Screening Of Nitrilase-producing Strains And Its Application In The Chemo-enzymatic Synthesis Of Optically Pure Baclofen/pregabalin

In this study, baclofen and pregabalin were prepared through chemo-enzymatic method.A high nitrilase-producing fungal strain WX12was isolated from80isolates preserved in ourlabortary using phenol-sodium hypochlorite method. The main research topics are biocatalyticsynthesis of (S)-cyano-(4-chlorophenyl)-butanoic acid and (R)-cyanomethyl-methyl hexanoicacid, which were key chiral intermediate of baclofen and pregabalin, respectively.Subsequently, optically pure baclofen and pregabalin was generated through chemicalmethod.The fungal strain WX12was isolated with3-(4-chlorophenyl)-glutaronitrile and3-isobutylpentanedinitrile as substrate. It was identified to be G. intermedia WX12. Thefermentation conditions and catalytic properties of this strain were investigated. The preferredcarbon and nitrogen sources for nitrilase production were lactose (30g/L) and peptone (20g/L), respectively. After being cultivated for96h, the cells were collected for3-(4-chlorophenyl)-glutaronitrile biotransformation. The hydrolysis reaction was performed at30°C in50mmol/L phosphate buffer (pH8.0) to produce4-cyano-3-(4-chlorophenyl)-butyricacid with specific activity of49.6U/g. After24h conversion, the production yield could reachup to90%. At the same conditions, the yield of3-(cyanomethyl)-5-methylhexanoic acid wasalso up to90%after72h of conversion.The nuclear magnetic resonance characterization (NMR) and high performance liquidchromatography (HPLC) were performed to determine the hydrolysis product. The hofmannrearrangement was further carried out to producte baclofen/pregabalin, which was detected bychiral HPLC. Results showed that (S)-4-cyano-3-(4-chlorophenyl)-butyric acid and (R)-3-(cyanomethyl)-5-methylhexanoic acid were obtained with ee value of>99%, which can beused for synthesis of optically pure (R)-/(S)-baclofen or (R)-/(S)-pregabalin.The recombinant strain E.coli BL21(DE3)/pET28a(+)-Nit was constructured usingrecombinant plasmid pET28a(+)-Nit. The production yield (90%) and ee value (﹥99%) ofrecombinant strain after24h conversion were similar to that of the wild-type strain. Inaddition, the production yield of recombinant strain was lower than that of standard nitrilasebll6402(100%), while ee value was better than this nitrilase (92%).