title: Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel.
abstract: 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 10.0 U/mg and 11.3 U/mg, respectively. The optimal CPC acylase productivity of mutant sCPCAcy(Ala675Gly) reached 5349 U/l after 24h in culture, which was a 35% increase over the activity of sCPCAcy.
authors.affiliation: Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.forename: Wensi
authors.initials: W
authors.lastname: Song
authors.forename: Ming
authors.initials: M
authors.lastname: An
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Hui
authors.initials: H
authors.lastname: Luo
authors.forename: Zhongyao
authors.initials: Z
authors.lastname: Shen
mesh_terms: D000215:Acylation; D002511:Cephalosporins / Q000737:chemistry*; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D002511:Cephalosporins; D003062:Codon; C081063:glutaryl-7-aminocephalosporanic acid; C025163:cephalosporin C; D010399:Penicillin Amidase