文献类型：期刊文献

英文题名：Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of (R)-o-Chloromandelic Acid

作者：Wang, Hualei[1];Gao, Wenyuan[1];Sun, Huihui[1];Chen, Lifeng[1];Zhang, Lujia[1];Wang, Xuedong[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：81

期号：24

起止页码：8469

外文期刊名：APPLIED AND ENVIRONMENTAL MICROBIOLOGY

收录：;EI(收录号：20155101687015);WOS:【SCI-EXPANDED(收录号:WOS:000365212800021)】；

基金：This work was supported by the National Natural Science Foundation of China (grant no. 21406068/B060804), China Postdoctoral Science Foundation-funded project (grant no. 2014M560308), National Major Science and Technology Projects of China (grant no. 2012ZX09304009), and National Basic Research Program of China (grant no. 2012CB721103).

语种：英文

外文关键词：Hydrolysis - Genetic engineering - Biochemical engineering - Mutagenesis - Proteins

摘要：The nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic alpha-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzing o-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained from Burkholderia cenocepacia strain J2315 (BCJ2315) in hydrolyzing o-chloromandelonitrile. Four hot spots (T49, I113, Y199, and T310) responsible for the enantioselectivity and activity of BCJ2315 were identified by random mutagenesis. An effective double mutant (I113M/Y199G [encoding the replacement of I with M at position 113 and Y with G at position 199]), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess [ee] compared to 89.2% ee for the wild type) and relative activity (360% of the wild type), was created by two rounds of site saturation mutagenesis, first at each of the four hot spots and subsequently at position 199 for combination with the selected beneficial mutation I113M. Notably, this mutant also demonstrated dramatically enhanced enantioselectivity and activity toward other mandelonitrile derivatives and, thus, broadened the substrate scope of this nitrilase. Using an ethyl acetate-water (1:9) biphasic system, o-chloromandelonitrile (500 mM) was completely hydrolyzed in 3 h by this mutant with a small amount of biocatalyst (10 g/liter wet cells), resulting in a high concentration of (R)-o-chloromandelic acid with 98.7% ee, to our knowledge the highest ever reported. This result highlights a promising method for industrial production of optically pure (R)-o-chloromandelic acid. Insight into the source of enantioselectivity and activity was gained by homology modeling and molecular docking experiments.