ABSTRACT
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 100 g L-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 eep 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.
Subject(s)
Amidohydrolases/metabolism , Aminohydrolases/metabolism , Arabis/enzymology , Pregabalin/metabolism , Aminohydrolases/genetics , Arabis/genetics , Biotransformation , Catalysis , Enzymes , Escherichia coli/genetics , Hydrolysis , Kinetics , Mutation , Pantoea/enzymology , Substrate SpecificityABSTRACT
To develop a highly efficient method for aprepitant chiral intermediate (S)-4-fluorophenylglycine, a continuous reaction system was established in packed bed bioreactor using amidase covalently immobilized on epoxy resin as biocatalyst. The epoxy resin was firstly modified by metal-chelate method and functional groups (Cu2+-IDA) generated were able to rapidly adsorb amidases, which were further covalently bound onto the modified resin with 90.1% immobilization yield and 80.2% activity recovery. The immobilized amidase exhibited excellent thermal stability with the longest half-life of 1456.8â¯h at 40⯰C ever reported. (S)-4-fluorophenylglycine was continuously produced using the reaction system with 49.9% conversion, 99.9% ee, and an outstanding space-time yield of 5.29â¯kgâ¯L-1â¯d-1. Moreover, the efficient reaction system exhibited a high operational stability and retained 86.3% catalytic activity after 25-day continuous operation. This efficient continuous bioprocess presents great industrial potential for large-scale production of (S)-4-fluorophenylglycine.