The Study Of Nitrilase Immobilization And Its Application Catalytic Synthesis Of Pregabalin Chiral Intermediates

Pregabalin[?S?-3-aminomethyl-5-methylhexanoic acid,PGB],is the substituendumof3-isobutyloftheneurotransmitterinhibitor?-aminobutyric acid?GABA?.PGB was first applied in the treatment of peripheral neuropathic pain and refractory partial-onset seizures,and then currently used for other indications.Nitrilase?EC3.5.5.1?is one of the important nitrile-converting enzymes that catalyze nitriles to their corresponding acids and ammonia in one step.The process of regio-stereo specific synthesis of ?S?-3-cyano-5-methylhexanoic acid from isobutylsuccinonitrile?IBSN?by nitrilase,followed by its reduction to PGB through hydrogenation exhibits the highest atom economy properties than other chemistry-enzymatic processes.The bottleneck of the enzymatic process is to obtain a nitrilase with high activity and selectivity towards IBSN.Based on the starting point of selecting immobilization carrier with excellent comprehensive performance,various immobilization methods were tried in this study.The nitrile hydrolase was finally immobilized on a chosen carrier and exihibited properties with high specific surface area,sturdy texture,and high reuse frequency.The culture and enzyme production conditions of the recombinant E.coli BL21/pET28b-NIT were studied.The results showed that the addition of glucose in the fermentation medium can improve enzyme activity by 1.96 times,and the addition of trace amount of Mg²⁺in culture can reduce the production of inclusion body of expressed protein.In the process of enzyme production,the initial pH value of the culture medium was set at7.5,the best induce temperature was 28°C,the lactose was used as inducer and it was added 3 h after inoculation at a concentration of 12g/L.Under this condition,the biomass of E.coli BL21/p ET28b-NIT reached 2.80 g/L,and the enzyme activity reached 95U/g dcw.The nitrilase was immobilized on different carrier surface bydifferent covalent immobilization methods.The LX-1000EP?C?epoxy resin was finally selected for nitrilase immobilization and its stabilities were studied.The results indicated that the optimal conditions of nitrilase immobilization were as follows:the appropriate temperature was 20°C,the immobilization time was 24 h,thepotassium phosphate was selected as immobilization buffer with a concentration of 0.1 M,the pH value was 8.0,and the amount of enzyme added was 10 mL crude enzyme solution?3 mg/ml?per gram of carrier.Based on above conditions,the recovery of the enzyme activity of the immobilized nitrilase reached 98.1%.The immobilized enzyme showed high selectivity to IBSN catalysis.The optimum temperature and pH value of the catalytic reaction was 35°C and 8.0,respectively.the temperature and pH stability of immobilized enzyme has obvious improvement compared with the free enzyme.Immobilized enzyme can be used in a row after 6 batches remained more than 30%of the initial enzyme activity,The racemization of R-IBSN yield reached more than 80.9%,after racemization,The selectively of IBSN converted to 3.6%from 90%.