Production Of (R)-4-chloro-3-Hydroxyethylbutyrate And (S)-3-hydroxy-γ-butyrolactone By Microbial Resolution Using Recombinant Esterase

Due to their high substrate specificity,regional selectivity and enantioselectivity,esterases were widely used in food,pharmaceuticals,fine chemicals,environmental protection and other fields.In this study,a novel esterase from B.megaterium WZ009 which exhibited high hydrolytic activityandexcellentenantioselectivitytowardsrac4-Chloro-3-hydroxyethylbutyratehasbeensuccessfullypurified.Subsequently,the gene encoding the esterase was cloned and functionally over-expressed in Escherichia coli.Its catalytic properties and the application in the resolution of rac 4-Chloro-3-hydroxyethylbutyrate have been studied.The strain named B.megaterium WZ009 exhibited excellent enantioselectivity was performed,and intracellular proteins were obtained after cells of B.megaterium WZ009 was disrupted by an ultrasonic disrupter.The esterase was purified to homogeneity using a three-step procedure:DEAE-anion exchange chromatography,phenyl-sepharose chromatography and hydroxyapatite columns chromatography and verified by zymography.Purification folds and specific activity of the purified enzyme were 34.5and 135.3 U/mg,respectively.The apparent molecular mass of the esterase was found to be 55 kDa according to SDS-PAGE analysis.The gene enconding esterase was amplified by PCR from genomic DNA of B.megaterium WZ009.Sequencing of the fragment revealed that the esterase gene was 1401 bp and encoded 466 amino acids.Meanwhile,the gene fragment was cloned into the vector pEASY-E1 and transformed into E.coli BL21（DE3）.The results about enzymatic properties of the recombined esterase showed that the recombined esterase exhibited optimal activity at a temperature of 50℃and in a pH of 9.0.The effects of metal ions on recombined esterase activity was mainly embodied as follows,all of metal ions inhibited the esterase activity,especially Co²⁺、Mn²⁺、Na⁺and Ca²⁺,which made the activity lossed more than 50%.Most surfactants and inhibitors had inhibition on the recombined esterase,if continue increasing the concentration of Tween 20,Tween 80 and Triton X-100 to 5%（w/v）,recombined esterase would be inhibited seriously.In addition,it also had a feature of preferences for short-chain fatty acid esters,especially toward the C₂-C₄,but did not exhibit any significant activity with long-chain fatty acids(C₁₀-C₁₈).The results from the substrate specificity characterization indicated that the enzyme is a true esterase and not a lipase.The kinetic parameters of est were determined using CHBE as a substrate.It can be seen that the recombinant esterase is selective for CHBE,presenting a K_m value of 317.5 mM.The values of K_cat/K_m indicated that est is very efficient in hydrolyzing CHBE.Based on the functions of the recombined esterase toward different substrates,it is concluded that the esterase could recognizeβ-hydroxy and had high stereoselectivity and catalytic activity,especiallytowardsto3-hydroxyethylbutyrateand4-Chloro-3-hydroxyethylbutyrate.Moreover,The recombinant enzyme exhibited optimal activity at a temperature of 25℃and at pH 11.5.when activated carbon（62 g/L）was added to the reaction,this enzymatic catalyzed reaction was performed with high substrate loading of the novel recombinant esterase,the production of（R）-CHBE was 337 mM.