Thermostability Enhancement And Immobilization Of D-allulose 3-epimerase From Clostridium Cellulolyticum

D-allulose,a rare sugar with high sweetness and low calories,is C-3 epimer of D-fructose.D-allulose has the function of regulating lipid metabolism,maintaining blood sugar levels and anti-obesity,which is one of the most promising sweeteners.It has broad application prospects in the fields of medicine and food.At present,D-allulose is mainly synthesized by D-allulose 3-epimerase（DAEase）,which can catalyze the epimerization reaction between D-fructose and D-allulose.However,industrial applications of DAEase are still limited by its poor thermostability and low reusability.In this study,directed evolution was applied to improve the enzyme activity and thermostability of DAEase derived from Clostridium cellulolyticum H10（CcDAEase）and combinatorial mutation was carried out to obtain combined mutants with improved thermostability.The enzymatic properties and conversion yield of D-fructose of the mutants were investigated.Finally,the combined mutants with improved thermostability were recombinant expressed in Bacillus subtilis and immobilized to gradually improve the potential in industrial application of CcDAEase.The main research contents of this study are as follows:（1）Improving the enzyme activity and thermostability of CcDAEase by directed evolution.The biosensor based on transcription factor PsiR was constructed and optimized in terms of medium components,and stability of plasmid used for screening.Mutant libraries were screened by the optimized ultrahigh-throughput screening method using flow cytometer and optimized microplate screening method.The mutants T119K,D281G and C289R with significantly improved thermostability were obtained.（2）Characterization of the mutants’enzymatic properties and analysis of structure-activity relationships.By characterizing the enzymatic properties of T119K,D281G and C289R,it was found that the optimum temperature(T_opt)of D281G and C289R was 70℃,which was 5℃higher than that of wild type,and the half-life(t_1/2)were 13.80-fold and 13.88-fold as that of wild type at 65℃,respectively.By prediction of protein structure and molecular dynamics simulation,it was found that the formation of hydrogen bonds or salt bridges between subunits or in subunits,the increased rigidity of loop region betweenα4 andβ4 and C-terminal of CcDAEase,the optimized surface charge distribution,and the weakened electrostatic repulsion may be the reasons for the improved thermostability of the mutants.（3）Construction of combined mutants with improved thermostability.D281G,C289R and T119K with improved thermostability were combined and combined mutant T119K/D281G/C289R was obtained.The T_opt of T119K/D281G/C289R was 65℃,the melting temperature（T_m）was 14.36℃higher than that of the wild type,and the t_1/2 at 65℃was82.14-fold as that of the wild type.D281G and C289R were combined with previously identified mutant H207L with improved specific activity but decreased thermostability and combined mutants H207L/D281G/C289R was generated.The specific activity of H207L/D281G/C289R was 2.24-fold as that of the wild type,the T_opt was 65℃,theΔT_m value was 11.48℃,and the t_1/2 value at 65℃was 23.80-fold as that of the wild type.The above experimental results indicated that D281G and C289R could improve thermostability.（4）Recombinant expression and whole cell immobilization of T119K/D281G/C289R in B.subtilis.The recombinant plasmid pHY300PLK-Δ109-T119K/D281G/C289R was constructed and transformed into B.subtilis WS9,WHS9,and WS11,respectively,and the expression of recombinant strains was determined.The enzymatic activity of B.subtilis WHS9/pHY300PLK-Δ109-T119K/D281G/C289R was the highest,which was 3.26-fold as that of B.subtilis WS11/pHY300PLK-Δ109-ccdae.The recombinant strains were immobilized by waterborne polyurethane（WPU）and diatomite（entrapment-adsorption）.The optimum immobilization conditions were as follows:the ratio of waterborne polyurethane to cells was1:1(w·w^-1),the concentration of 40μm diatomite was 5 g·L^-1,the concentration of immobilized cells added in the enzyme conversion system was 50 g·L^-1,and the rotational speed was50 r·min^-1.The immobilized cells had highest enzyme activity at 80℃and pH 7.5.The T_opt of immobilized cells was 10℃higher than that of unimmobilized cells.The conversion yield of immobilized cells remained 80%of the highest conversion yield after reusing for 64 times.