Information on EC 1.1.2.4 - D-lactate dehydrogenase (cytochrome)

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.1.2.4
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RECOMMENDED NAME
GeneOntology No.
D-lactate dehydrogenase (cytochrome)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(R)-lactate + 2 ferricytochrome c = pyruvate + 2 ferrocytochrome c + 2 H+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
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redox reaction
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-
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
methylglyoxal degradation VI
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methylglyoxal degradation
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Pyruvate metabolism
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SYSTEMATIC NAME
IUBMB Comments
(R)-lactate:ferricytochrome-c 2-oxidoreductase
A flavoprotein (FAD).
CAS REGISTRY NUMBER
COMMENTARY hide
37250-79-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Jerusalem artichoke
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-
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
No. 7, growth photoanaerobically on lactate
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
prostate cancer cells metabolize D-lactate inside mitochondria via a D-lactate dehydrogenase which is more active and highly expressed than in normal cells. D-Lactate can enter prostate mitochondria and cause the export of newly synthesized malate in a carrier-mediated manner, with the rate of malate efflux from mitochondria being twofold higher in cancer compared to healthy cells, overview
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c
show the reaction diagram
(R)-lactate + 2 ferricytochrome c
pyruvate + ferrocytochrome c
show the reaction diagram
(R)-lactate + ferricyanide
pyruvate + ferrocyanide
show the reaction diagram
(R)-lactate + ferricytochrome c
pyruvate + ferrocytochrome c
show the reaction diagram
D-2-hydroxybutyrate + cytochrome c
2-oxobutanoate + ferrocytochrome c
show the reaction diagram
D-2-hydroxybutyrate + ferricytochrome c
?
show the reaction diagram
135% relative activity compared to (R)lactate, highest catalytic efficiency, prefered substrate
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-
?
D-2-hydroxyglutarate + ferricytochrome c
?
show the reaction diagram
0.15% relative activity compared to (R)lactate
-
-
?
D-2-hydroxyvalerate + cytochrome c
2-oxovalerate + ferrocytochrome c
show the reaction diagram
-
very poor substrate
-
-
?
D-glycerate + ferricytochrome c
?
show the reaction diagram
9.2% relative activity compared to (R)lactate
-
-
?
D-lactate + 2 ferricytochrome
pyruvate + 2 ferrocytochrome
show the reaction diagram
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-
-
-
?
D-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c
show the reaction diagram
D-lactate + 2,6-dichlorophenolindophenol
pyruvate + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
D-lactate + cytochrome c2ox
pyruvate + cytochrome c2red
show the reaction diagram
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-
-
-
?
D-lactate + ferricytochrome c
pyruvate + ferrocytochrome c
show the reaction diagram
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-
-
-
?
DL-2-hydroxybutyrate + ferricytochrome c
2-oxobutanoate + ferrocytochrome c
show the reaction diagram
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40% of the activity with (R)-lactate
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-
?
DL-glycerate + ferricyanide
?
show the reaction diagram
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10% of the rate of D-lactate oxidation
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DL-glycerate + ferricytochrome c
?
show the reaction diagram
10.3% relative activity compared to (R)lactate
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-
?
glycolate + ferricytochrome c
?
show the reaction diagram
0.15% relative activity compared to (R)lactate, O2 does not function as the acceptor for glycolate electrons
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?
L-lactate + ferricytochrome c
?
show the reaction diagram
10.8% relative activity compared to (R)lactate
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(R)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c
show the reaction diagram
(R)-lactate + 2 ferricytochrome c
pyruvate + ferrocytochrome c
show the reaction diagram
additional information
?
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study of the D-lactate transport and metabolism in Helianthus tuberosus, overview
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,6-dichlorophenolindophenol
cytochrome c
ferricytochrome c
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phenazine methosulfate
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
inhibitory
Co2+
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restores activity after inhibition with o-phenanthroline
Cu2+
-
rapid inactivation, can be restored by addition of dithiothreitol
Fe2+
-
restores activity after inhibition with o-phenanthroline
ferricyanide
Mg2+
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inhibitory
Mn2+
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restores activity after inhibition with o-phenanthroline
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-hydroxybutanoate
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irreversible inactivation, modification of FAD
2-oxobutanoate
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inactivation under anaerobic conditions
DL-gycerate
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strong inhibition
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High ionic strength
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o-phenanthroline
oxalate
oxaloacetate
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competitive
p-chloromercuribenzoate
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weak
p-Mercuriphenylsulfonate
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phenylsuccinate
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additional information
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antimycin A does not affect the enzyme activity. Myxothiazol binds to the quinone-binding site and blocks electron transfer from ubiquinol to cytochrome b. At 20 mM myxothiazol the sensitivity of wild-type plants to D-lactate is not affected. Inhibition of complex III does not affect D-lactate oxidation
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
carboxylic acid
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activates, stimulation is non-competitive, hyperbolic and its dimension is directly related to the number of carboxylic groups on the activator. Stimulation does not affect the yield of oxidative phosphorylation
D-lactate
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stimulation with D-lactate as substrate, is insensitive to malate or pyruvate addition
EDTA
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slightly stimulating
L-malate
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non-competitive activation
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.264
(R)-lactate
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30C, pH 7.5
0.0054
cytochrome c
0.061 - 1.4
D-2-hydroxybutyrate
2.5
D-2-hydroxyvalerate
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8.871
D-glycerate
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0.0026 - 26
D-lactate
0.23
ferricyanide
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0.432
glycolate
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4.486
L-lactate
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4.45
phenazine methosulfate
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additional information
additional information
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kinetics of D-lactate metabolism, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.467
D-2-hydroxybutyrate
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0.1
D-glycerate
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1.083 - 1500
D-lactate
0.00167
glycolate
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0.1167
L-lactate
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
24.17
D-2-hydroxybutyrate
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0.0167
D-glycerate
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6.583
D-lactate
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0.0033
glycolate
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0.033
L-lactate
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0016 - 0.12
oxalate
0.01
oxaloacetate
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.203
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after purification by 4-hydroxy-alpha-cyanocinnamic acid-AH-Sepharose column chromatography, pH and temperature not specified in the publication
0.342
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after purification by hydroxylapatite column chromatography and 4-hydroxy-alpha-cyanocinnamic acid-AH-Sepharose column chromatography, pH and temperature not specified in the publication
3.38
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enzyme purified on H-Sepharose with covalently bound 4-hydroxy-alpha cyanocinnamic acid
5.71
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combination of chromatography on hydroxylapatite and H-Sepharose with covalently bound 4-hydroxy-alpha cyanocinnamic acid
20.3
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purified enzyme
87
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purified enzyme
1670
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purified enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
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with ferricyanide
7.8
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assay at
8 - 9
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pH optimum for D-lactate, L-lactate and glycolate oxidation
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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no activity at pH 5, almost maximum activity at pH 9
5.5 - 7.5
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very low activity with ferricyanide
5.5 - 8.5
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low activity with cytochrome c at pH 5.5, almost maximum activity at pH 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
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assay at, in permeabilized cells
37
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
the enzyme is located on the inner face of the inner mitochondrial membrane
Manually annotated by BRENDA team
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localization of recombinantly expressed GFP-tagged enzyme
Manually annotated by BRENDA team
additional information
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equally distributed between soluble and membrane fraction
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55000
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2 * 55000, sedimentation equilibrium centrifugation and SDS-PAGE
57000
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4 * 57000, SDS-PAGE
59000
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2 * 59000, sequence analysis
100000
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sedimentation equilibrium centrifugation
105000
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sedimentation equilibrium centrifugation
110000
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recombinant enzyme, native PAGE
135000
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gel filtration
235000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 55000, sedimentation equilibrium centrifugation and SDS-PAGE
homodimer
tetramer
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4 * 57000, SDS-PAGE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3
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stable for short term exposure (30 s)
389623
6.5
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very stable
389623
7.25
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retains at least 25% activity down to pH 7.25, whereas it rapidly loses activity at lower pH values
698932
7.5 - 9.5
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655064
7.5
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complete inactivation above pH 7.5
389623
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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30 min, about 10% loss of activity
55
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10 min, about 90% loss of activity
60
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rapid inactivation above
75
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2 min,about 90% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dithiothreitol and bovine serum albumin required for stability
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very stable in cold
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, as ammonium sulfate precipitate
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4C, 0.1 M Tris-buffer, pH 7.3, 20% loss of activity
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4C, in presence of dithiothreitol, stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by affinity chromatography
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by centrifugation, sonication and on Ni2+-NTA column, to near homogeneity
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by fast protein liquid chromatography on hydroxylapatite and on AH-Sepharose with covalently bound 4-hydroxy-a cyanocinnamic acid; hydroxylapatite column chromatography and 4-hydroxy-alpha-cyanocinnamic acid-AH-Sepharose column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene amplification in Escherichia coli
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gene DLD, expression of C-terminally GFP-tagged full-length enzyme under control of the CaMV35S promoter in Nicotiana benthamiana leaf epidermi. Overexpression of D-LDH ameliorates the growth of wild-type plants in the presence of D-lactate and methylglyoxal and reverses the increased sensitivity of dldh loss-of-function lines to these compounds
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gene DLD1, 6fold overexpression of DLDH under the strong constitutive promoter prAOX in Hansenula polymorpha strain DELTAcyb2 tr6, a strain with impairment in glucose repression of AOX promoter and devoid of catalase and L-lactate-cytochrome c oxidoreductase activities
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gene DLD1, recombinant expression in L-lactate-cytochrome c oxidoreductase CYB2 deficient Hansenula polymorpha strain NCYC495 leu1-1 is used for recombinant overexpression of the His6-tagged enzyme using the gene with a terminator region driven by the alcohol oxidase gene promoter. The mutant strain shows increased DlDH activity and abolished activity of flavocytochrome b2, EC 1.1.2.3
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in Saccharomyces cerevisiae, gene amplification in Escherichia coli
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into vector pET16 and expressed in Escherichia coli BLR DE3 pLysS
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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Pseudomonas stutzeri containing D-lactate dehydrogenase can act as a novel biocatalyst for pyruvate production from DL-lactate
additional information
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carbon paste electrode-based biosensor with immobilized D-LCR seems to be optimal for analysis, representing significant advantage due to simplification of the whole device, i.e., the sensor exerts lower limit of detection under supposed concentration of D-lactate in real samples, low material demands, simplicity of D-LCR biosensor and short total time of analysis of about 2 min, pointing at the sensor possibilities in commercial applications
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