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PEDS Advance Access originally published online on August 27, 2004
Protein Engineering Design and Selection 2004 17(7):571-579; doi:10.1093/protein/gzh070
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Protein Engineering, Design & Selection vol. 17 no. 7 © Oxford University Press 2004; all rights reserved

Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis

Esther M. Gabor and Dick B. Janssen1

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

1 To whom correspondence should be addressed. E-mail: d.b.janssen{at}chem.rug.nl

A semi-random mutagenesis approach was followed to increase the performance of penicillin acylase PAS2 in the kinetically controlled synthesis of ampicillin from 6-aminopenicillanic acid (6-APA) and activated D-phenylglycine derivatives. We directed changes in amino acid residues to positions close to the active site that are expected to affect the catalytic performance of penicillin acylase: {alpha}R160, {alpha}F161 and ßF24. From the resulting triple mutant gene bank, six improved PAS2 mutants were recovered by screening only 700 active mutants with an HPLC-based screening method. A detailed kinetic analysis of the three most promising mutants, T23, TM33 and TM38, is presented. These mutants allowed the accumulation of ampicillin at 4–5 times higher concentrations than the wild-type enzyme, using D-phenylglycine methyl ester as the acyl donor. At the same time, the loss of activated acyl donor due to the competitive hydrolytic side reactions could be reduced to <20% with the mutant enzymes compared >80% wild-type PAS2. Although catalytic activity dropped by a factor of 5–10, the enhanced synthetic performance of the recovered penicillin acylase variants makes them interesting biocatalysts for the production of ß-lactam antibiotics.

Received May 25, 2004; revised August 7, 2004; accepted August 18, 2004.

Edited by Jacques Fastrez


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