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PEDS Advance Access published online on August 27, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh070
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Received May 25, 2004
Revised August 7, 2004
Accepted August 18, 2004

Article

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

Esther M. Gabor 1 Dick B. Janssen 1*

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

* To whom correspondence should be addressed. E-mail: D.B.Janssen{at}chem.rug.nl.


  Abstract

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 {beta}F24. From the resulting triple mutant gene bank, 6 improved PAS2 mutants were recovered by only screening 700 active mutants with a 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 to 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 less than 20% with the mutant enzymes compared to > 80% with wild-type PAS2. Although catalytic activity dropped by a factor 5 to 10, the enhanced synthetic performance of the recovered penicillin acylase variants makes them interesting biocatalysts for the production of {beta}-lactam antibiotics.

Keywords: {beta}-lactam antibiotics; semi-random mutagenesis; penicillin acylase; synthesis.
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