Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel

Cephalosporin C (CPC) acylase converts CPC into 7-aminocephalosporanic acid (7-ACA) by single-step enzymatic catalysis. An optimized CPC acylase gene with substituted codons and a reduced GC content was artificially designed, synthesized and overexpressed in recombinant Escherichia coli. The synthetic CPC acylase (sCPCAcy) exhibited 2.3 times more CPC specific deacylation activity with substrate CPC than with substrate glutaryl-7-ACA (GL-7-ACA). Site-directed mutagenesis of the residues around the active center showed that not only the residues that were adjacent to the CPC D-α-aminoadipyl moiety, but also the residues that were in the substrate transport tunnel (Leu666, Ala675, Leu677), played crucial roles in catalysis as the ones locating in active center. Mutant sCPCAcy ^Leu666Phe and sCPCAcy ^Leu677Ala exhibited significantly reduced specific enzymatic activity, while mutant sCPCAcy ^Ala675Gly demonstrated enhanced activity. The specific activity of purified sCPCAcy and sCPCAcy ^Ala675Gly was 10.0U/mg and 11.3U/mg, respectively. The optimal CPC acylase productivity of mutant sCPCAcy ^Ala675Gly reached 5349U/l after 24h in culture, which was a 35% increase over the activity of sCPCAcy.