| Institution: | 1. Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan;2. Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan;3. Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan;4. Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan;5. Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa;6. Japan |
| Abstract: | 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 kcat value of 0.0485 s-1. Its activity has been enhanced by site directed and random mutagenesis. The kcat/Km 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 γ-glutamyltranspeptidase of B. subtilis. |
