Cloned (Comment) | Organism |
---|---|
expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Bifidobacterium adolescentis |
Protein Variants | Comment | Organism |
---|---|---|
A498H | site-directed mutagenesis, the mutant shows reduced activity and thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
D445P | site-directed mutagenesis, the mutant shows slightly decreased activity and increased thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
D445P/D446G | site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
D445P/D446P | site-directed mutagenesis, the mutant shows reduced activity and unaltered thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
D445P/D446T | site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
D446G | site-directed mutagenesis, the mutant shows similar activity and thermostability as the wild-type enzyme | Bifidobacterium adolescentis |
D446P | site-directed mutagenesis, the mutant shows slightly increased activity and the same thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
D446T | site-directed mutagenesis, the mutant shows reduced activity and slightly reduced thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
L306H | site-directed mutagenesis, the mutant shows similar activity and thermostability as the wild-type enzyme | Bifidobacterium adolescentis |
additional information | increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis, substitution of the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, and substitutions to promote electrostatic interactions | Bifidobacterium adolescentis |
N325D/V473H | site-directed mutagenesis, the mutant shows reduced activity and thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
N414D | site-directed mutagenesis, the mutant shows reduced activity and thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
Q331E | site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
Q460E/E485H | site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
R393N | site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme | Bifidobacterium adolescentis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
6.5 | - |
sucrose | pH 7.0, 60°C, recombinant wild-type enzyme | Bifidobacterium adolescentis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
sucrose + phosphate | Bifidobacterium adolescentis | - |
D-fructose + alpha-D-glucose 1-phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bifidobacterium adolescentis | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Bifidobacterium adolescentis |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
213 | - |
purified recombinant wild-type enzyme, pH 7.0, 37°C | Bifidobacterium adolescentis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | assay method with production of alpha-D-glucose-1-phosphate is coupled to the reduction of NAD+ in the presence of phosphoglucomutase and glucose-6-phosphate dehydrogenase | Bifidobacterium adolescentis | ? | - |
? | |
sucrose + phosphate | - |
Bifidobacterium adolescentis | D-fructose + alpha-D-glucose 1-phosphate | - |
? |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Bifidobacterium adolescentis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
purified wild-type enzyme, pH 7.0, 24 h, 30% activity remaining, purified mutants show 15.3-42.9% remaining activity, overview | Bifidobacterium adolescentis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
201 | - |
sucrose | pH 7.0, 60°C, recombinant wild-type enzyme | Bifidobacterium adolescentis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Bifidobacterium adolescentis |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to glycoside hydrolase family GH 13 and follows the typical doubledisplacement mechanism of retaining glycosidases | Bifidobacterium adolescentis |