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Literature summary for 3.5.1.93 extracted from

  • Zhang, W.; Liu, Y.; Zheng, H.; Yang, S.; Jiang, W.
    Improving the activity and stability of GL-7-ACA acylase CA130 by site-directed mutagenesis (2005), Appl. Environ. Microbiol., 71, 5290-5296.
    View publication on PubMedView publication on EuropePMC

Application

Application Comment Organism
synthesis enzymatic production of 7-aminocephalosporanate. Construction of site-directed mutants with enhanced activity and stability Pseudomonas sp.

Protein Variants

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 [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

Organism UniProt Comment Textmining
Pseudomonas sp. P07662 130, recombinant enzyme
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme Pseudomonas sp.

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

Synonyms Comment Organism
GL-7-ACA acylase
-
Pseudomonas sp.

Temperature Stability [°C]

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 [1/s]

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

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 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.