The Thermostability Modification Of Thermotoga Petrophila Tagatose 4-epimerase And Application

D-tagatose,as a rare sugar,not only has the characteristics of low calorie and good taste,but also has physiological functions such as zero glycemic index and prebiotic effect,which has become a research hotspot.At present,D-tagatose is mainly synthesized by chemical and biological methods.However,the existing biological methods have the disadvantages of high substrate cost and limited conversion rate.Therefore,it is of great research significance and application value to find an efficient alternative way to synthesize D-tagatose.D-fructose is the differential isomer of C-4 of D-tagatose,which is low in price and is an ideal substrate for the production of D-tagatose.Therefore,finding new enzymes that can catalyze the synthesis of D-tagatose becomes the key to break down the barriers to the synthesis of D-tagatose.After molecular modification of Tagaturonate 3-epimerase from Thermotoga petrophila,D-tagatose can be synthesized from D-fructose.The mutant was named Tagatose 4-epimerase（T4E）.In this study,T4E was taken as the research object to explore its recombinant expression and whole-cell immobilization.Then,it was modified for thermal stability through rational design and directional evolution strategy,and the obtained dominant mutants were qualitative and applied in enzymology,which laid a certain foundation for the industrial application of T4E.The main research contents of this study are as follows:（1）Recombinant expression,enzymatic properties and whole cell immobilization of T4E in Escherichia coli.The recombinant expression of T4E in E.coli was successfully achieved by fusion of T7 tag on the N-terminal of T4E,and the enzymatic properties of T4E were determined,and the whole cell immobilization of T4E was explored.Results show that the optimum p H of T4E is 8.5,the optimum temperature is 70℃,the half-life of 2.9 h.The optimal immobilization conditions were as follows:sodium alginate concentration(1%,w·v^-1),cell embedding amount(75 g·L^-1),Ca Cl₂ concentration(2%,w·v^-1),diatomite concentration(1.5%,w·v^-1).Under these conditions,the enzyme recovery rate of immobilized cells was 66.7%.（2）Rational design improves thermal stability of T4E.Through software analysis and prediction,T4E structure analysis and combination of B-factor,free energy（?G）and other parameters for mutation prediction,according to disulfide bond strategy to construct mutants,I120C/G157C,A93C/L117C and T276C/L300C,according to B-factor strategy to construct mutants,D213E,D250V,E322H and E334G.The thermal stability and specific activity of the mutant were measured respectively.The thermal stability of the mutant E322H was significantly improved,but its specific activity decreased to a certain extent.The conversion rate of the mutant was lower than that of the wild-type T4E.The results show that it is still difficult to carry out thermal stability transformation of T4E through rational design strategy,and other means should be found to carry out thermal stability transformation of T4E.（3）Directed evolution improves the thermal stability of T4E.A novel T4E high-throughput screening method was constructed and used for high-throughput screening of T4E mutation libraries.A mutant I430P with significantly improved thermal stability was obtained through screening.The mutant G90S/T272A/I430P with significantly increased specific activity was obtained through the second round of screening using this mutant as the parent.Enzymatic properties and application effects of the mutant were also studied.The half-life(t_1/2)of the mutant I430P was 1.83 times that of the wild type,and the unfolding temperature（T_m）was5.1℃higher than that of the wild type.The specific activity of the mutant G90S/T272A/I430P was 21.4%higher than that of the wild type,and the half-life(t_1/2)was 1.69 times higher than that of the wild type.The decompression temperature（T_m）was 3.4℃higher than that of wild type T4E.The enzyme conversion and immobilization effects of the mutant I430P,G90S/T272A/I430P were better than those of the wild type.