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Published online before print October 31, 2005, 10.1110/ps.051671705
Protein Science (2005), 14:3064-3076. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Evolution of an acylase active on cephalosporin C

Loredano Pollegioni1, Simona Lorenzi1, Elena Rosini1, Giorgia Letizia Marcone1, Gianluca Molla1, Roberto Verga2, Walter Cabri2 and Mirella S. Pilone1

1 Department of Biotechnology and Molecular Sciences, University of Insubria, 21100 Varese, Italy
2 Antibioticos S.p.A., 20090 Rodano, Italy

(RECEIVED June 27, 2005; FINAL REVISION September 2, 2005; ACCEPTED September 12, 2005)

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.

Keywords: cephalosporin C; 7-amino cephalosporanic acid; protein engineering; directed evolution; site-saturation mutagenesis; enzymes; active sites; structure/function studies; protein sequencing; modification; mass spectrometry; protein structure prediction; kinetics

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051671705.

Reprint requests to: Loredano Pollegioni, Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell’Insubria, Via J.H. Dunant, 3, 21100 Varese, Italy; e-mail: loredano.pollegioni{at}uninsubria.it; fax: +39-0332-421-500.


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