Biocatalytic Synthesis Of The Key Chiral Intermediate Of Pregabalin By Stenotrophomonas Maltophilia ZJB-14132

Pregabalin[?S?-3-?aminomethyl?-5-methylhexanoic acid]is a lipophilic derivative of the inhibitory neurotransmitter?-aminobutyric acid?GABA?.Due to its good curative effect on neuropathic pain and epilepsy,Pregabalin?PGB?has been one of the fastest growing drugs.It is reported that PGB has a promising market with sales reaching nearly$5.19 billion in 2014.The most successful scalable chemoenzymatic strategy from the Pfizer company for?S?-Pregabalin involved lipase-catalyzed resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester?CNDE?to?3S?-2-carboxyethyl-3-cyano-5-methylhexan-oic acid,but the used enzyme in this strategy is too expensive to be applied in the factory.In this thesis,we focused on the development a novel robust biocatalyst for kinetic resolution of CNDE so that we could find a new strategy of synthesizing?S?-Pregabalin.Then the strain ZJB-14132 which has good stereoselectivity and activity towards CNDE was isolated from soil samples.Based on morphology,physiological characteristics,16S rDNA analysis and phylogenetic tree analysis,the isolated strain was identified as Stenotrophomonas maltophilia.It was revealed that the enzyme from S.maltophilia ZJB-14132 was esterase because of its substrate specificity.The medium components and cultivation conditions of S.maltophilia ZJB-14132 were optimized by single factor method.The optimal medium composition was as follows?g/L?:glucose 15.0,peptone7.0,MgCl₂ 0.1,Na₂HPO₄ 1.0.The optimal fermentation conditions for cell growth and formation of esterase were as follows:medium volumetric ratio,100 mL/500 mL;inoculum volume,4.0%?v/v?;initial pH value,8.0;30 ^oC;150 rpm.Under these conditions,the esterase activity and biomass of ZJB-14132 reached 188.47 U/L and 6.88 g/L,which was 2.18-fold and 2.75-fold as the starting medium,respectively.The chiral resolution of CNDE using whole cells of ZJB-14132 was also studied.The data showed that the optimal temperature and pH was40°C and 8.0,respectively and 15%Triton X-100 was added.Under these conditions,after 72 hour-reaction,the conversion reached 47%and188 mM production was achieved by catalyzing 400 mM CNDE.It was showed that this S.maltophilia ZJB-14132 could be potential for the large scale production of chiral intermediate of Pregabalin.