Enhancement of glutaryl-7-aminocephalosporanic acid acylase activity of gamma-glutamyltranspeptidase of Bacillus subtilis.
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 gamma-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type gamma-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 kcat 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 gamma-glutamyltranspeptidase of B. subtilis.
Suzuki
Hideyuki
H
Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan. hideyuki@kit.ac.jp
Yamada
Chiaki
C
Kijima
Kyoko
K
Ishihara
Sayaka
S
Wada
Kei
K
Fukuyama
Keiichi
K
Kumagai
Hidehiko
H
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007218:Industrial Microbiology / Q000379:methods*; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011487:Protein Conformation; D016415:Sequence Alignment; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002511:Cephalosporins; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; D005723:gamma-Glutamyltransferase; D000581:Amidohydrolases