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Hybrid penicillin acylases with improved properties for synthesis of β-lactam antibiotics

Paper ID Volume ID Publish Year Pages File Format Full-Text
18183 42713 2007 10 PDF Available
Title
Hybrid penicillin acylases with improved properties for synthesis of β-lactam antibiotics
Abstract

Penicillin acylase (PA) from Escherichia coli can catalyze the acylation of 6-aminopenicillanic acid (6-APA), a conversion that is applicable in the biocatalytic preparation of semi-synthetic β-lactam antibiotics such as ampicillin. The efficiency of this kinetically controlled conversion, in which an amide or ester acts as the acyl donor, is dependent on the kinetic properties of the enzyme. To further improve the synthetic properties of PAs, family gene shuffling was performed with the PA-encoding genes of the PAs from E. coli, Kluyvera cryocrescens and Providencia rettgeri. Of these three PAs, the E. coli enzyme possessed the best properties for the synthesis of ampicillin. Shuffled recombinant libraries were pre-screened for activity by growth selection, followed by testing the catalytic performance in ampicillin synthesis using HPLC. Three clones with improved synthetic properties were selected and sequence analysis showed that the shuffled genes were hybrids of the PA-encoding genes from E. coli and K. cryocrescens, with additional point mutations. The hybrid enzymes displayed a 40–90% increase in the relative rate of acyl transfer to the β-lactam nucleus during ampicillin synthesis. This increase was not accompanied by a reduction of synthetic activity that has previously been reported for mutants of E. coli PA constructed by site-directed mutagenesis. Similar improvements in acyl transfer were obtained for the synthesis of amoxicillin, cephalexin and cefadroxil, making the new hybrid enzymes interesting candidates for the biocatalytic synthesis of several β-lactam antibiotics.

Keywords
Penicillin acylase; Ampicillin; Cephalexin; Synthesis; Biocatalysis; Gene shuffling; Directed evolution
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Hybrid penicillin acylases with improved properties for synthesis of β-lactam antibiotics
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 5, 3 April 2007, Pages 1335–1344
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us