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

Semisynthetic cephalosporins, the best-selling antibiotics worldwide, are derived from 7-amino-cephalosporanic acid (7-ACA) Currently, in the pharmaceutical Industrie, 7-ACA is mainly produced from cephalosporin C by sequential application oft-amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme gamma-glutamyltranspeptidase from Bacillus subtilts for 7-ACA production, since the wild-type gamma-glutamyltranspeptidase of B. sub-tilis has inherent glutaryl-7-aminocephalosporanic acid acylase activity with a k(cat) value of 0 0485 s(-1) Its activity has been enhanced by site directed and random mutagenesis The k(cat)/K(m) value was increased to 3 41 s(-1) mM(-1) for a E423Y/E442Q/D445N mutant enzyme and the k(cat) 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 gamma-glutamyltranspeptidase of B subtilis