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Applied and Environmental Microbiology, June 2008, p. 3400-3409, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.02693-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Improvement of the Glutaryl-7-Aminocephalosporanic Acid Acylase Activity of a Bacterial {gamma}-Glutamyltranspeptidase{triangledown}

Chiaki Yamada,1 Kyoko Kijima,1 Sayaka Ishihara,1 Chinatsu Miwa,1 Kei Wada,2 Toshihiro Okada,2 Keiichi Fukuyama,2 Hidehiko Kumagai,3 and Hideyuki Suzuki4*

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan,1 Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan,2 Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-cho, Ishikawa 921-8836, Japan,3 Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan4

Received 29 November 2007/ Accepted 29 March 2008

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 kcat/Km 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.

* Corresponding author. Mailing address: Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. Phone: 81-75-724-7763. Fax: 81-75-724-7766. E-mail: hideyuki{at}kit.ac.jp

{triangledown} Published ahead of print on 4 April 2008.

Applied and Environmental Microbiology, June 2008, p. 3400-3409, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.02693-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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