Protein Variants | Comment | Organism |
---|---|---|
H57betaS/H70betaS | site-directed mutagenesis, the mutant enzyme shows increased activity on cephalosporin compared to the wild-type enzyme | Pseudomonas sp. N176 |
H57betaS/H70betaS/F72betaR | site-directed mutagenesis, the mutant enzyme shows increased activity on cephalosporin compared to the wild-type enzyme, this mutant is the most active acylase on glutaryl-7-aminocephalosporanic acid | Pseudomonas sp. N176 |
H57betaS/H70betaS/L154betaY | site-directed mutagenesis, the mutant enzyme shows increased activity on cephalosporin compared to the wild-type enzyme | Pseudomonas sp. N176 |
additional information | one-pot conversion of cephalosporin C by using an optimized two-enzyme process, producing 7-aminocephalosporanic acid (7-ACA) and involving D-amino acid oxidase (DAAO, EC 1.4.3.3) and glutaryl-7-aminocephalosporanic acid acylase (GA, EC 3.5.1.93). Stability of the employed biocatalysts when incubated at 25°C in 20 mM phosphate buffer, pH 8.0, overview. The two enzymes are used in one-pot and in a soluble form. By adding 4.5 kU/l of both RgDAAO and wild-type VAC, 50 mM cephalosporin C is fully converted in about 7 hours although only 30 mM 7-ACA is produced | Pseudomonas sp. N176 |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
glutaryl-7-aminocephalosporanic acid | substrate inhibition of wild-type enzyme and mutant H57betaS/H70betaS/F72betaR, but not of mutants H57betaS/H70betaS and H57betaS/H70betaS/L154betaY | Pseudomonas sp. N176 |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.5 | - |
glutaryl-7-aminocephalosporanic acid | pH 8.0, 25°C, recombinant wild-type enzyme | Pseudomonas sp. N176 | |
2 | - |
glutaryl-7-aminocephalosporanic acid | pH 8.0, 25°C, recombinant mutant H57betaS/H70betaS/F72betaR | Pseudomonas sp. N176 | |
2 | - |
glutaryl-7-aminocephalosporanic acid | pH 8.0, 25°C, recombinant mutant H57betaS/H70betaS/L154betaY | Pseudomonas sp. N176 | |
4.5 | - |
cephalosporin C | pH 8.0, 25°C, recombinant mutant H57betaS/H70betaS/L154betaY | Pseudomonas sp. N176 | |
6.9 | - |
glutaryl-7-aminocephalosporanic acid | pH 8.0, 25°C, recombinant mutant H57betaS/H70betaS | Pseudomonas sp. N176 | |
9.5 | - |
cephalosporin C | pH 8.0, 25°C, recombinant wild-type enzyme | Pseudomonas sp. N176 | |
12.2 | - |
cephalosporin C | pH 8.0, 25°C, recombinant mutant H57betaS/H70betaS | Pseudomonas sp. N176 | |
16.4 | - |
cephalosporin C | pH 8.0, 25°C, recombinant mutant H57betaS/H70betaS/F72betaR | Pseudomonas sp. N176 |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
cephalosporin C + H2O | Pseudomonas sp. N176 | - |
7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate | - |
? | |
glutaryl-7-aminocephalosporanic acid + H2O | Pseudomonas sp. N176 | - |
7-aminocephalosporanic acid + glutarate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas sp. N176 | A0A1D8GRD5 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
cephalosporin C + H2O | - |
Pseudomonas sp. N176 | 7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate | - |
? | |
glutaryl-7-aminocephalosporanic acid + H2O | - |
Pseudomonas sp. N176 | 7-aminocephalosporanic acid + glutarate | - |
? | |
glutaryl-7-aminocephalosporanic acid + H2O | GL-7-ACA | Pseudomonas sp. N176 | 7-aminocephalosporanic acid + glutarate | - |
? | |
additional information | one-pot conversion of cephalosporin C to 7-aminocephalosporanic acid (7-ACA) using an optimized two-enzyme process, involving D-amino acid oxidase (DAAO, EC 1.4.3.3) from Rhodotorula gracilis and glutaryl-7-aminocephalosporanic acid acylase (GA, EC 3.5.1.93) from Pseudomonas sp. N176. The flavoenzyme DAAO oxidizes the D-2-aminoadipyl moiety of CephC to give 2-oxoadipyl-7-ACA, which is converted to glutaryl-7-ACA (Gl-7-ACA) nonenzymatically. Concerning the simultaneous action of DAAO and GA for 7-ACA production in a single reactor, the main problems are the presence of H2O2 during the reaction process (produced by the DAAO reaction and required to push decarboxylation of oxo-7-ACA into Gl-7-ACA), which can inactivate the enzymes being employed, especially DAAO, and the substrate/product inhibition effects observed with GA | Pseudomonas sp. N176 | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
cephalosporin C acylase | - |
Pseudomonas sp. N176 |
CephC acylase | - |
Pseudomonas sp. N176 |
glutaryl-7-aminocephalosporanic acid acylase | - |
Pseudomonas sp. N176 |
VAC | - |
Pseudomonas sp. N176 |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Pseudomonas sp. N176 |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Pseudomonas sp. N176 |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
6.3 | - |
glutaryl-7-aminocephalosporanic acid | pH 8.0, 25°C, recombinant mutant H57betaS/H70betaS/F72betaR | Pseudomonas sp. N176 | |
21 | - |
glutaryl-7-aminocephalosporanic acid | pH 8.0, 25°C, recombinant wild-type enzyme | Pseudomonas sp. N176 |