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Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio

Paper ID Volume ID Publish Year Pages File Format Full-Text
17253 42654 2012 6 PDF Available
Title
Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio
Abstract

α-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of β-lactam antibiotics with α-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of α-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes’ S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained.

► AEH catalyzes undesired substrate hydrolysis when used to synthesize cefaclor. ► epPCR was used to create diversity of AEH. ► Two spectrophotometric assays were developed and applied to library screening. ► Three improved variants are obtained with decreased hydrolysis.

Keywords
AEH, amino acid ester hydrolase; 7-ACCA, 7-amino-3-chloro-desacetoxycephalosporanic acid; 7-ADCA, 7-amino-desacetoxycephalosporanic acid; d-PGM, d-phenylglycine methyl esterα-Amino acid ester hydrolase; S/H ratio; Cefaclor; Spectrophotometric assay; Paral
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Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 51, Issue 2, 15 July 2012, Pages 107–112
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