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There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalo-sporin antibiotics. In this study, the active site of the glutaryl acylase from PseudomonasSY-77 was randomized rationally. | ễFEBS Journal A highly active adipyl-cephalosporin acylase obtained via rational randomization Linda G. Otten Charles F. Sio t Carlos R. Reis Gudrun Koch Robbert H. Cool and Wim J. Quax PharmaceuticalBiology University Centre for Pharmacy University of Groningen the Netherlands Keywords biocatalysis cephalosporin acylase directed evolution protein engineering 7-amino-cephalosporanic acid Correspondence W. J. Quax PharmaceuticalBiology Antonius Deusinglaan 1 9713 AV Groningen the Netherlands Fax 31 50 3633000 Tel 31 50 3632558 E-mail w.j.quax@rug.nl These authors contributed equally to this work Present address Biocatalysis Organic Chemistry Delft University of Technology the Netherlands BioMolecular Engineering Philips Research Eindhoven the Netherlands Received 10 June 2007 revised 13 August 2007 accepted 30 August 2007 doi 10.1111 j.1742-4658.2007.06081.x There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalosporin antibiotics. In this study the active site of the glutaryl acylase from Pseudomonas SY-77 was randomized rationally. Several mutations that were found in previous studies to enhance the activity of the enzyme towards adi-pyl-7-aminodesacetoxycephalosporanic acid ADCA and cephalosporin C have now been combined and libraries have been made in which random amino acid substitutions at these positions are joined. The mutants were expressed in a leucine-deficient Escherichia coli strain and subjected to growth selection with adipyl-leucine or amino-adipyl-leucine as sole leucine source. The mutants growing on these media were selected and purified and their hydrolysis activities towards adipyl-7-ADCA and cephalosporin C were tested. Several mutants with highly improved activities towards the desired substrates were found in these rationally randomized libraries. The best mutant was selected .
