Enhancement of glutaryl-7-aminocephalosporanic acid acylase activity of γ-glutamyltranspeptidase of Bacillus subtilis

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 wild-type γ-glutamyltranspeptidase of B. subtilis. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.