Characterization And Thermostability Modification Of D-allulose 3-epimerase

D-Allulose,a C-3 epimer of D-fructose,has many advantages such as high sweetness,low energy,hypolipidemic,and hypoglycemic.It is an ideal substitute for sucrose and has great application value and market prospect in the fields of medicine and food.D-Allulose3-epimerase(DAEase)can catalyze the epimerization at the C-3 position between D-fructose and D-allulose.It is the key enzyme for biological production of D-allulose.However,most DAEases from microorganisms were generally reported to show poor thermostability,which makes them difficult to meet the needs of industrial applications.To promote the industrial application of DAEase,a novel DAEase derived from thermophilic microorganism Thermoclostridium caenicola(Thca-DAEase)was screened by gene mining in this study.The enzymatic properties of Thca-DAEase and its ability to biosynthesize D-allulose were investigated in detail.Additionally,the computer-assisted modification of Thca-DAEase was also carried out to improve its thermostability.The main research contents were summarized as follows:(1)Based on sequence homology searches,sequence clustering analysis,and feature annotations,Thca-DAEase was screened.Its protein accession number is SHI77623.1 and it is composed of 289 amino acids.The gene encoding the enzyme was synthesized and connected to the expression vector p ET28a-Cred to construct the recombinant plasmid.The target enzyme was produced using E.coli BL21(DE3)as the host,and purified by Ni²⁺affinity chromatography.SDS-PAGE detection and gel filtration chromatography analysis were performed.The results showed that the target enzyme was correctly expressed.Its subunit molecular weight was about 33 k Da and it existed in dimeric forms.(2)By studying the optimum conditions of Thca-DAEase,it was found that the optimum reaction temperature,optimum p H and optimum metal ions were 65?,p H 7.5,and Co²⁺,respectively.Metal ions are indispensable for Thca-DAEase to show the enzyme activity.In the range of p H 6.5-8.0,its enzyme activity was less affected,and more than 89.0%of the relative activity can be maintained.The results of kinetic and thermodynamic stability studies showed that the half-life(t_1/2)of Thca-DAEase at 50?was 13.64 h,and the melting temperature(T_m)was 62.42?.Co²⁺incubation could improve its thermostability.(3)By studying the substrate specificity and kinetics of Thca-DAEase,it was found that when D-fructose was used as the substrate,the specific activity of Thca-DAEase was 242.4±2.3 U/mg,and K_m,k_cat,and k_cat/K_m were 4.5±8.0 m M,204.5±0.4 s^-1,and 2.2±0.2 m M^-1 s^-1,respectively.Thca-DAEase was used to biosynthesize D-allulose and the results showed that the epimerization reactions reached equilibrium after 5 h with a conversion yield of 29.2%at p H 7.5 using 500 g/L D-fructose as the substrate.While under the condition of weak acid(p H6.5),the equilibrium conversion could also reach 28.0%.(4)The hot spots for mutagenesis were rational selected based on the prediction of protein folding free energy change,and four single-point mutants A70P,G107P,F155Y,and D162T with improved thermostability were obtained.The optimum temperature of mutant G107P was increased by 5?,and the T_m value was increased by 5.70?.Its t_1/2 value at 60?was 40.6 times that of the wild-type Thca-DAEase.The results of ordered combinatorial mutation showed that four positive single-point mutations had additive effects on the thermostability of Thca-DAEase.The T_m value of the four-point combinatorial mutant Var3reached 79.48?,and its optimum temperature was increased by 10?.After incubation at60?for 24 h,more than 80%of the residual enzyme activity was maintained.Molecular dynamics simulation and structural analysis showed that the improvement of thermostability of the mutant might be related to the strengthening of overall rigidity,the formation of hydrogen bonds between subunits and the optimization of surface charge distribution.