The Screening Of Stereoselective Nitrilase And Its Catalytic Synthesis Of Pregabalin Chiral Intermediates

Nitrilase hydrolyze nitriles to their corresponding carboxylic acid and ammonia.Due to its high stereoselectivity,mild and environment-friendly reaction condition,nitrilase is widely used in the pharmaceutical industry.Pregabalin(Pregabalin,PGB),(S)-(+)-3-methyl-5-methyl-amino acid,is used for the treatments of neuropathic pain,epilepsy,anxiety and other central nervous system damage.The asymmetric synthesis of pregabalin had become a hotspot in recent years since(S)-(+)-3-methyl-5-methyl-amino acid has stronger pharmacological activity.Nitrilase hydrolyzed IBSN to(S)-3-cyano-5-methylhexanoic acid,which was further convered to(S)-(+)-3-amino-5-methyl-hexanoic acid through hydrogenation.The above chem-enzymatic route was the optimal route for synthesis of Pregabalin with highest atom economy.However the difficulty was to obtain nitrilase with high catalytic activity and stereoselectivity.Here,we screened a nitrilase(BrNIT)from Brassica rapa with high activity and stereoselectivity for IBSN catalysis.The reaction conditions of recombinant E.coli BL21/pET-28b(+)-BrNIT were investigated.The result indicated that hydrolysis of 100 g/L ISBN by 50 g/L(wcw)E.coli BL21/pET28b-BrNIT under 30℃,pH 8.0(Tris-HCl buffer,100 mM)for 4 h resulted in 45.6% yield and 95.6% e.e,respectively,which demonstrated the potential of BrNIT for industrail application.Diatomite and polyethyleneimine glutaraldehyde cross-linked method was applied to immobilize E.coli BL21/pET28b-BrNIT.Catalytic properties of immobilized cells were studied and the results showed that the optimum temperature and pH of the recombinant strain was 30℃ and 8.0,respectively.The immobilized cells showed good operational stability.With a substrate concentration of 100 g/L,the immobilized cells remained 80% of its initial activity after continuous reaction for more than 15 batches.The amplification of fermentation system and immobilized system was further studied.The fermentation process amplificated progressively to 15 L,500 L and 5000 L fermenter from 5 L laboratory fermentor.The results showed no significant differences in biomass and enzyme activity among different size of fermenters.Under a 5000 L fermentor culture condition,the biomass of E.coli BL21/pET28b-BrNIT reached 11.5 g DCW/L and the activity reached 3375 U/L.