文献类型：期刊文献

英文题名：Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction

作者：Wang, Hualei[1];Sun, Huihui[1];Wei, Dongzhi[1]

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

年份：2013

卷号：13

外文期刊名：BMC BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000317105300001)】；

基金：This work was supported by the National Major Science and Technology Projects of China (2012ZX09304009) and the Fundamental Research Funds for the Central Universities (WF1013008).

语种：英文

外文关键词：(R)-(-)-mandelic acid; Nitrilase; Burkholderia cenocepacia J2315; Substrate specificity prediction; Enantioselective hydrolysis

摘要：Background: A nitrilase-mediated pathway has significant advantages in the production of optically pure (R)(-)- mandelic acid. However, unwanted byproduct, low enantioselectivity, and specific activity reduce its value in practical applications. An ideal nitrilase that can efficiently hydrolyze mandelonitrile to optically pure (R)(-)- mandelic acid without the unwanted byproduct is needed. Results: A novel nitrilase (BCJ2315) was discovered from Burkholderia cenocepacia J2315 through phylogeny-based enzymatic substrate specificity prediction (PESSP). This nitrilase is a mandelonitrile hydrolase that could efficiently hydrolyze mandelonitrile to (R)-(-)-mandelic acid, with a high enantiomeric excess of 98.4%. No byproduct was observed in this hydrolysis process. BCJ2315 showed the highest identity of 71% compared with other nitrilases in the amino acid sequence. BCJ2315 possessed the highest activity toward mandelonitrile and took mandelonitrile as the optimal substrate based on the analysis of substrate specificity. The kinetic parameters V-max, K-m, K-cat, and K-cat/K-m toward mandelonitrile were 45.4 mu mol/min/mg, 0.14 mM, 15.4 s(-1), and 1.1x10(5) M(-1)s(-1), respectively. The recombinant Escherichia coli M15/BCJ2315 had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100 mM) with fewer amounts of wet cells (10 mg/ml) within 1 h. Conclusions: PESSP is an efficient method for discovering an ideal mandelonitrile hydrolase. BCJ2315 has high affinity and catalytic efficiency toward mandelonitrile. This nitrilase has great advantages in the production of optically pure (R)-(-)-mandelic acid because of its high activity and enantioselectivity, strong substrate tolerance, and having no unwanted byproduct. Thus, BCJ2315 has great potential in the practical production of optically pure (R)-(-)-mandelic acid in the industry.