Biosynthesis Of The Key Chiral Intermediate Of Pregabalin By Morgarella Morganii ZJB-09203

The chemical name of Pregabalin(PGB)is S-3-(aminomethyl)-5-methylhexanoic acid,mainly developed for the treatment of epilepsy,neuropathic pain,et al.Because of its good effects,less dose to take and slight side effects,etc.,so the research of Pregabalin grow rapidly,many new methods for the synthesis of Pregabalin had emerged.Pfizer developed a chemoenzymatic production process for Pregabalin,using Lipolase,a commercially available lipase,rac-2-carboxyethyl-3-cyano-5-methyl hexanoic acid ethyl ester(CNDE)can be resolved to form S-2-carboxyethyl-3-cyano-5-methylhexanoic acid,the key chiral intermediates of Pregablin,after decarboxylation,hydrolysis,hydrogenation,finally obtained Pregabalin.This manufacturing process using commercial Lipolase which belongs to Novozymes as biocatalyst,requires high costs.Screening and exploration of the new enzyme which can kinetic resolve CNDE to form S-2-carboxymethyl-3-cyano-5-methyl hexanoic acid have been hot issues and another key area of interest in chemoenzymatic process synthesis of Pregabalin.Firstly,in this paper we established a gas chromatography analysis of racemic 2-carboxyethyl-3-cyano-5-methyl ethyl caproate,as well as the product of S-2-carboxymethyl-ethyl-3-cyano-5-methyl-hexanoic acid.Then the strain ZJB-09203,a bacteria with good stereselectivity and activity towards CNDE was isolated from soil samples,the optical purity was up to 94%.Based on morphology,physiological characteristics,16S rDNA analysis and phylogenetic tree analysis,ZJB-09203 was identified as Morgarella morganii.The results of substrate specificity of the enzyme from ZJB-09203 revealed that it was esterase.Secondly,production of esterase by Morgarella morganii ZJB-09203 was optimized by single factor method and orthogonal test.The optimal medium composition was as follows(g/L):glucose 15.0,beef extract 7.0,Na2HP04 1.0,Fe2(S04)3 0.1 and Tween-80 10.0.The optimial initial pH was 7.0.The esterase activity and the biomass of ZJB-09203 reached 1071.0 U/L and 4.5 g DCW/L,which was 2.5-fold and 1.5-fold as the starting medium,respectively.Thirdly,kinetic resolution of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester using whole cells of M.morganii ZJB-09203 was also studied.The data showed that the optimal temperature and pH was 45? and 8.5,respectively.In addition,we added ethanol as cosolvent,its optimal concentration was 15%.At last,we found that when the D201 resin was added,the product concentration reached 450 mM with 1.0 M substrate concentration,E reached 92.2.The results we mentioned above revealed that Morgarella morganii ZJB-09203 performed a good effect on kinetic resolution of CNDE ester to form S-2-carboxymethyl-ethyl-3-cyano-5-methylhexanoic acid,this study showed that this Morgarella morganii ZJB-09203 could be potential for the large scale production of chiral intermediate of Pregabalin.