Characterization And Molecular Modification Of D-lyxose Isomerase

D-mannose is a functional sugar,which has a variety of beneficial physiological effects,such as prebiotic and antihypertensive functions.It has been widely used in food,medicine and cosmetics industries.The traditional extraction method and chemical method in the preparation of D-mannose will add a lot of organic reagents,and the reaction conditions are very strict,therefore,the preparation of D-mannose by biological method has become a recent research hotspot,in which D-lyxose isomerase is used to catalyze the conversion of D-fructose to D-mannose.D-LIase is an aldose ketose isomerase with a broad substrate spectrum,which can catalyze the conversion of D-lyxose to D-xylulose and D-fructose to D-mannose.In this study,we used gene mining technology to find a new D-lysose isomerase,and used molecular modification technology to acid modify the enzyme.We studied the properties of the enzyme and the change of Maillard reaction degree after acid modification,and successfully obtained a acid resistant and high temperature resistant D-lysose isomerase,which significantly reduced the Maillard degree in the reaction process.In this study,a D-LIase from Thermoprotei archaeon was identified(Thar-D-LIase).The results showed that the optimum temperature of the enzyme was 80?,and the optimum p H was 6.5.The enzyme showed significant dependence on metal ions,and its optimum metal ion was 0.5 mM Ni²⁺.It showed good thermostability,and could maintain more than 50%of the relative activity after incubating at 70?and 80?for 48 h and 33 h,respectively.The T_m(melting temperature)value of the enzyme is 88.30?,which is the highest compared to other reported D-LIases.When using D-fructose,D-mannose,D-lyxose and L-ribose as the substrate,respectively,the K_mvalues of Thar-D-LIase were determined to be 10.1,111.5,76.1 and1427.9 mM,and the catalytic efficiency(k_cat/K_m)values were determined to be 14.4,25.2,45.4 and 19.6 mM^-1·min^-1,respectively.The ability of the enzyme to catalyze the synthesis of D-mannose from different D-fructose concentrations and at different temperatures were also studied.The results showed that high temperature could shorten the time enzyme takes to reach reaction equilibrium,and the increased substrate concentration could lead to substrate inhibition.At 80?,the conversion rate of D-mannose decreased from 23.9%to 17.1%after12 h.Using 25 g/L?100 g/L fructose as the substrate,the conversion rate of D-mannose decreased from 23.9%to 17.1%after 10 h in 80?.Because the Maillard reaction can be inhibited significantly in acid conditions,site-directed mutagenesis technology was used to modify the enzyme to decrease its optimal p H and increase the catalytic activity at acid conditions.The designed variants include E8K,E9K,E19K,E28K,D37K,D42K,D81K,E82K,K83E,P105K,E119K,E165K,E179K,E182K,E82K/P105K,E82K/E165K and P105K/E165K,and the combinational variant E82K/P105K showed the best catalytic capability.The results showed that the optimal p H of E82K/P105K shifted from 6.5 to 5.5,and its enzymatic activity was 3.4 times higher than that of wild-type enzyme at p H 5.5.The kinetic parameters K_mof E82K/P105K to D-fructose and D-mannose were 78.77 mM and 328.12 mM,respectively,and the k_cat/K_mwere 15.37mM^-1·min^-1and 48.92 mM^-1·min^-1,respectively.Under the conditions of 80?and p H 5.5,the conversion rate of D-fructose reached 18%after 10 h using 80 g/L D-fructose as substrate,which was higher than that of wild-type enzyme(17%).In addition,the Maillard degree at p H5.5 is much lower than that at p H 6.5.Therefore,the molecular modification in this study successfully improved the catalytic capability of Thar-D-LIase in acidic environment,and greatly reduced the Maillard reaction.