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Literature summary extracted from

  • Wang, Y.; Yu, H.; Song, W.; An, M.; Zhang, J.; Luo, H.; Shen, Z.
    Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel (2012), J. Biosci. Bioeng., 113, 36-41.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.93 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.
3.5.1.93 expressed in Escherichia coli BL21(DE3) cells Pseudomonas sp.

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.93 A675G the mutant exhibits 112.8% activity with cephalosporin C compared to the wild type enzyme Pseudomonas sp.
3.5.1.93 A675G the mutant exhibits significantly enhanced specific enzymatic activity compared to the wild type enzyme (35% increase) Pseudomonas sp.
3.5.1.93 A675G site-directed mutagenesis, the mutant shows 13% increased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 A677A site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 A677F site-directed mutagenesis, the mutant shows 24.6% decreased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 H296A site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 H309A site-directed mutagenesis Pseudomonas sp.
3.5.1.93 H309L site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 H309S site-directed mutagenesis, the mutant shows slightly decreased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 H309V the mutant exhibits 88.9% activity with cephalosporin C compared to the wild type enzyme Pseudomonas sp.
3.5.1.93 H309V site-directed mutagenesis, the mutant shows 11.1% decreased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 L666F the mutant exhibits significantly reduced specific enzymatic activity (75.4%) with cephalosporin C compared to the wild type enzyme Pseudomonas sp.
3.5.1.93 L666F site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 L677A the mutant exhibits significantly reduced specific enzymatic activity compared to the wild type enzyme Pseudomonas sp.
3.5.1.93 M270F site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 additional information three-dimensional structure model of the wild-type enzyme for semi-rational determination of mutation residues, overview Pseudomonas sp.
3.5.1.93 P295A the mutant exhibits 20% activity with cephalosporin C compared to the wild type enzyme Pseudomonas sp.
3.5.1.93 P295A site-directed mutagenesis, the mutant shows 80% decreased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 R263A site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme Pseudomonas sp.
3.5.1.93 R263L site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme Pseudomonas sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.93 37
-
cephalosporin C mutant enzyme L677F, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication Pseudomonas sp.
3.5.1.93 37
-
cephalosporin C pH 8.5, 37°C, recombinant mutant L677F Pseudomonas sp.
3.5.1.93 43
-
cephalosporin C wild type enzyme, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication Pseudomonas sp.
3.5.1.93 43
-
cephalosporin C pH 8.5, 37°C, recombinant wild-type enzyme Pseudomonas sp.
3.5.1.93 86
-
cephalosporin C mutant enzyme P295A, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication Pseudomonas sp.
3.5.1.93 86
-
cephalosporin C pH 8.5, 37°C, recombinant mutant P295A Pseudomonas sp.
3.5.1.93 105
-
cephalosporin C mutant enzyme A675G, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication Pseudomonas sp.
3.5.1.93 105
-
cephalosporin C pH 8.5, 37°C, recombinant mutant A675G Pseudomonas sp.
3.5.1.93 121
-
cephalosporin C mutant enzyme H309V, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication Pseudomonas sp.
3.5.1.93 121
-
cephalosporin C pH 8.5, 37°C, recombinant mutant H309V Pseudomonas sp.

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.5.1.93 28000
-
1 * 58000 + 1 * 28000, SDS-PAGE Pseudomonas sp.
3.5.1.93 58000
-
1 * 58000 + 1 * 28000, SDS-PAGE Pseudomonas sp.

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.93 Pseudomonas sp.
-
-
-
3.5.1.93 Pseudomonas sp. P15558
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.93 Ni2+-HiTrap chelating column chromatography and Cu2+-immobilized iminodiacetic acid affinity chromatography Pseudomonas sp.
3.5.1.93 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography Pseudomonas sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.93 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
-
?
3.5.1.93 cephalosporin C + H2O
-
Pseudomonas sp. (7R)-7-aminocephalosporanate + 2-aminoadipate
-
?
3.5.1.93 glutaryl-7-aminocephalosporanic acid + H2O
-
Pseudomonas sp. 7-aminocephalosporanate + glutarate
-
?

Subunits

EC Number Subunits Comment Organism
3.5.1.93 heterodimer 1 * 58000 + 1 * 28000, SDS-PAGE Pseudomonas sp.

Synonyms

EC Number Synonyms Comment Organism
3.5.1.93 cephalosporin acylase
-
Pseudomonas sp.
3.5.1.93 cephalosporin acylase II
-
Pseudomonas sp.
3.5.1.93 cephalosporin C acylase
-
Pseudomonas sp.
3.5.1.93 CPC acylase
-
Pseudomonas sp.
3.5.1.93 CPCAcy
-
Pseudomonas sp.
3.5.1.93 sCPCAcy
-
Pseudomonas sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.93 37
-
assay at Pseudomonas sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.93 8.5
-
assay at Pseudomonas sp.

General Information

EC Number General Information Comment Organism
3.5.1.93 additional information three-dimensional structure model of wild-type enzyme, overview Pseudomonas sp.