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

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• 作者：Lu Wang^a ; ^b ; ¹ ; Li-Juan Ye^a ; ^b ; ¹ ; Yue Pan^b ; Yi Cao^a ; ^{geneium@scu.edu.cn}
• 关键词：AEH ; amino acid ester hydrolase ; 7-ACCA ; 7-amino-3-chloro-desacetoxycephalosporanic acid ; 7-ADCA ; 7-amino-desacetoxycephalosporanic acid ; d-PGM ; d-phenylglycine methyl ester
• 刊名：Enzyme and Microbial Technology
• 出版年：2012
• 期刊代码：26_01410229
• 类别：ce
• 出版时间：15 July, 2012
• 卷：51
• 期：2
• 页码：107-112
• 文件大小：694 K

摘要

伪-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of 尾-lactam antibiotics with 伪-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of 伪-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes鈥?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.