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Enhancement of glutaryl‐7‐aminocephalosporanic acid acylase activity of γ‐glutamyltranspeptidase of Bacillus subtilis
Author(s) -
Suzuki Hideyuki,
Yamada Chiaki,
Kijima Kyoko,
Ishihara Sayaka,
Wada Kei,
Fukuyama Keiichi,
Kumagai Hidehiko
Publication year - 2010
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201000015
Subject(s) - bacillus subtilis , cephalosporin c , mutant , mutagenesis , chemistry , d amino acid oxidase , directed evolution , enzyme , cephalosporin , cephalosporin antibiotic , biochemistry , site directed mutagenesis , oxidase test , wild type , biology , antibiotics , bacteria , genetics , gene
Semisynthetic cephalosporins, the best‐selling antibiotics worldwide, are derived from 7‐aminocephalosporanic acid (7‐ACA). Currently, in the pharmaceutical industrie, 7‐ACA is mainly produced from cephalosporin C by sequential application of D ‐amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme γ‐glutamyltranspeptidase from Bacillus subtilis for 7‐ACA production, since the wild‐type γ‐glutamyltranspeptidase of B. subtilis has inherent glutaryl‐7‐aminocephalosporanic acid acylase activity with a k cat value of 0.0485 s ‐1 . Its activity has been enhanced by site directed and random mutagenesis. The k cat / K m value was increased to 3.41 s ‐1 mM ‐1 for a E423Y/E442Q/D445N mutant enzyme and the k cat value was increased to 0.508 s ‐1 for a D445G mutant enzyme. Consequently, the catalytic efficiency and the turnover rate were improved up to about 1000‐fold and 10‐fold, compared with the wildtype γ‐glutamyltranspeptidase of B. subtilis .