Site-Directed Mutagenesis of Cephalosporin C Acylase and Enzymatic Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid

Objective: A cephalosporin C acylase catalyzes hydrolysis of cephalosporin C to 7-aminocephalosporanic acid directly. This work was considered helpful for the further study of the cephalosporin C acylase and also useful for the strain improvement. Methods: Its mutant (G139αS/F58βN/I75βT/I176βV/S471βC) named A12 was cloned into pET28a (+) vector and expressed in E.coli BL21 (DE3). The three dimentional structure of A12 was constructed by the homology modeling and its’ catalytic sites was analyzed by the DOCK software. Results: The mutant A12 was expressed in E.coli BL21 (DE3) with the molecular weight 87kDa containing two subunits of 58kDa α-subunit and 25kDa β-subunit. The activity of A12 was 291 U/L which was lower than that of AcyII (322 U/L) because of the low expression level. The specific activity of A12 was 6.011 U/mg which was higher than that of the AcyII (2.868 U/ mg). Catalytic analysis suggested that A12 had the improved catalytic efficiency (kcat/Km) to convert cephalosporin C to 7-ACA at the beginning of the reaction. These results combined with the model analysis indicated that Phe58β、Ile75β and Ile176β were involved in the catalysis from CPC to 7-ACA. Conclusion: In this work, the gene of cephalosporin C acylase AcyII was synthesized, mutated and expressed successfully in the E.coli BL21 (DE3). The specific activity and the catalytic efficiency of A12 increased 2-fold and 3-fold respectively. Compared with the study of cephalosporin C acylase in N176, this work was considered helpful for the further study of the catalytic mechanism of cephalosporin C acylase and also useful for the strain improvement for the cephalosporin C acylase production.