title: Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis.
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, 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 beta-lactam antibiotics.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Esther M
authors.initials: EM
authors.lastname: Gabor
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
mesh_terms: D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D016296:Mutagenesis; D009154:Mutation; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D010399:Penicillin Amidase / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013997:Time Factors; D047090:beta-Lactams / Q000737:chemistry
chemical_list: D047090:beta-Lactams; C033393:methyl phenylglycine; D010397:Penicillanic Acid; D010399:Penicillin Amidase; C004408:aminopenicillanic acid; D005998:Glycine