Overexpression,characterization And Molecular Modification Of D-psicose 3-epimerase

D-Allulose is an epimer of D-fructose,which is a rare monosaccharide and exists in extremely small quantities in nature.It is an ideal substitute for sucrose,because it has 70%of the sweetness of sucrose and ultra-low the energy.In addition,it has many unique physiological functions and potential health benefits,including the ability to reduce the accumulation of intra-abdominal fat,reduce postprandial glycemic elevation,suppress hepatic lipase activity,scavenge reactive oxygen species?ROS?,treat atherosclerotic diseases,increase insulin resistance and protect the nervous system.However,it is scarce in nature,and difficult to chemically synthesize.As this result,the bioconversion of D-allulose has attracted researchers'attention.It has been confirmed that ketose 3-epimerase plays an important role in the bioconversion of D-fructose to D-allulose.In this study,the encoding genes of Dorea sp.DPEase,F.bacterium DPEase,Rhizobium sp.DPEase and Roseburia sp.DPEase were cloned and expressed in E.coli BL21?DE3?.Then,the recombinant enzymes was purified,identified and characterized.Dorea sp.DPEase displayed the optimum activity at acidic condiction,and showed the highest specific activity.As this result,Dorea sp.DPEase was selected as the main target for further study.The A.cellulolyticus GIase and Dorea sp.DPEase genes were co-expressed,which could convert D-glucose to D-allulose in one step.In addition,Dorea sp.DPEase gene was inserted into pUB-P43-dore-dal without antibiotic resistance genes,and then transformed into B.subtilis1A751?dal^-?for food-grade expression.Furthermore,the molecular modifications of catalytic efficiency and thermostability were carried out,and homology modeling was used to analyze the mutation results.The main results are as follows:?1?The encoding genes of Dorea sp.DPEase,F.bacterium DPEase,Rhizobium sp.DPEase and Roseburia sp.DPEase were were synthesized and inserted into the vector pET-22b?+?.Then the recombinant plasmids were transformed into E.coli BL21?DE3?and induced by IPTG.Then the recombinant proteins were purified by nickel-affinity chromatography.The SDS-PAGE results showed that the monomer molecular weights were around 32 to 34 kDA,which were corresponding to the theory values.The HPLC results confirmed that all these enzymes could conver D-fructose to D-allulose,and showed best substrate specificity to D-allulose.?2?The optimum conditions for Dorea sp.DPEase?F.bacterium DPEase?Rhizobium sp.DPEase?Roseburia sp.DPEase were pH 6.0 and 70°C,pH 9.0 and 70°C,pH 9.0 and 65°C,and pH 7.0 and 65°C respectively.The specific activity of Dorea sp.DPEase was 803.5 U/mg,which was 1.6 to 5.3 fold higher than other DPEases.All the four DPEases were metal-depedent,and the optimum ions were Co²⁺.Besides,Co²⁺could also improve the structure stability of the proteins.After adding Co²⁺,the T_m values of Dorea sp.DPEase,F.bacterium DPEase,Rhizobium sp.DPEase and Roseburia sp.DPEase were increased by 8.6,10.2,11.5 and 13.2?,respectively.All the four DPEases showed specificity activity to D-allulose,D-fructose and D-tagatose,while they showed no specificity activity to L-sorbose except F.bacterium DPEase.In the acidic conditions(70°C,pH 6.0,1 mmol/L Co²⁺),when using 500 g/L D-fructose as the substrate,Dorea sp.DPEase could produce 114 g/L D-allulose,which is much faster and higher than any other reported DPEases.?3?The Dorea sp.DPEase and A.cellulolyticus GIasegenes were inserted into the Nco I/Hind III and Nde I/Xho I restriction sites of pETDuet-1 vector,respectively.Then the co-expression plasmid was transformed into E.coli BL21?DE3?for cultivation.The optimum condition for the co-expression cells was pH 6.5 and 75°C,and it showed relatively high thermostability below 60°C.Co²⁺and Mn²⁺improved the catalytic activity significiantly,and the optimum Co²⁺concentration was 0.4 mmol/L.After reaching the reaction equilibrium,the equilibrium ratio of D-glucose,D-fructose and D-allulose was approximately 6.5:7:3.Using500 g/L D-glucose as the substrate,the co-expression cells could produce 205 g/L D-fructose and 89.1 g/L D-allulose,respectively.?4?A food-grade expression system was constructed,using the dal gene deficiency variant B.subtilis 1A751?dal^-?as the expression host.The Dorea sp.DPEase gene was inserted into the pUB-P43-dore-dal,which replaced the antibiotic resistance gene by dal gene.After 27 h fermentation in Super-Rich medium,the fermentation activity could reach 10.9U/mL.The optimum condition for the recombinant B.subtilis was pH 6.5 and 75°C,with the presence of Co²⁺.After 40%ethanol permeabilization,the activity was increased by up to2.65-fold,with the fermentation activity of 28.9 U/mL.?5?The amino acid residues aroud the O-4,O-5 and O-6 of D-fructose were selected for catalytic efficiency modification.The positive mutant A38E,G105A and A38E/G105A were otained,which increased 22.3%,28.5%and 38.6%in the relative activity.However,the thermostability of the mutations decreased to some extent.The K_m values for D-fructose decreased,and k_cat/K_m values increased,indicating that the affinity to D-fructose was enhanced.After docking,it was found that mutant A38E and G105A could form new H bond,and strengthen the original H bond nets.?6?The amino acid residues located at the interface of subunits were selected for thermostability modification.The positive mutant V22C,F154Y,E191D,I193F,I193L and F154Y/E191D/I193F were otained.The optimum temperature of I193F and F154Y/E191D/I193F was increased by 5°C.The stability of all mutants was improved,including t_1/2 at 50 and 60°C,and T_m values.For the large scale production,F154Y/E191D/I193F mutant could produce 148.2 g/L D-allulose from 500 g/L D-fructose,which was 34.2 g/L than the wild type enzyme.The homology modeling results showed that,V22C could form slight disulfide bond with Cys54 from the neighbouring?-helix.F154Y could form new H bond with Asn152 from the other subunit,while E191D could also form new H bond with Asn213 from the other subunit.The new bond between different subunits could improve the structure stability of multimeric enzyme.I193F and I193L tended to reduce the flexibility and increase the structure stability of the hydrophobic pocket around the surface.