Cloned (Comment) | Organism |
---|---|
production of a recombinant plasmid, pT7-GO by insertion of the DNA encoding for glycine oxidase into the multiple cloning site of the expression vector pT7.7. The pT7-glycine oxidase encodes a fully active fusion protein with six additional residues at the N-terminus of glycine oxidase. In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric glycine oxidase is expressed up to 1.14 U per mg of cell. An N-terminally His-tagged glycine oxidase is also successfully expressed in Escherichia coli as a soluble protein and a fully active holoenzyme | Bacillus subtilis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
46600 | - |
4 * 46600, recombinant glycine oxidase, SDS-PAGE | Bacillus subtilis |
49400 | - |
4 * 49400, recombinant His-tagged chimeric glycine oxidase | Bacillus subtilis |
166400 | - |
recombinant His-tagged chimeric glycine oxidase, gel filtration | Bacillus subtilis |
187600 | - |
recombinant glycine oxidase, gel filtration | Bacillus subtilis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | - |
- |
- |
Purification (Comment) | Organism |
---|---|
wild-type enzyme and His-tagged chimeric glycine oxidase | Bacillus subtilis |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.568 | - |
wild-type enzyme | Bacillus subtilis |
1.06 | - |
His-tagged chimeric glycine oxidase | Bacillus subtilis |
Storage Stability | Organism |
---|---|
-80°C, pH 7.5, both glycine oxidase and His-tagged glycine oxidase are stable for months | Bacillus subtilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-2-aminobutyrate + H2O + O2 | as active as sarcosine | Bacillus subtilis | 2-oxobutyrate + H2O2 | - |
? | |
D-Ala + H2O + O2 | about 115% of the activity with sarcosine | Bacillus subtilis | pyruvate + NH3 + H2O2 | - |
? | |
D-Arg + H2O + O2 | about 35% of the activity with sarcosine | Bacillus subtilis | ? + H2O2 | - |
? | |
D-His + H2O + O2 | about 25% of the activity with sarcosine | Bacillus subtilis | ? + H2O2 | - |
? | |
D-Ile + H2O + O2 | about 30% of the activity with sarcosine | Bacillus subtilis | 3-methyl-2-oxopentanoate + H2O2 | - |
? | |
D-Leu + H2O + O2 | about 35% of the activity with sarcosine | Bacillus subtilis | 4-methyl-2-oxopentanoate + H2O2 | - |
? | |
D-pipecolate + H2O + O2 | about 85% of the activity with sarcosine | Bacillus subtilis | ? + H2O2 | - |
? | |
D-Pro + H2O + O2 | about 120% of the activity with sarcosine | Bacillus subtilis | ? + H2O2 | - |
? | |
D-Val + H2O + O2 | about 35% of the activity with sarcosine | Bacillus subtilis | 3-methyl-2-oxobutanoate + H2O2 | - |
? | |
glycine + H2O + O2 | as active as sarcosine | Bacillus subtilis | glyoxylate + NH3 + H2O2 | - |
? | |
additional information | the enzyme is strictly stereospecific as it only catalyzes the oxidation of the D-isomer | Bacillus subtilis | ? | - |
? | |
N-ethylglycine + H2O + O2 | about 65% of the activity with sarcosine | Bacillus subtilis | glyoxylate + ethylamine + H2O2 | - |
? | |
N-methyl-D-Ala + H2O + O2 | about 110% of the activity with sarcosine | Bacillus subtilis | pyruvate + methylamine + H2O2 | - |
? | |
sarcosine + H2O + O2 | - |
Bacillus subtilis | glyoxylate + methylamine + H2O2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
tetramer | 4 * 46600, recombinant glycine oxidase, SDS-PAGE | Bacillus subtilis |
tetramer | 4 * 49400, recombinant His-tagged chimeric glycine oxidase | Bacillus subtilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
increase in activity with no evidence for any plateau or decrease up to 60°C, reaction with sarcosine | Bacillus subtilis |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
15 | 60 | increase in activity with no evidence for any plateau or decrease up to 60°C, reaction with sarcosine | Bacillus subtilis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
35 | - |
pH 7.0, 30 min, stable up to | Bacillus subtilis |
45 | - |
pH 7.0, 30 min, about 30% loss of activity | Bacillus subtilis |
46 | - |
Tm-value, after 30 min | Bacillus subtilis |
50 | - |
pH 7.0, 30 min, about 75% loss of activity | Bacillus subtilis |
60 | - |
pH 7.0, 30 min, complete loss of activity | Bacillus subtilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
10 | - |
reaction with sarcosine | Bacillus subtilis |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
8 | 11 | pH 8.0: about 55% of maximal activity, pH 11.0: about 55% of maximal activity, reaction with sarcosine | Bacillus subtilis |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
7 | 8.5 | 25°C, 300 min, maximal stability | Bacillus subtilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | tight binding of coenzyme to the protein moiety, addition of exogenous FAD or FMN to the assay mixture does not increase enzyme activity | Bacillus subtilis |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Bacillus subtilis | theoretical value, His-tagged chimeric enzyme | - |
6 |
Bacillus subtilis | theoretical value, wild-type enzyme | - |
6 |