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Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio.
MedLine Citation:
PMID:  22664195     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
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.
Authors:
Lu Wang; Li-Juan Ye; Yue Pan; Yi Cao
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Publication Detail:
Type:  Journal Article     Date:  2012-05-03
Journal Detail:
Title:  Enzyme and microbial technology     Volume:  51     ISSN:  1879-0909     ISO Abbreviation:  Enzyme Microb. Technol.     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-06-05     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8003761     Medline TA:  Enzyme Microb Technol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  107-12     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier Inc. All rights reserved.
Affiliation:
College of Life Sciences, Sichuan University, Chengdu 610064, China; Department of Biotechnology, China National Pharmaceutical Group Corporation Sichuan Industrial Institute of Antibiotics, Chengdu 610052, China.
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