The Purification,Cloning,Expressing And Application Of A Esterase From Bacillus Cereus

Esterases are a group of biocatalysts that have ability to catalyze the hydrolysis,esterification,alcoholysis,transesterification and other chemical reactions.Due to their high substrate specificity,regional selectivity and enantioselectivity,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 a-ethyl-2-oxo-pyrrolidineacetic acid methyl ester that is key chiral intermediates of levetiracetam,has been successfully purified from B.cereus WZZ001,and then characterized.Subsequently,the gene encoding the esterase was cloned and functionally over-expressed in the Escherichia coli.Finally,the application of the recombinant E.coli on levetiracetam synthesis was explored.An esterase was purified to homogeneity from B.cereus WZZ001 using a three-step procedure:ion-exchange chromatography,hydrophobic chromatography and Ceramic Hydroxyapatite chromatography.The purification folds and yield of esterase were 12.54 and 11.20%,respectively.The specific activity of the purified enzyme was 7.5 U/mg and the molecular weight of the purified enzyme determined by SDS-PAGE was shown to be approximately 55 kDa.The Optimal pH-value and temperature for esterase activity were 8.5 and 60℃,respectively.The activity of esterase was more than 60%of its original values after 1 h incubation at pH 7.0-9.0,and completely stable after 2 h pre-incubation at 50℃.Most metal ions inhibited the esterase activity,especially Cu2+ and Al3+.Organic solvents had a great impact on the enzyme activity,such as glycerol,methanol and n-hexane can activate the enzyme activity,while others inhibited.Most surfactants and inhibitors had inhibition on the enzyme,especially PMSF and DEPC.Meanwhile,the activation of the reductant DTT indicated the presence of disulfide bonds in esterase.The gene enconding esterase was amplified by PCR from genomic DNA of B.cereus WZZ001.Sequencing of the fragment revealed that the esterase gene is 1458 bp in size and encodes 485 amino acids.Then,the gene fragment was cloned into the vector pEASY-E1 and transformed into E.coli BL21(DE3).After IPTG induction,the recombinant esterase showed the activity of 1631.5U/L,which was approximately 10-fold higher than that of the wild B.cereus strain.Sequences and structure analysis revealed that the enzyme show the highest similarity to class Cβ-lactamase,which indicated the esterase belong to the family Ⅷ of bacterial esterase.The resolution of racemic a-ethyl-2-oxo-pyrrolidineacetic acid methyl ester catalyzed by the recombinant esterase was also studied.The catalytic reaction was performed under the optimized conditions of pH 8.0,35 ℃,and 500 mM of initial substrate.A successful enzymatic chiral resolution was achieved with an enantiomeric excess of the substrate of 99.5%and 49%of conversion.At last,the levetiracetam was obtained by ammonolysis and the structure was identified by GC-MS.