The Purification,Cloning,Expression And Application Of A Esterase From Pseudochrobactrum Asaccharolyticum WZZ003

Esterases are a group of biocatalysts that have the ability to catalyze hydrolysis,esterification,alcoholysis,transesterification and other chemical reactions.Due to their high substrate specificity,regio-selectivity and enantio-selectivity,they were widely used in food,pharmaceuticals,fine chemicals,environmental protection and other fields.In this study,a novel esterase,which exhibited high hydrolytic activity and excellent enantioselectivity towards methy(R,S)-N-(2,6-dimethylphenyl)alaninate(R,S)-DMPM that is key chiral intermediates of metalaxyl,has been successfully purified from PSEUDOCHROBACTRUM ASACCHAROLYTICUM WZZ003,Subsequently,the gene encoding the esterase was cloned and functionally over-expressed in the Escherichia coli.Finally,The effects(pH,temperature and substrate concentration)on enzymatic catalyzed enantioselective hydrolysis of(R,S)-DMPM was investigated and the reusability of recombinant E.coli was evaluated.The whole cells of PSEUDOCHROBACTRUM ASACCHAROLYTICUM WZZ003 were disrupted using an ultrasonic disrupter and then centrifuged,the cell lysis liquid was obtained.An esterase was purified from PSEUDOCHROBACTRUM ASACCHAROLYTICUM WZZ003 using a two-step procedure:ion-exchange chromatography and hydrophobic chromatography.The purification folds and yield of esterase were 96.7 and 12.4%,respectively.The specific activity of the purified enzyme was 1.2 U/mg and the molecular weight of the purified enzyme determined by SDS-PAGE was shown to be approximately 31 kDa.The gene encoding esterase was amplified by PCR from genomic DNA of PSEUDOCHROBACTRUM ASACCHAROLYTICUM WZZ003.Sequencing of the fragment revealed that the esterase gene is813 bp in size and encodes 270 amino acids.Then the gene fragment was cloned into the vector pEASY-bluntE1 and transformed into E.coli BL21(DE3).After IPTG induction,the recombinant esterase Cell showed the activity of 123.3 U/g,which was approximately 10-fold higher than that of the wild PSEUDOCHROBACTRUM ASACCHAROLYTICUM strain.The resolution of racemic(R,S)-DMPM catalyzed by the recombinant E.coli was also studied.The catalytic reaction was performed under the optimized conditions of pH 6.0,30?,and 350 mMof initial substrate.A successful enzymatic chiral resolution was achieved with an enantiomeric excess of the substrate of 99.5% and 49% of conversion.The optical purity of(R)-MAP-acid was improved up to 98%.The recombinant E.coli retained 63% residual activity on the third cycle,and 32% of its initial activity after repeating the reaction five times.