期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 103, 期 14, 页码 5617-5626出版社
SPRINGER
DOI: 10.1007/s00253-019-09857-1
关键词
Nitrilase; Amidase; Cascade reaction; Pregabalin; (S)-3-Cyano-5-methyl hexanoic acid
资金
- Natural Science Foundation of Zhejiang Province [LR19B060001]
- Public Welfare Project of Zhejiang Province [2014C33223]
- Program for Changjiang Scholars and Innovative Research Team in University [IRT13096]
Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. In this study, a robust nitrilase from Arabis alpina (AaNIT) was screened and engineered. The N258D mutant was obtained with high catalytic activity and excellent enantioselectivity (E>300) towards IBSN at a high substrate concentration of 100gL(-1). Byproduct (S)-3-cyano-5-methyl hexanoic amide ((S)-CMHM) was detected and identified for the first time during the catalytic process. By employing a feasible one-pot bienzymatic cascade of mutant N258D and amidase from Pantoea sp. (Pa-Ami) expressed separately in recombinant Escherichia coli cells, the byproduct (S)-CMHM was eliminated and (S)-CMHA was obtained with a conversion of 45.0% and ee(p) of 99.3%. These results provided the novel plant-derived nitrilase as a promising biocatalyst for (S)-CMHA biosynthesis and demonstrated the feasibility of one-pot bienzymatic cascade reaction for large-scale production of the pregabalin precursor.
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