Application | Comment | Organism |
---|---|---|
synthesis | enzymatic production of 7-aminocephalosporanate. Construction of site-directed mutants with enhanced activity and stability | Pseudomonas sp. |
Protein Variants | Comment | Organism |
---|---|---|
D286betaA | beta-subunit mutant enzyme, KM-value is 1.1fold lower than the wild-type value, turnover-number is 1.03fold higher than the wild-type value. Half-life at 37°C is 53.9 h compared to 68.1 h for wild-type enzyme. Optimal pH is pH 9.0 compared to pH 7.0 for wild-type enzyme | Pseudomonas sp. |
K198betaA | beta-subunit mutant enzyme, KM-value is 1.04fold higher than the wild-type value, turnover-number is 1.1fold lower than the wild-type value. Half-life at 37°C is 107.5 h compared to 68.1 h for wild-type enzyme. Optimal pH is pH 8.0 compared to pH 7.0 for wild-type enzyme. Mutant enzyme shows higher stability at alkaline pH than wild-type enzyme | Pseudomonas sp. |
Q50betaN/K198betaA | beta-subunit mutant enzyme, KM-value is 2.14fold lower than the wild-type value, turnover-number is 1.45fold higher than the wild-type value, immobilized mutant enzyme shows 34.2% increase in specific activity compared to immobilized wild-type enzyme. Ability of the mutant enzyme to hydrolyze adipoyl 6-aminopenicillinic acid is improved, compared to activity of wild-type enzyme | Pseudomonas sp. |
Q50N | beta-subunit mutant enzyme, KM-value is 2.05fold lower than the wild-type value, turnover-number is 1.45fold higher than the wild-type value. Ability of the mutant enzyme to hydrolyze adipoyl 6-aminopenicillinic acid is improved, compared to activity of wild-type enzyme | Pseudomonas sp. |
R121A | beta-subunit mutant enzyme, KM-value is identical to the wild-type value, turnover-number is nearly identical to wild-type value. Half-life at 37°C is 88.3 h compared to 68.1 h for wild-type enzyme. Optimal pH is pH 6.0 compared to pH 7.0 for wild-type enzyme | Pseudomonas sp. |
Y151F | alpha-subunit mutant enzyme, KM-value is 1.9fold lower than the wild-type value, turnover-number is 1.17fold higher than the wild-type value. Ability of the mutant enzyme to hydrolyze adipoyl 6-aminopenicillinic acid is improved, compared to activity of wild-type enzyme | Pseudomonas sp. |
Y151F/Q50N | mutant enzyme with mutation Y151F in alpha-subunit and mutation Q50N in beta-subunit, KM-value is 1.2fold lower than the wild-type value, turnover-number is 1.09fold higher than the wild-type value | Pseudomonas sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
11.7 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme K198A | Pseudomonas sp. | |
12.2 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme R121A | Pseudomonas sp. | |
12.3 | - |
glutaryl-7-aminocephalosporanic acid | wild-type enzyme | Pseudomonas sp. | |
12.7 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme D286A | Pseudomonas sp. | |
13.4 | - |
glutaryl-7-aminocephalosporanic acid | mutant enzyme with mutation Y151F in alpha-subunit and mutation Q50N in beta-subunit | Pseudomonas sp. | |
14.4 | - |
glutaryl-7-aminocephalosporanic acid | alpha-subunit mutant enzyme Y151F | Pseudomonas sp. | |
17.8 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme Q50N/K198A | Pseudomonas sp. | |
17.9 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme Q50N | Pseudomonas sp. |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas sp. | P07662 | 130, recombinant enzyme | - |
Purification (Comment) | Organism |
---|---|
recombinant enzyme | Pseudomonas sp. |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
11.3 | - |
beta-subunit mutant enzyme K198A | Pseudomonas sp. |
11.6 | - |
beta-subunit mutant enzyme R121A | Pseudomonas sp. |
11.8 | - |
wild-type enzyme | Pseudomonas sp. |
12 | - |
beta-subunit mutant enzyme D286A | Pseudomonas sp. |
12.3 | - |
mutant enzyme with mutation Y151F in alpha-subunit and mutation Q50N in beta-subunit | Pseudomonas sp. |
14.4 | - |
alpha-subunit mutant enzyme Y151F | Pseudomonas sp. |
17.4 | - |
beta-subunit mutant enzyme Q50N/K198A | Pseudomonas sp. |
17.5 | - |
beta-subunit mutant enzyme Q50N | Pseudomonas sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
adipyl-aminopenicillanic acid + H2O | wild-type enzyme shows 70.6% of the activity with glutaryl-7-aminocephalosporanic acid | Pseudomonas sp. | adipate + aminopenicillanic acid | - |
? | |
cephalosporin C + H2O | wild-type enzyme shows 2.3% of the activity with glutaryl-7-aminocephalosporanic acid | Pseudomonas sp. | cephalosporanic acid + 2-amino-5-hydroxypentanoate | - |
? | |
glutaryl-7-aminocephalosporanic acid + H2O | - |
Pseudomonas sp. | 7-aminocephalosporanate + glutarate | - |
? | |
glutaryl-aminopenicillanic acid + H2O | wild-type enzyme shows 90.6% of the activity with glutaryl-7-aminocephalosporanic acid | Pseudomonas sp. | glutarate + aminopenicillanic acid | - |
? |
Synonyms | Comment | Organism |
---|---|---|
GL-7-ACA acylase | - |
Pseudomonas sp. |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
half-life: 68.1 h (wild-type enzyme), 88.3 h (mutant enzyme R121betaA), 107.5 h (mutant enzyme K198betaA), 53.9 h (mutant enzyme D286betA) | Pseudomonas sp. |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.21 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme Q50N/K198A | Pseudomonas sp. | |
0.22 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme Q50N | Pseudomonas sp. | |
0.24 | - |
glutaryl-7-aminocephalosporanic acid | alpha-subunit mutant enzyme Y151F | Pseudomonas sp. | |
0.38 | - |
glutaryl-7-aminocephalosporanic acid | mutant enzyme with mutation Y151F in the alpha-subunit and Q50N in the beta-subunit | Pseudomonas sp. | |
0.41 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme D286A | Pseudomonas sp. | |
0.45 | - |
glutaryl-7-aminocephalosporanic acid | wild-type enzyme | Pseudomonas sp. | |
0.45 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme R121A | Pseudomonas sp. | |
0.47 | - |
glutaryl-7-aminocephalosporanic acid | beta-subunit mutant enzyme K198betaA | Pseudomonas sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
beta-subunit mutant enzyme R121A | Pseudomonas sp. |
7 | - |
wild-type enzyme | Pseudomonas sp. |
8 | - |
beta-subunit mutant enzyme K198A | Pseudomonas sp. |
9 | - |
beta-subunit mutant enzyme D286A | Pseudomonas sp. |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
7 | - |
37°C, 125 min, 10% loss of wild-type activity, 7% loss of activity of mutant enzyme K198betaA | Pseudomonas sp. |
8 | - |
37°C, 125 min, 13% loss of wild-type activity, 7% loss of activity of mutant enzyme K198betaA | Pseudomonas sp. |
9 | - |
37°C, 125 min, 20% loss of wild-type activity, 3% loss of activity of mutant enzyme K198betaA | Pseudomonas sp. |
10 | - |
37°C, 125 min, 35% loss of wild-type activity, 10% loss of activity of mutant enzyme K198betaA | Pseudomonas sp. |