Cloning、Expression And Application Of A Thermophilic Esterase Tnap-1739

Esterases, a kind of hydrolases, which can catalyze the cleavage and formation ofester bond, are generally distributed in many species like plants, animals andmicroorganisms. Esterases can participate in a variety of chemical reactions, such as,transesterification, esterification, and hydrolysis. Because of their excellent advantagesof good thermal stability and wide range of organic tolerance, thermophilic esteraseswith special source attract more attention recently.This paper aims to isolate two new gene tnap-1739and tnap-1753via PCR fromThermotoga naphthophila RUK-10，which were successfully expressed in E. coli BL21(DE3) and E. coli BLP, respectively. Enzyme activity test results showed that Tnap-1739has high hydrolysis activity while Tnap-1753has low hydrolysis activity. Amino-acid sequence alignment showed that the recombinant esterase Tnap-1739had a typicalα/β hydrolysis structure, which belongs to hydrolases Ⅲ. After optimization ofexpression and purification, the pure target protein was achieved (GelPro purity91%)with the purification fold and recovery ratio3.22and20%respectively. The molecularmass of this thermophilic esterase was about30kDa confirmed by SDS-PAGE andGelpro analysis. Substrate specificity shows that Tnap-1739has much higher activitytoward short chain p-nitrophenol ester than longer ones. The optimum temperature ofTnap-1739was80oC for hydrolysis of p-NPC3. Compared to other esterases, Tnap-1739has a wide temperature range, it showed activity form45oC to95oC. The half-life of Tnap-1739at70oC and80oC were15hours and3hours respectively. Theoptimum pH of this enzyme was7.5. Tnap-1739was not a metal ion reliable enzyme,but Fe3+can greatly inhibit its activity. It was stable in many organic solvents, acetonehas the least virulence toward the Tnap-1739. The values of Km、Vmax、Kcatwere0.39mM、2440.21μM.min-1.mg-1、7391.50S-1with p-NPC3as substrate, showing itsexcellent substrate affinity and catalytic efficiency. Tnap-1739can catalyze aldol reaction using acetone and aldehyde as substratewith the presence of water. Compared to chemical synthesis, enzyme catalytic reactionattracts more attention because of the advantages such as mild reaction conditions, lowenergy consumption, environmental friendly, easy to separation and purification.According the references, therophilic esterases could catalyze the reaction under ahigher temperature than normal temperature enzymes, which can accelerate the reactionrate and had a good stereoselectivity toward the production of chiral drugs.