Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity |
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Authors: | H Niersbach A Kühne W Tischer M Weber F Wedekind R Plapp |
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Institution: | (1) Department of Microbiology, University of Kaiserslautern, PO box 3049, D-67663 Kaiserslautern, Germany;(2) Boehringer Mannheim GmbH, Nonnenwald 2, D-82377 Penzberg, Germany;(3) Present address: Department of Microbiology, New York University Medical School, 550 First Avenue, 10016 New York, NY, USA |
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Abstract: | Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl--alanyl-l-proline (PhAc-Ala-Pro) phthalyl-l-leucine (Pht-Leu) or phthalylglycyl-l-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-gbAla-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The K
m of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45° C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold. |
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