Characterization And Thermal Stability Modification Of D-Mannose Isomerase

D-Mannose is an isomer at the C2 position of D-glucose and also an aldose isomer of D-fructose.It has been widely applied in the cosmetic,nutritional,and pharmaceutical industries.D-Mannose has been proposed to be used as a glyconutrient supplement for infants and children.It also holds the protential to be used as nutritional supplement with various health benefits,such as prebiotic effect.Diabetes,inflammation,and urinary tract infections can be prevented and treated with D-mannose.D-Mannose is also frequently used as a key ingredient or raw material in the manufacture of vitamins,D-mannitol and anti-tumor drugs.D-Mannose can inhibit the development of tumors and may be used to treat cancer.In recent years,D-mannose has gained prominence due to its attractive applications.Currently,there are three methods to produce D-mannose,namely extraction,chemical,and bioenzymatic methods.Bioenzymatic methods attract more attention for their low energy consumption and environmental friendliness compared to the other methods.D-Mannose isomerase（D-MIase）can catalyze the conversion of D-fructose to D-mannose.It is a relatively ideal catalyst for D-mannose production.The main content of this thesis:（1）In this paper,three D-MIases from different sources were mined from the NCBI database,namely Deba-D-MIase from Deltaproteobacteria bacterium,Caji-D-MIase from Caballeronia jiangsuensis,and Strh-D-MIase from Stenotrophomonas rhizophil.（2）The experimental results showed that the optimum p H of all three D-MIases was 7.0,the optimum temperature was 55°C,50°C,and 50°C,respectively.Their activities were not dependent on metal ions and their optimum metal ions were Mg²⁺,Mg²⁺,and Ca²⁺,respectively.The T_mvalues of three enzymes were 61.51°C,50.57°C,and 60.69°C,respectively.The specific enzyme activities measured under optimal conditions were 322.3 U mg^-1,275.6 U mg^-1,and 267.2 U mg^-1,respectively.The half-lives of Deba-D-MIase were376.7 min and 231.8 min at 50°C and 55°C,respectively.The corresponding half-lives of Caji-D-MIase were 28 min and 6.5 min,respectively.The values for Strh-MIase were 66 min and 7.8 min,respectively.The kinetic parameters k_catfor Deba-D-MIase was 623.44 s^-1,K_mwas 539.75 m M,and k_cat/K_mwas 1.16 s^-1m M^-1;k_catfor Caji-D-MIase was 734.53 s^-1,K_mwas515.47 m M,and k_cat/K_mwas 1.42 s^-1m M^-1;k_catfor Strh-D-MIase was 352.84 s^-1,K_mwas260.89 m M,and k_cat/K_mwas 1.35 s^-1.The D-fructose with concentrations of 50 g/L and 100g/L were used to produce D-mannose.The conversion rates catalyzed by these D-MIases were all around 25%.（3）The best-performing Deba-D-MIase was selected for thermal stability molecular modification.The mutants R37K,A76V,N89M,N109F,S125Y,G164L,S178M,N234D,and I239L were designed using Fire Prot website.The half-life value of the double-point mutant R37K/G164L at 60°C was measured to be 4 times that of the wild-type Deba-D-MIase,and the optimum temperature of 55°C was not changed.There was almost no decrease in its catalytic activity.