Evolution of an acylase active on cephalosporin C

• Published in: Protein science 2005-12, Vol.14 (12), p.3064-3076
• Main Authors: Pollegioni, Loredano, Lorenzi, Simona, Rosini, Elena, Marcone, Giorgia Letizia, Molla, Gianluca, Verga, Roberto, Cabri, Walter, Pilone, Mirella S.
• Format: Article
• Language: English
• Subjects: 7‐amino cephalosporanic acid; active sites; Amidohydrolases - chemistry; Amidohydrolases - genetics; Amidohydrolases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Base Sequence; cephalosporin C; Cephalosporins - metabolism; directed evolution; Directed Molecular Evolution; enzymes; Escherichia coli; Gene Library; Kinetics; mass spectrometry; Models, Molecular; modification; Molecular Sequence Data; Molecular Structure; Mutation - genetics; protein engineering; protein sequencing; protein structure prediction; Protein Structure, Quaternary; site‐saturation mutagenesis; structure/function studies; Substrate Specificity
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.051671705

Semisynthetic cephalosporins are synthesized from 7‐amino cephalosporanic acid, which is produced by chemical deacylation or by a two‐step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl‐7‐amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl‐7‐amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error‐prone PCR mutagenesis, a molecular modeling approach combined with site‐saturation mutagenesis, and site‐directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl‐7‐amino cephalosporanic acid catalytic efficiency that was increased up to 100‐fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl‐7‐amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y‐H296S‐H309S mutant). Our data in a bioreactor indicate an ∼90% conversion of cephalosporin C to 7‐amino‐cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7‐amino cephalosporanic acid.