Cloned (Comment) | Organism |
---|---|
cloning in Escherichia coli strain DH5alpha, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strains JM109(DE3) and BL21(DE3), optimization of the culture conditions for Escherichia coli JM109(DE3)/pET28-sCPCAcyA675G, overview | Pseudomonas sp. |
expressed in Escherichia coli BL21(DE3) cells | Pseudomonas sp. |
Protein Variants | Comment | Organism |
---|---|---|
A675G | the mutant exhibits 112.8% activity with cephalosporin C compared to the wild type enzyme | Pseudomonas sp. |
A675G | the mutant exhibits significantly enhanced specific enzymatic activity compared to the wild type enzyme (35% increase) | Pseudomonas sp. |
A675G | site-directed mutagenesis, the mutant shows 13% increased activity compared to the wild-type enzyme | Pseudomonas sp. |
A677A | site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
A677F | site-directed mutagenesis, the mutant shows 24.6% decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
H296A | site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
H309A | site-directed mutagenesis | Pseudomonas sp. |
H309L | site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme | Pseudomonas sp. |
H309S | site-directed mutagenesis, the mutant shows slightly decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
H309V | the mutant exhibits 88.9% activity with cephalosporin C compared to the wild type enzyme | Pseudomonas sp. |
H309V | site-directed mutagenesis, the mutant shows 11.1% decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
L666F | the mutant exhibits significantly reduced specific enzymatic activity (75.4%) with cephalosporin C compared to the wild type enzyme | Pseudomonas sp. |
L666F | site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
L677A | the mutant exhibits significantly reduced specific enzymatic activity compared to the wild type enzyme | Pseudomonas sp. |
M270F | site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme | Pseudomonas sp. |
additional information | three-dimensional structure model of the wild-type enzyme for semi-rational determination of mutation residues, overview | Pseudomonas sp. |
P295A | the mutant exhibits 20% activity with cephalosporin C compared to the wild type enzyme | Pseudomonas sp. |
P295A | site-directed mutagenesis, the mutant shows 80% decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
R263A | site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
R263L | site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme | Pseudomonas sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
37 | - |
cephalosporin C | mutant enzyme L677F, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication | Pseudomonas sp. | |
37 | - |
cephalosporin C | pH 8.5, 37°C, recombinant mutant L677F | Pseudomonas sp. | |
43 | - |
cephalosporin C | wild type enzyme, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication | Pseudomonas sp. | |
43 | - |
cephalosporin C | pH 8.5, 37°C, recombinant wild-type enzyme | Pseudomonas sp. | |
86 | - |
cephalosporin C | mutant enzyme P295A, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication | Pseudomonas sp. | |
86 | - |
cephalosporin C | pH 8.5, 37°C, recombinant mutant P295A | Pseudomonas sp. | |
105 | - |
cephalosporin C | mutant enzyme A675G, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication | Pseudomonas sp. | |
105 | - |
cephalosporin C | pH 8.5, 37°C, recombinant mutant A675G | Pseudomonas sp. | |
121 | - |
cephalosporin C | mutant enzyme H309V, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication | Pseudomonas sp. | |
121 | - |
cephalosporin C | pH 8.5, 37°C, recombinant mutant H309V | Pseudomonas sp. |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
28000 | - |
1 * 58000 + 1 * 28000, SDS-PAGE | Pseudomonas sp. |
58000 | - |
1 * 58000 + 1 * 28000, SDS-PAGE | Pseudomonas sp. |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas sp. | - |
- |
- |
Pseudomonas sp. | P15558 | - |
- |
Purification (Comment) | Organism |
---|---|
Ni2+-HiTrap chelating column chromatography and Cu2+-immobilized iminodiacetic acid affinity chromatography | Pseudomonas sp. |
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography | Pseudomonas sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
cephalosporin C + H2O | the recombinant enzyme exhibits 2.3times more cephalosporin C specific deacylation activity compared to glutaryl-7-aminocephalosporanic acid | Pseudomonas sp. | cephalosporamic acid + 2-amino-5-hydroxypentanoate | - |
? | |
cephalosporin C + H2O | - |
Pseudomonas sp. | (7R)-7-aminocephalosporanate + 2-aminoadipate | - |
? | |
glutaryl-7-aminocephalosporanic acid + H2O | - |
Pseudomonas sp. | 7-aminocephalosporanate + glutarate | - |
? |
Subunits | Comment | Organism |
---|---|---|
heterodimer | 1 * 58000 + 1 * 28000, SDS-PAGE | Pseudomonas sp. |
Synonyms | Comment | Organism |
---|---|---|
cephalosporin acylase | - |
Pseudomonas sp. |
cephalosporin acylase II | - |
Pseudomonas sp. |
cephalosporin C acylase | - |
Pseudomonas sp. |
CPC acylase | - |
Pseudomonas sp. |
CPCAcy | - |
Pseudomonas sp. |
sCPCAcy | - |
Pseudomonas sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Pseudomonas sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
assay at | Pseudomonas sp. |
General Information | Comment | Organism |
---|---|---|
additional information | three-dimensional structure model of wild-type enzyme, overview | Pseudomonas sp. |