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Literature summary for 3.5.1.93 extracted from

  • Conti, G.; Pollegioni, L.; Rosini, E.
    One-pot conversion of cephalosporin C by using an optimized two-enzyme process (2015), Catal. Sci. Technol., 5, 1854-1863 .
No PubMed abstract available

Protein Variants

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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Pseudomonas sp. N176 A0A1D8GRD5
-
-

Substrates and Products (Substrate)

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

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 [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Pseudomonas sp. N176

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Pseudomonas sp. N176

Ki Value [mM]

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