Document Detail

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase.
MedLine Citation:
PMID:  18390671     Owner:  NLM     Status:  MEDLINE    
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.
Chiaki Yamada; Kyoko Kijima; Sayaka Ishihara; Chinatsu Miwa; Kei Wada; Toshihiro Okada; Keiichi Fukuyama; Hidehiko Kumagai; Hideyuki Suzuki
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-04-04
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  74     ISSN:  1098-5336     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2008 Jun 
Date Detail:
Created Date:  2008-05-28     Completed Date:  2008-06-26     Revised Date:  2013-06-05    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3400-9     Citation Subset:  IM    
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
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MeSH Terms
Amino Acid Sequence
Amino Acid Substitution / genetics
Cephalosporins / metabolism*
Escherichia coli K12 / enzymology*
Escherichia coli Proteins / genetics*,  isolation & purification,  metabolism*
Models, Molecular
Molecular Sequence Data
Mutation, Missense
Sequence Alignment
gamma-Glutamyltransferase / genetics*,  isolation & purification,  metabolism*
Reg. No./Substance:
0/Cephalosporins; 0/Escherichia coli Proteins; 27920-90-7/glutaryl-7-aminocephalosporanic acid; EC
Erratum In:
Appl Environ Microbiol. 2008 Aug;74(15):4983

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