文献类型：期刊文献

英文题名：Directed evolution of nitrilase PpL19 from Pseudomonas psychrotolerans L19 and identification of enantiocomplementary mutants toward mandelonitrile

作者：Sun, Huihui[1];Wang, Hualei[1];Gao, Wenyuan[1];Chen, Lifeng[1];Wu, Kai[1];Wei, Dongzhi[1]

机构：[1]E China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2015

卷号：468

期号：4

起止页码：820

外文期刊名：BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000367868500048)】；

基金：This work was supported by the National Natural Science Foundation of China (No. 21406068/B060804), a China Postdoctoral Science Foundation funded project (No. 2014M560308), and National Major Science and Technology Projects of China (2012ZX09304009).

语种：英文

外文关键词：Directed evolution; Enantiocomplementary; Nitrilase; Pseudomonas psychrotolerans L19

摘要：Nitrilase PpL19 from Pseudomonas psychrotolerans L19 can hydrolyze racemic mandelonitrile to (S)-mandelic acid with an enantiomeric excess (ee) value of 52.7%. In this study, random mutagenesis combined with site-directed mutagenesis was performed to identify the key residues responsible for nitrilase enantioselectivity. Five enzyme mutants exhibiting distinct selectivity were generated and four "hot spots" (M113, R128, A136, and 1168) responsible for enantioselectivity toward mandelonitrile were identified and characterized. Furthermore, through saturation mutagenesis, positions 113 and 128 were confirmed to substantially influence the enantioselectivity of PpL19, and certain replacements of the methionine at position 113, in particular, were found to reverse the enantioselectivity of PpL19 from S- to R-selectivity. Two other single mutants of the enzyme, PpL19-A136Y and -I168Y, also showed reversed selectivity and preferentially produced (R)-mandelic acid (ee values: 66.7% and 74.3%, respectively). By combining the beneficial mutations, two enantiocomplementary nitrilase mutants, PpL19-LH and PpL19-GYY, were created, which exhibited high 5- and R-selectivity toward mandelonitrile, respectively: PpL19-LH showed the highest S-selectivity toward mandelonitrile ever reported (91.1% ee), and, notably, the PpL19-GYY mutant was identified to be highly R-selective (90.1% ee) and thus an unexpected enantiocomplementary mutant for mandelonitrile. (C) 2015 Elsevier Inc. All rights reserved.