Abstract: Cephalosporin acylase can catalyze cephalosporin C (CPC) to produce 7-amino cephalosporanic acid (7-ACA) which is the important pharmaceutical intermediate. However, the cephalosporin C acylase has low catalytic activity for CPC and is far from the requirements of industrial production. The gene of cephalosporin acylase (CPCase) from Pseudomonas sp. SE83 was synthesized, optimized and cloned into the vector pET28a to achieve high expression of CPCase, named WT. By comparing WT with the sequences of cephalosporin acylase from Pseudomonas SY-77, Pseudomonas N176 and CAD, 25 sites were selected near the conserved amino acids and 25 mutants were obtained by the site-directed mutagenesis. The results from enzyme activity showed that the catalytic activity of the mutant F311 was increased by 50% and others had decreased activity to CPC. The specific activity of F311 was 4.302 U/mg and the kcat/Km was 1.7 times to that of the WT. The structural analysis revealed that the F311A mutant made the binding pocket larger, the binding to the substrate CPC was stronger, and the catalytic efficiency was improved.

Key words: cephalosporin acylase, 7-aminocephalosporanic acid, site-directed mutagenesis, cephalosporin C