Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase.
7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.
Yamada
Chiaki
C
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
Kijima
Kyoko
K
Ishihara
Sayaka
S
Miwa
Chinatsu
C
Wada
Kei
K
Okada
Toshihiro
T
Fukuyama
Keiichi
K
Kumagai
Hidehiko
H
Suzuki
Hideyuki
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002511:Cephalosporins / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D016415:Sequence Alignment; D005723:gamma-Glutamyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D002511:Cephalosporins; D029968:Escherichia coli Proteins; C081063:glutaryl-7-aminocephalosporanic acid; D005723:gamma-Glutamyltransferase