Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

Appl Environ Microbiol. 2008 Jun;74(11):3400-9. doi: 10.1128/AEM.02693-07. Epub 2008 Apr 4.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Cephalosporins / metabolism*
  • Escherichia coli K12 / enzymology*
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / isolation & purification
  • Escherichia coli Proteins / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation, Missense
  • Sequence Alignment
  • gamma-Glutamyltransferase / genetics*
  • gamma-Glutamyltransferase / isolation & purification
  • gamma-Glutamyltransferase / metabolism*

Substances

  • Cephalosporins
  • Escherichia coli Proteins
  • glutaryl-7-aminocephalosporanic acid
  • gamma-Glutamyltransferase