Synthesis Of D-allulose From D-fructose By Mutienzyme Reaction

D-allulose is a kind of rare sugar with health care function,which plays an important role in food and medicine industry.In this study,a phosphorylation-dephosphorylation reaction was established by coupling D-psicose-3-epimerase（DPE）from Burkholderia Insecticola,L-rhamnulose kinase（Rha B）from Shigella flexneri 2a str.301 and polyphosphate kinase（PPK）from Sinorhizobium meliloti.The conversion rates of D-allulose were both increased by pure enzyme reaction and whole-cell reaction.At the same time,the enzymatic properties of DPE and Rha B were investigated.The main research results are as follows:（1）Expression,purification and enzymatic properties of DPE.DPE from B.Insecticola was expressed in Escherichia coli BL21（DE3）.DPE protein was purified using a nickel ion affinity chromatography column and its enzymatic properties were determined.The results showed that DPE was a dimer protein,and its optimum reaction conditions were 65℃,p H 9with Mn²⁺.Moreover,DPE exhibited strong stability in various temperatures and p H values.DPE showed broad substrate specificity and the kinetic parameters of DPE toward different rare sugars were measured.Among the tested rare sugars,the affinity and the catalytic efficiency of DPE for D-allulose were the highest.When DPE was applied in D-alluose production,500 g×L^-1 D-fructose could be converted to 180 g×L^-1 D-allulose in 30 min and the conversion rate was 36%.This study provides more options for the production of D-allulose by conventional biological methods.（2）Expression,purification and enzymatic properties of Rha B.The p ET28a-rha B and p ET28a-rha B^E437Q plasmids were constructed and expressed in E.coli BL21（DE3）.The proteins were purified using a nickel affinity chromatography column.Compared with the wild type enzyme,the enzymatic activity of the mutant Rha B^E437Q was increased by 10 times,and most of the inclusion body proteins became soluble proteins.The enzymatic properties of Rha B^E437Qwere studied.The results showed that Rha B^E437Q was a monomer with a molecular weight of 54 k Da and the optimum reaction conditions were 40℃,p H 8.5,Mn²⁺.Rha B^E437Qonly has catalytic activity for C-3 sugars with the structure of R configuration.D-allulose was docked into Rha B^E437Q and the preliminary catalytic mechanism was investigated.（3）D-allulose production using multienzyme reaction.The pure enzymes DPE,Rha B^E437Q,PPK were coupled for D-allulose production and the optimum reaction conditions for the cascade reaction was 40℃,p H 8.5,Mn²⁺.When 50 mM of D-fructose,5 mM of ATP,25 mM of Poly P₆ was used under optimized condtion,D-fructose was almost converted into D-allulose;When the concentration of D-fructose was 100,200,300,400 mM,the conversion rates were 80%,75%,65%and 59%,respectively under the same conditions.Then,D-allulose production was performed using E.coli BL21（DE3）whole cells co-expressing DPE,Rha B^E437Q and PPK.The optimal reaction conditions were 40℃,p H 8.5,Mg²⁺,40 g×L^-1wet cells.When 222 mM(40 g×L^-1)of D-fructose,5 mM of ATP,25 mM of Poly P₆ was used,the conversion rate was 65%.