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

BMC Biotechnology

Hualei Wang¹  Huihui Sun¹  Dongzhi Wei¹ 

[1] State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, People’s Republic of China

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

Others  :  1123262
DOI  :  10.1186/1472-6750-13-14

 received in 2012-09-19, accepted in 2013-02-05,  发布年份 2013

【 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 μmol/min/mg, 0.14 mM, 15.4 s^-1, and 1.1×10⁵ M^-1s^-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.

【 授权许可】

   
2013 Wang et al; licensee BioMed Central Ltd.

【 图 表 】

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