically derivatized as N(O,S)-iso-butyloxycarbonyl iso-butyl esters using iso-butyl chloroformate in an aqueous iso-butanol/pyridine environment. S-Adenosylmethionine, S-adenosylhomocysteine, S-ribosylhomocysteine, homocysteine, methionine, cystathionine, cysteine, and homoserine are quantified by liquid chromatography-positive ion tandem electrospray ionization mass spectrometry. Internal standards are isotopically labeled [13CD3]methionine and S-adenosylcysteine. Linearity of the assay is established up to a concentration of 700 microg/g cell dry weight for each analyte 4.4.1.36 hercynylcysteine S-oxide lyase analysis a synthetic route toward ergothioneine pathway intermediates for the preparation of stable isotopically labelled hercynine-d3, which is enzymatically converted to ergothioneine-d3 4.6.1.1 adenylate cyclase analysis technical problems of AC detection, some of which caused by poor quality-control of commercially supplied antibodies. Intracellular targeting of ACs may be isoform-specific and also dependent on the cellular context of expression 4.6.1.1 adenylate cyclase analysis the enzyme activity is useful as a marker for determination of G-protein-coupled receptor function mediating adenylyl cylcase activation by extracellular baculovirus particles, usage of this budded virus display system to detect G-protein-coupled receptor signaling, method development, overview 4.6.1.1 adenylate cyclase analysis development of a fluorescence resonance energy transfer (FRET) sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain. There is a strong linear correlation between catalytic domain complementation and cyclase activity upon stimulation with forskolin and the Galphas subunit. The sensor is functional in live cells 4.6.1.2 guanylate cyclase analysis purified sGC can act as detector for NO-like bioactivity generated from nitrite and glycerol trinitrate and for NO released by heart and liver mitochondria 4.6.1.12 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase analysis high-throughput method for the screening of 2C-methyl-D-erythritol synthase, IspC protein, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD protein, 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, IspE protein, and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, IspF protein, against large compound libraries 4.6.1.13 phosphatidylinositol diacylglycerol-lyase analysis use of the enzyme and specific antibodies for the enzyme for the examination of the growth inhibition, morphological change and ectoenzyme release of the LLC-PK1 cells from pig, effective for the investigation of the function of the glycosyl-phosphatidylinositol-anchor protein 4.6.1.18 pancreatic ribonuclease analysis preparation of polymeric nanoparticles imprinted with RNase A via miniemulsion polymerization using methyl methacrylate and ethylene glycol dimethacrylate. The addition of poly(vinyl alcohol) as a co-surfactant is effective in preserving the protein structural integrity. Imprinted nanoparticles produces by the optimized method show increased target specificity 4.6.1.18 pancreatic ribonuclease analysis pressure tuning hole burning experiments using the UV-absorbing tyrosine residues. Ribonuclease A protein stays intact upon cooling to 2 K. Its various tyrosine sites show characteristic features which can be resolved in pressure tuning hole burning spectra. Reducing the sulfur bridges leads to a loss of the individual features, and the sites become alike. The respective compressibility is reduced by more than a factor of 2 and comes close to the value of free tyrosine in solution. Compared to the reduction of the sulfur bridges, the influence of guanidinium hydrochloride on the pressure tuning behavior is less pronounced 4.6.1.18 pancreatic ribonuclease analysis studies on application to purify ribonuclease from eggs of Rana catesbeiana with an aqueous–aqueous polymer phase system by using a small-scale cross-axis coil planet centrifuge 4.6.1.18 pancreatic ribonuclease analysis study on spermidine modulation of enzyme activity via individual RNA plasmon rulers which combine high throughput with high temporal resolution at the single molecule level and are able to retrieve otherwise obscured information about weak structural stabilizations 4.6.1.18 pancreatic ribonuclease analysis synthesis of molecularly imprinted polymers from the monomers styren and polyethyleneglycol 400 dimethacylate with high rebinding efficiency of RNase A to polymer. Polymers show high selectivity for RNase A and high stability 4.6.1.18 pancreatic ribonuclease analysis synthesis of monodispersed RNase A surface-imprinted particles with good magnetic property for practical bioseparation. Use of methyl methacrylate and ethylene glycol dimethacrylate as the functional and cross-linker monomers to produce particles of 700-800 nm diameter imprinted with ribonuclease A and encapsulated with nanosized Fe3O4 particles. Surface-imprinted particles show good selectivity toward the RNase template over control protein 4.6.1.20 ribonuclease U2 analysis bacteriophage lambda protein phosphatase and recombinant RNase U2 can be used to generate RNase digestion products amenable to liquid chromatography-tandem mass spectrometry analysis, which can be used to determine the presence and location of modified ribonucleosides in RNA samples 4.6.1.20 ribonuclease U2 analysis improvement of RNA modification mapping sequence coverage by LC-MS. The non-specific ribonuclease activity RNase U2 E49A substitution mutant provides increased sequence coverage of substrate RNA during modification mapping. Data analysis software is modified to account for non-specific digestion. This combination allows efficient and accurate RNA modification mapping 4.6.1.21 Enterobacter ribonuclease analysis - 4.98.1.1 protoporphyrin ferrochelatase analysis Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase, ferrochelatase mutants with improved tolerance towards N-methylprotoporphyrin may be potentially used in cell assay systems to study physiological responses to haem deficiency 4.99.1.8 heme ligase analysis development of a high resolution correlative combination of cryo soft X-ray tomography to obtain 3D parasite ultrastructure with cryo X-ray fluorescence microscopy to measure heme concentrations 5.1.1.7 diaminopimelate epimerase analysis simple and sensitive spectrophotometric method for the determination of meso-alpha,epsilon-diaminopimelate with meso-2,6-diaminopimelate D-dehydrogenase and its application to the assay of diaminopimelate epimerase 5.1.1.7 diaminopimelate epimerase analysis a high-performance liquid chromatography method for the simultaneous assay of diaminopimelate epimerase and decarboxylase 5.1.1.8 4-hydroxyproline epimerase analysis spectrophotometric assay for hydroxyproline in collagenous tissue hydrolysates, with an enzymatic method using 4-hydroxyproline 2-epimerase,EC 5.1.1.8, D-amino acid oxidase, EC 1.4.3.3, and a colorimetric reagent of the mixture of Ti(IV) and 4-(2-pyridylazo)-resorcinol 5.1.1.8 4-hydroxyproline epimerase analysis estimation of the content of L-hydroxyprolines using coupling systems with metabolic enzymes of the trans-4-hydroxy-L-proline pathway (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and the trans-3-hydroxy-L-proline pathway (trans-3-hydroxy-L-proline dehydratase (T3LHypD) and DELTA1-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. A functional expression system of recombinant HypDH with a heterooligomeric structure is constructed in Escherichia coli cells. Enzymological characterization reveals that the beta-subunit acts as a catalytic subunit, and also that assembly with other subunit(s) improves the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline are successfully estimated within the ranges of 0.004-1 mM and 0.05-1 mM, respectively, and are consistent with the contents determined by HPLC. This enzymatic method is used to measure the content of trans-4-hydroxy-L-proline in the acid-hydrolysate of collagen, and blood plasma 5.1.1.10 amino-acid racemase analysis simple procedure for in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with amino acid racemase, EC 5.1.1.10, and L-leucine dehydrogenase, EC 1.4.1.9 5.1.1.13 aspartate racemase analysis estimation of age from dentin by using the racemization reaction of Asp 5.1.2.2 mandelate racemase analysis direct kinetic assay for mandelate racemase using circular dichroic measurement 5.1.3.1 ribulose-phosphate 3-epimerase analysis spectrophotometric assay for D-ribulose-5-phosphate 3-epimerase and D-ribose-5-phosphate ketol-isomerase 5.1.3.1 ribulose-phosphate 3-epimerase analysis direct assay procedure which exploits differences in CD spectrum between ribulose 5-phosphate and xylulose 5-phosphate 5.1.3.1 ribulose-phosphate 3-epimerase analysis spectrophotometric determination of ribulose 5-phosphate 3-epimerase in tissue extract 5.1.3.3 Aldose 1-epimerase analysis kinetic assay method for aldose 1-epimerase based upon fast in situ generation of alpha-D-glucose employing hydrolysis of sucrose by beta-fructofuranosidase and a subsequent reporter reaction involving the aerobic oxidation of beta-D-glucose via glucose o 5.1.3.3 Aldose 1-epimerase analysis rapid determination of glucose by aldose 1-epimerase 5.1.3.3 Aldose 1-epimerase analysis conductometric biosensor for sucrose determination using a membrane containing enzymes invertase, mutarotase, and glucose oxidase as a sensitive element immobilized on the conductometric interdigitated planar electrodes. The biosensor demonstrates high selectivity, operational stability and reproducibility 5.1.3.17 heparosan-N-sulfate-glucuronate 5-epimerase analysis assay procedure for heparosan-N-sulfate-glucuronate 5-epimerase, which is based on the use of a two-phase system for liquid scintillation counting 5.1.3.19 chondroitin-glucuronate 5-epimerase analysis assay procedure for chondroitin-glucuronate 5-epimerase which is based on the use of a two-phase system for liquid scintillation. 3H2O formed during the reaction, is extracted into an organic phase containing fluorine and isoamyl alcohol, while unreacted pol 5.1.3.37 mannuronan 5-epimerase analysis development of a high-throughput screening method to discriminate between different alginate structures 5.1.3.37 mannuronan 5-epimerase analysis in vitro assay for mannuronan C5 epimerization wherein extracts of Escherichia coli expressing high levels of AlgG are incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer is detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsieila aerogenes 5.1.3.37 mannuronan 5-epimerase analysis rapid and reproducible assay for the analysis of epimerase activity in the fermentation broth using [3H]-alginate as a substrate. The effects of potential interfering compounds are characterized and the uncertainty of the analysis system has been determined. A standard method suitable for use directly on the fermentation broth with a standard deviation within a single analysis of 2.5% has been developed 5.1.99.1 methylmalonyl-CoA epimerase analysis a simple direct assay for DL-methylmalonyl-coenzyme A racemase which is based on the fact that the proton on C-2 of methylmalonyl-CoA is replaced by a proton in the medium during racemization 5.1.99.4 alpha-methylacyl-CoA racemase analysis fluorescent enzyme-linked aptamer assay for alpha-methylacyl-CoA racemase. The assay shows a dynamic range from 0.1 to 1000 nM of AMACR, a low detection limit of 0.44 nM (19.5 ng/ml), and high AMACR specificity 5.2.1.8 peptidylprolyl isomerase analysis the enzyme weakly catalyzes some protein processes which are rate-limited by proline isomerization, but probably exhibits no measurable catalysis towards others. This somewhat limits the usefulness of the enzyme as a diagnostic reagent for proline isomeri 5.2.1.8 peptidylprolyl isomerase analysis high-throughput screening method for isoform FKBP12 binding based on the decrease of a fluorescence signal generated by a small molecule fluorescent FKBP12 ligand bound to the protein and measured in the presence of inhibitor 5.2.1.8 peptidylprolyl isomerase analysis fluorescent probe 6-(dimethylamino)-2-naphthoyl-Ala-Ala-(cis)-Pro-Phe-4-nitroanilide can be used for imaging active peptidyl-prolyl cis/trans isomerases in live cells 5.3.1.1 triose-phosphate isomerase analysis specific polymerase chain reaction protocols used to determine the prevalence of toxigenic Clostridium difficile in Vhembe, South Africa. The study confirms the usefulness of PCR methodologies in the detection of toxigenic Clostridium difficile and suggests that Clostridium difficile is responsible for a small, but underappreciated, proportion of diarrheal cases in the region 5.3.1.1 triose-phosphate isomerase analysis systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins 5.3.1.8 mannose-6-phosphate isomerase analysis development and optimization of a transformation method using the phosphomannose-isomerase gene pmi as a selectable marker for Brassica napus transformation via Agrobacterium tumefaciens, overview 5.3.1.9 glucose-6-phosphate isomerase analysis Pgi is an adaptive marker, rather than providing insights into individual genetic health, Pgi appears to have a role in conservation genetics by providing insights into gene by environment interactions, local adaptation and evolutionary significant units, and potentially even morphologically cryptic dispersal phenotypes, method development, overview 5.3.1.9 glucose-6-phosphate isomerase analysis phosphoglucose isomerase variability of Cerastoderma glaucum as a model for testing the influence of environmental conditions and dispersal patterns through quantitative ecology approaches, overview. The PGI locus is an appropriate marker for revealing the spatial distribution of genetic variation and for establishing the relationships between allelic composition and the environmental influencing variables 5.3.1.9 glucose-6-phosphate isomerase analysis development of a bioautographic assay based on thin layer chromatography for inhibition of PDI by phosphoenolpyruvate. The detection limit for phosphoenolpyruvate as an inhibitor of PGI is 226 microg per spot/zone 5.3.3.7 aconitate DELTA-isomerase analysis a simple sensitive and specific method for measuring aconitate isomerase in plants, which depends on the release of tritium from labeled trans-aconitate 5.3.3.7 aconitate DELTA-isomerase analysis method for estimation of enzyme activity in plant material 5.3.4.1 protein disulfide-isomerase analysis development of a method to determine quantitatively the redox state of active-site cysteines found in the Cys-Xaa-Xaa-Cys motif in living cells. Method is based on the alkylation of cysteines by methoxy polyethylene glycol 5000 maleimide. In vivo, protein disulfide isomerase is present in two semi-oxidized forms in which either the first active site in the a domain or the second active site in the a' domain is oxidized. In HEK-293 cells, about 50% of enzyme is fully reduced, in 18% a domain is oxidized, a' reduced, in 15%, the a domain is reduced, a' oxidized, and 16% of enzyme are fully oxidized 5.3.4.1 protein disulfide-isomerase analysis study on the critical influence of reference genes used for data normalization, shown for protein disulfide isomerase 5.3.4.1 protein disulfide-isomerase analysis PDIA3 and C/EBP? may be valuable markers in fish for exposure and effect to environmental stress 5.3.99.2 Prostaglandin-D synthase analysis particle concentration fluorescence immunoassay for prostaglandin D synthase is suitable for determining the content of prostaglandin D synthetase in various regions of the rat CNS. The method allows to assay a large number of samples with reasonable sensitivity 5.3.99.2 Prostaglandin-D synthase analysis PGD synthase is a useful marker for identifying the differentiation stage of human megakaryocytic cells 5.3.99.5 thromboxane-A synthase analysis application of monoclonal antibodies to develop a tandem immunoradiometric assay 5.3.99.5 thromboxane-A synthase analysis immobilized enzyme is sufficiently stable to be used as a model for studying the properties of the enzyme 5.4.2.6 beta-Phosphoglucomutase analysis a specific method for the quantitative determination of beta-glucose 1-phosphate 5.4.2.7 phosphopentomutase analysis a coupled optical enzyme assay 5.4.3.7 leucine 2,3-aminomutase analysis separation by high-performance liquid chromatography of alpha-amino acids and beta-amino acids and the application to the assay of leucine 2,3-aminomutase 5.4.99.2 methylmalonyl-CoA mutase analysis method for separation of methylmalonyl-CoA and succinyl-CoA by capillary electrophoresis suitable for evaluation of total and holo-enzyme activity in biological matrices. Application of method for the differential diagnosis of methylmalonic acidemia, in relation to protein or coenzyme defects 5.4.99.2 methylmalonyl-CoA mutase analysis establishment of a method to measure the concentration of succinyl-CoA with UPLC-MS/MS after enzyme reaction using peripheral lymphocytes, and investigation of the MCM enzyme activity of patients with methylmalonic acidemia 5.4.99.5 chorismate mutase analysis As an intramolecular reaction that appears to be catalyzed without intermediate steps, covalent catalysis, or modification of the reaction pathway, the chorismate-prephenate rearrangement has become an important model system for theoretical approaches to the study of enzyme catalysis 5.4.99.9 UDP-galactopyranose mutase analysis high-throughput fluorescence polarization assay for indentification of inhibitors of enzyme and homologues 5.5.1.6 chalcone isomerase analysis improvements in assay procedure 5.6.2.1 DNA topoisomerase analysis the absence of cysteine residues in MtTOP1 makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening assays and can be utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate 5.6.2.2 DNA topoisomerase (ATP-hydrolysing) analysis use of triple-helical DNA structures for targeting enzyme-mediated cleavage to DNA specific sequences and use of drug-triplex-forming oligonucleotides to investgate enzyme mechanism 6.1.1.21 histidine-tRNA ligase analysis alternating catalysis requires a mechanism for coupling events between active sites, presumably through conformational changes propagated between these active sites, a version of HisRS is developed that features the site-specific incorporation of extrinsic environmentally sensitive fluorescent probes, allowing the adenylation reaction to be followed by stopped-flow fluorometry 6.2.1.1 acetate-CoA ligase analysis colorimetric assay method to measure acetyl-CoA synthetase activity 6.2.1.7 cholate-CoA ligase analysis a coupled assay for bile acid:CoA ligase and glycination of bile acid-CoA catalyzed by bile acid-CoA:glycine N-acetyltransferase 6.2.1.7 cholate-CoA ligase analysis the ligase assay can be used as a sensitive enzymic marker for endoplasmic reticulum in rat liver 6.2.1.11 biotin-CoA ligase analysis enzymatic assay for biotin 6.2.1.17 propionate-CoA ligase analysis rapid radiochemical assay method with high sensitivity and specificity 6.2.1.30 phenylacetate-CoA ligase analysis determination of of microbody-borne enzymes by analysis of putative microbody targeting singals and a proteomics based inventory of Penicillium chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis 6.3.1.2 glutamine synthetase analysis immunoassay that detects synthetase protein samples where the enzyme has been inactivated by repeated cycles of freezing and thawing, and in serum and cerebrospinal fluid where glutamine synthetase is undetectable by the enzyme activity assay 6.3.1.5 NAD+ synthase analysis the enzyme is used for an enzymatic cycling method for ammonia assay using a system consisting of three enzymes: EC 6.3.1.5, EC 1.1.1.47 and EC 1.6.99.2 6.3.1.5 NAD+ synthase analysis method for measurement of allantoin in human serum. Serum allantoin is converted to allantoate by the action of allantoinase,and endogenous ammonia is simultaneously removed by the action of glutamine synthetase II. In the second step, L-methionine sulfoximine is used to inhibit glutamine synthetase II, and ammonia is liberated from allantoate by the activity of allantoate amidohydrolase. The ammonia is then converted to NAD by NAD synthetase. Subsequent action of glucose dehydrogenase and diaphorase acts to cycle the formed NAD between its oxidized and reduced forms, resulting in the production of WST-1 formazan, which is monitored at 450 nm. The assay standard curve is linear from 0 to 70 microM allantoin 6.3.1.5 NAD+ synthase analysis use of substrate 2-fluro-ATP as tool for 18F NMR-based activity screening 6.3.2.3 glutathione synthase analysis assay of glutathione synthetase in erythrocytes by HPLC with fluorimetric detection, useful for rapid screening of erythrocytes glutathione synthetase activity in various pathological conditions 6.3.2.4 D-Alanine-D-alanine ligase analysis assay method for screening for effectors of alanine racemase and/or D-alanine:D-alanine ligase 6.3.2.9 UDP-N-acetylmuramoyl-L-alanine-D-glutamate ligase analysis assay for monitoring enzyme activity based on the accumulation of adenosine 5'-diphosphate, a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, with counterscreen assay to eliminate false positive results 6.3.2.12 dihydrofolate synthase analysis a simple radioassay for dihydrofolate synthetase activity and its application to an inhibition study of new pteroate analogs 6.3.2.17 tetrahydrofolate synthase analysis assay procedure for the measurement of FPGS in up to 50 or 100 samples of partially purified enzyme at a time 6.3.2.25 tubulin-tyrosine ligase analysis utilization of ligase to determine the state of tyrosination of tubulin 6.3.3.2 5-formyltetrahydrofolate cyclo-ligase analysis development of a functional complementation assay for 5-CHO-THF metabolism in Escherichia coli, based on deleting the gene encoding 5-FCL. The deletion mutant accumulates 5-formyltetrahydrofolate and,with glycine as sole nitrogen source, shows a growth defect, both phenotypes are complemented by bacterial or archaeal genes encoding glutamate formiminotransferase. Glutamate formiminotransferases functionally replace 5-formyltetrahydrofolate cyclo-ligases in certain prokaryotes 6.3.4.2 CTP synthase (glutamine hydrolysing) analysis fast assay allows the processing of a large number of samples 6.3.4.6 urea carboxylase analysis enzymatic assay with urea amidolyase for determination of potassium in serum 6.3.4.10 biotin-[propionyl-CoA-carboxylase (ATP-hydrolysing)] ligase analysis 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity by biotinylation of the polypeptide p67, which comprises the 67 C-terminal amino acids in human propionyl-CoA carboxylase, including the biotin-binding site lysine-669, using IRDye-streptavidin and infrared spectroscopy. The minimal concentration of recombinant HCS that can be detected by this assay is less than 1.08 nmol/l. Jurkat cells contain 0.14 U of HCS activity [in micromol of biotinylated p67 formed/(nmol/l HCS h)] in 400 microg of total protein 6.3.4.10 biotin-[propionyl-CoA-carboxylase (ATP-hydrolysing)] ligase analysis fusion of full-length HCS to DNA adenine methyltransferase Dam and subsequent transfection into breast cancer cells MCF-7 and normal breast cells MCF-10A for identification of chromatin binding sites 6.3.4.12 glutamate-methylamine ligase analysis assay for the enzymatic synthesis of gamma-glutamylmethylamide 6.3.4.15 biotin-[biotin carboxyl-carrier protein] ligase analysis biotin-mediated ATP-diphosphate assay which does not require the isolation of the apoenzyme and is simple and convenient for use on a routine assay. The procedure is specifically designed for assay of enzyme from adipose tissue of biotin-deficient rats. W 6.3.4.15 biotin-[biotin carboxyl-carrier protein] ligase analysis biotinylation of apo-carboxyl carrier protein, a subunit of acetyl-CoA carboxylase from E. coli is a sensitive and convenient assay method for biotin-[acetyl-CoA-carboxylase] ligase 6.3.4.15 biotin-[biotin carboxyl-carrier protein] ligase analysis SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex. An SH2 domain from lymphocyte-specific tyrosine kinase is genetically fused to a truncated biotin carboxyl carrier protein, and the resulting fusion protein is labeled through biotinylation with biotin protein ligase carrying multiple copies of a luminescent Tb3+ complex. The labeled SH2 fusion proteins are employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide is produced by phosphorylation to the substrate peptide by Src tyrosine kinase. The assay allows for a reliable determination of the activity of Src kinase lower than 10 ng/mL 6.3.4.15 biotin-[biotin carboxyl-carrier protein] ligase analysis development of a high-throughput assay building on the principle of differential scanning fluorimetry of GFP-tagged proteins 6.3.4.15 biotin-[biotin carboxyl-carrier protein] ligase analysis fluorescent probe (4S)-4-[5-(1-[3-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]propyl]-1H-1,2,3-triazol-4-yl)pentyl]tetrahydro-1H-thieno[3,4-d]imidazol-2(3H)-one is synthesized by Bpl in vivo, accumulates in cytoplasm and can be used to gain insights into the mechanism of uptake, efflux and metabolism of BPL inhibitors 6.3.4.16 carbamoyl-phosphate synthase (ammonia) analysis development of a CPS1-reporter system for the assessment of ammonia metabolism. Labeling of CPS1 gene in cell lines HepG2, and LO2, with fluorescence protein. Cellular detoxification enhancers are selected among a collection of 182 small molecules. In both CPS1 reporter cell lines, the fluorescence intensity is positively correlated with cellular CPS1 mRNA expression, ammonia elimination and secreted urea, and reflects ammonia detoxification in a dose-dependent manner 6.3.4.19 tRNAIle-lysidine synthase analysis development of a ultrahigh-throughput, fluorescence anisotropy-based assay for the incorporation of lysine into Ile2 tRNA 6.3.5.4 asparagine synthase (glutamine-hydrolysing) analysis high-performance liquid chromatography assay for Asn synthetase is an extremly sensitive and reliable method for assaying Asn synthetase 6.3.5.4 asparagine synthase (glutamine-hydrolysing) analysis a rapid, inexpensive micro assay that can be adapted for large numbers of samples 6.3.5.4 asparagine synthase (glutamine-hydrolysing) analysis mass spectrometry-based procedure for the direct quantification of asparagine synthetase protein concentration in complex sample mixtures. Assay is able to distinguish samples from transformed cell lines that express the enzyme over a wide dynamic range of concentration. The method directly detects asparagine synthetase protein, use in blast samples from patients with acute lymphoblastic leukemia 6.3.5.4 asparagine synthase (glutamine-hydrolysing) analysis development of a sensitive, non-radioactive assay for AsnS, based on incubation of desalted enzyme and substrates and then direct detection of either product asparagine or glutamate by HPLC 6.3.5.7 glutaminyl-tRNA synthase (glutamine-hydrolysing) analysis simple system for monitoring the inhibition of GatCAB activity using Escherichia coli Top10 co-expressing the non-discriminating glutamyl-tRNA synthetase ndGluRS and GatCAB genes from Staphylococcus aureus Mu50. Growth repression is confirmed by introducing ndgluRS from Staphylococcus aureus Mu50 into Escherichia coli. Co-expression of the gatCAB operon alleviates growth repression in the host Escherichia coli. The screening system consists of these two transformants and non-expressing Escherichia coli Top10. The transformant harbors both ndGluRS gene and GatCAB operon can be co-expressed in the presence and in the absence of chemical compounds of interest. There is no inhibitor that inactivates GatCAB activity, but upon expression of two inactive GatCAB deletion variants, GatCAB-10 and GatCAB-CHD, together with ndGluRS in Escherichia coli Top10, the cells show repressed growth as well as ndGluRS is expressed 6.4.1.3 propionyl-CoA carboxylase analysis method to analyze propionyl-CoA carboxylase activity in phytohemagglutinin stimulated lymphocytes using high performance liquid chromatography. Propionyl-CoA carboxylase activity is unaffected even when lymphocytes are isolated and phytohemagglutinin stimulated after a whole blood sample has been stored at 4°C for 5 days, and the method is useful for the confirmation of propionic acidemia in individuals, and prenatal diagnosis and genetic counseling for the affected families 6.4.1.8 acetophenone carboxylase analysis construction of a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome. mCherry production is proportional to the applied acetophenone concentrations. The reporter strain allows quantification of acetophenone within a concentration range of 50 microM (detection limit) to 250 microM after 12 and 24 h. Production of the Apc-mCherry fusion protein in the reporter strain is highly specific and responds to acetophenone and both enantiomers of 1-phenylethanol 6.5.1.1 DNA ligase (ATP) analysis essential reagent in studies on nucleic acid structure and metabolism. In combination with polynucleotide kinase end-group labeling, DNA ligase can be used to identify 3'- and 5'-end groups at single-strand interruptions by nearest neighbor analysis. DNA 6.5.1.1 DNA ligase (ATP) analysis a procedure for fast, sensitive and quantitative measurement of DNA ligase activity in crude cell extract 6.5.1.1 DNA ligase (ATP) analysis DNA automaton based on FokI without ligase has similar efficiency as with ligase in the context of automaton reactions. Other enzymes (HgaI, BsmFI, BbsI, and BseMII) show more discrepancy between with and without ligase 6.5.1.1 DNA ligase (ATP) analysis piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probe 6.5.1.2 DNA ligase (NAD+) analysis DNA ligase is an essential reagent in studies on nucleic acid structure and metabolism 6.5.1.2 DNA ligase (NAD+) analysis DNA ligase, in combination with polynucleotide kinase, can be used to identify 3'- and 5'-end groups at single-strand interruptions by nearest neighbor analysis 6.5.1.2 DNA ligase (NAD+) analysis DNA ligase can be used to determine the ability of other enzymes to act at nicks and gaps in duplex DNA molecules 6.5.1.2 DNA ligase (NAD+) analysis DNA ligase can be used to study the primary and secondary structure of DNA molecules 6.5.1.2 DNA ligase (NAD+) analysis high-throughput assay for the adenylation reaction of bacterial DNA ligase 7.1.1.2 NADH:ubiquinone reductase (H+-translocating) analysis kinetic analysis of enzyme using a seff-assembled proteoliposome system containing complex I, Q10, and quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2 to Q10. Complex I is completely rate determining and the Q10 pool is kept oxidized under steady-state catalysis 7.1.1.7 quinol oxidase (electrogenic, proton-motive force generating) analysis development of a mCherry-based fluorescent reporter for detection of cytochrome bd expression in Mycobacterium marinum. The expression reporter is suitable for monitoring mycobacterial cytochrome bd expression during mouse macrophage infection and in a zebrafish embryo infection model 7.1.1.9 cytochrome-c oxidase analysis cytochrome c oxidase modified electrodes can be used to distinguish amino acid sequence variations in proteins such as cytochrome c. This has potential relevance as a diagnostic for disease states, characterization of electron transfer reactions of cytochrome c isolated from ischemic and control hearts 7.2.2.8 P-type Cu+ transporter analysis method to probe Cu flow through human Atox1 and copper chaperone ATP7B proteins when expressed in yeast. 7.5.2.1 ABC-type maltose transporter analysis the maltose/maltodextrin transport system of Escherichia coli/Salmonella serves as a model for canonical ATP-binding cassette importers in general 7.6.2.1 P-type phospholipid transporter analysis assay for phospholipid flippase activities of plasma membrane-localized P4-ATPases using human cell lines stably expressing isoforms ATP8B1, ATP8B2, ATP11A, and ATP11C 7.6.2.1 P-type phospholipid transporter analysis phosphatidylserine-coumarin O-[[(2R)-2-[(9-[5-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]-1H-1,2,3-triazol-4-yl]nonanoyl)oxy]-3-(hexadecanoyloxy)propoxy](hydroxy)phosphoryl]-L-serine exhibits bright fluorescence and good photostability. It should be useful as a blue-emitting fluorescent translocation substrate for extended imaging studies of flippase action in living cells using laser confocal microscopy 7.6.2.2 ABC-type xenobiotic transporter analysis evaluation of high-speed ATPase activity assay method by comparing with in vitro transport assay systems using MDR1-transfected MDR1-MDCK cells. The kinetic parameters obtained in ATPase activity assay are not necessarily corresponding with those in in vitro transport assay. The combination of the ATPase activity assay and in vitro transport technologies gives insight into mechanisms 7.6.2.2 ABC-type xenobiotic transporter analysis use of in vitro transporter assays for analysis of the role of P-glycoprotein mediated efflux activity. The transwell assay is a valuable tool to evaluate human P-glycoprotein interaction with compounds for assessing brain penetration of new chemical entities 7.6.2.2 ABC-type xenobiotic transporter analysis use of in vitro transporter assays for analysis of the role of P-glycoprotein mediated efflux activity. The transwell assay is a valuable tool to evaluate P-glycoprotein interaction with compounds for assessing brain penetration of new chemical entities 7.6.2.2 ABC-type xenobiotic transporter analysis use of pharmacophore models for the prediction of binding to P-glycoprotein