Application | Comment | Organism |
---|---|---|
synthesis | faster substrate turnover, reduced glutaryl-7-aminocephalosporanic acid inhibition and improved thermostability of the F54Y mutant compared to the wild-type enzyme render it a useful candidate in industrial production of semi-synthetic cephems | Trigonopsis variabilis |
Cloned (Comment) | Organism |
---|---|
expression of wild-type enzyme and mutants F54Y in Escherichia coli BL21(DE3) | Trigonopsis variabilis |
Protein Variants | Comment | Organism |
---|---|---|
F54A | site-directed mutagenesis | Trigonopsis variabilis |
F54S | site-directed mutagenesis | Trigonopsis variabilis |
F54Y | site-directed mutagenesis, the mutant shows 6fold improvement in kcat,app and about 2.5fold increase in Ki of glutaryl-7-aminocephalosporanic acid, the substitution improves the catalytic activity and thermostability of mutant DAAO compared to the wild-type enzyme. Heat treatment at 55° for 60 min does not decrease the activity of F54Y. The Tyr substitution might initiate hydrogen bond formation with the amino group of CPC and facilitate deamination | Trigonopsis variabilis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
glutaryl-7-aminocephalosporanic acid | - |
Trigonopsis variabilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.9 | - |
cephalosporin C | recombinant mutant F54S, pH 7.5, 22°C | Trigonopsis variabilis | |
1.2 | - |
cephalosporin C | recombinant mutant F54A, pH 7.5, 22°C | Trigonopsis variabilis | |
1.6 | - |
cephalosporin C | recombinant wild-type enzyme, pH 7.5, 22°C | Trigonopsis variabilis | |
4.8 | - |
cephalosporin C | recombinant mutant F54Y, pH 7.5, 22°C | Trigonopsis variabilis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Trigonopsis variabilis | Q6R4Q9 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type enzyme and mutants from Escherichia coli BL21(DE3) | Trigonopsis variabilis |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
8.5 | - |
purified recombinant wild-type enzyme, substrate cephalosporin C, pH 7.5, 22°C | Trigonopsis variabilis |
22.3 | - |
purified recombinant mutant F54S, substrate cephalosporin C, pH 7.5, 22°C | Trigonopsis variabilis |
26.4 | - |
purified recombinant mutant F54A, substrate cephalosporin C, pH 7.5, 22°C | Trigonopsis variabilis |
30.6 | - |
purified recombinant mutant F54Y, substrate cephalosporin C, pH 7.5, 22°C | Trigonopsis variabilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
cephalosporin C + H2O + O2 | conversion of cephalosporin C to 7-aminocephalosporanic acid and spontaneous decarboxylation of oxoadipyl-7-amino cephalosporanic acid is promoted by the H2O2 formed in the oxidase reaction of TvDAO | Trigonopsis variabilis | 7-aminocephalosporanic acid + ? + H2O2 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
DAAO | - |
Trigonopsis variabilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at | Trigonopsis variabilis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | 65 | - |
Trigonopsis variabilis |
55 | - |
TvDAAO is active after heat treatment for 30 min | Trigonopsis variabilis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
6.167 | - |
cephalosporin C | recombinant wild-type enzyme, pH 7.5, 22°C | Trigonopsis variabilis | |
16.67 | - |
cephalosporin C | recombinant mutant F54S, pH 7.5, 22°C | Trigonopsis variabilis | |
18.33 | - |
cephalosporin C | recombinant mutant F54A, pH 7.5, 22°C | Trigonopsis variabilis | |
36.67 | - |
cephalosporin C | recombinant mutant F54Y, pH 7.5, 22°C | Trigonopsis variabilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Trigonopsis variabilis |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
6 | 10 | - |
Trigonopsis variabilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Trigonopsis variabilis |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.7 | - |
glutaryl-7-aminocephalosporanic acid | recombinant mutant F54A, pH 7.5, 22°C | Trigonopsis variabilis | |
1.1 | - |
glutaryl-7-aminocephalosporanic acid | recombinant mutant F54S, pH 7.5, 22°C | Trigonopsis variabilis | |
1.5 | - |
glutaryl-7-aminocephalosporanic acid | recombinant wild-type enzyme, pH 7.5, 22°C | Trigonopsis variabilis | |
3.8 | - |
glutaryl-7-aminocephalosporanic acid | recombinant mutant F54Y, pH 7.5, 22°C | Trigonopsis variabilis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
3.83 | - |
cephalosporin C | recombinant wild-type enzyme, pH 7.5, 22°C | Trigonopsis variabilis | |
7.83 | - |
cephalosporin C | recombinant mutant F54Y, pH 7.5, 22°C | Trigonopsis variabilis | |
15.67 | - |
cephalosporin C | recombinant mutant F54A, pH 7.5, 22°C | Trigonopsis variabilis | |
18.33 | - |
cephalosporin C | recombinant mutant F54S, pH 7.5, 22°C | Trigonopsis variabilis |