Research On Cloning,expression And Enzymatic Characterization Of Thermophilic Esterase Est11 And Its Application In Sugar Ester Synthesis

Esterases could catalyze many reactions including transesterification, ester synthesis and peptide synthesis. These esterases played important roles in food industry, chemical industry and environmental protection. Compared with the normal esterases, thermophilic esterases showed great potential with a strong thermal stability and organic solvent resistance in biological catalysis synthesis. The purpose of this study was to clone and express a new novel thermophilic esterase and to explore its applications in catalysing the synthesis of sugar esters. Fistly, the target gene Tm1350 from Thermotoga maritima MSB8 was cloned and expressed. The recombinant protein Est11 was got. Then the related enzymatic characteristics were studied. Finally, the application of Est11 in catalysing the synthesis of sugar esters was explored.The results were as follows:(1)The esterase gene Tm1350 from T.maritima MSB8 was 780 bp and 259 amino acid were encoded. Ser103, Asp183 and His249 possiblely were the key catalytic site of Tm1350 through the analysis of amino acids homologous sequences using Blast software and conservative domain and sites using software Clustal W.(2)The target gene Tm1350 was cloned by the method of overlapping PCR and the recombinant plasmid p ET15b-Tm1350 were constructed successfully. Then the recombinant plasmid was transformed into Escherichia.coli BL21 The recombinant enzyme Est11 was expressed stably in recombinant bacteria E.coli BL21. The target protein was purified and the molecular mass was about 28 KDa showed by SDS-PAGE. The purification recovery rate of target protein was 27.13% and the specific activity of Est11 was 993.05 U/mg.(3)The characterization of Est11 were as follows:(a) The optimal function temperature and the optimum p H of Est11 were respectively 70℃ and 6.5. The residual enzyme activity was still above 60% after 5 h treatment at 90℃and the thermal esterase Est11 held the higher resistant ability to the high temperature.(b) Est11 showed stronger hydrolysis ability to nitro phenol ester whose carbon chain length was less than 10 and the optimal hydrolysis substrate of Est11 was p NP-C5.(c) 0.5 m M metal cation chelator EDTA, Ba2+ and Mg2+ had no significant effect on the activity of Est11. The enzyme activity of Est11 was significantly increased by about 20% by Na+ and K+. Ni2+, Mn2+, Ca2+, Zn2+ and Pb2+ had about 50% inhibitions to the activity of Est11. The residual activity of Est11 was down to 14% by Cu2+. Specially, the activity of Est11 was completely inhibited by Al3+.(d) The thermophilic esterase Est11 had strong resistant ability to most organic solvents. Such as the enzymatic activity of Est11 had no significant changes after 2 h treatment by DMSO/methanol/ethanol/acetone/1,4-butanediol/n-butyl alcohol/DMF. And the residual activity of Est11 still held 50% after 2 h treatment by tertiary amyl alcohol/ chloroform/dichloromethane/hexane/toluene/xylene.(e) The enzyme activity of the thermophilic esterase Est11 in the ionic liquids was reduced, such as the residual activity was only 60% after 2 h treatment in [OPy]BF4.(f) SDS, Tween-20, Tween-80 and Triton X-100 had no significant effect on the enzyme activity. The residual enzyme activity was respectively down to 13% and 51% treated by 5 m M DTT and 5 m M PMSF. The activity was completely inhibited by 5m M DEPC.(g) The activity of Est11 was closely related to its secondary structure of alpha-helix and beta-folding through the IR ananlysis.(4)The thermophilic esterase Est11 could catalyze the esterification reaction of glucose and acetic anhydride and the product catalyzed by Est11 was mainly the alpha-D-Glucose pentaacetate. Which indicated that the thermophilic esterase Est11 had some potentials in catalysing the synthesis of sugar esters.