Heterologous Expression And Immobilization Of D-allulose 3-epimerase In Bacillus Subtilis

D-allulose is an epimer at the C-3 position of D-fructose.It is a rare sugar and is widely used in food,medicine,health care products and other fields.D-allulose 3-epimerase(DPEase,EC5.1.3.30)is the key enzyme that isomerizes D-fructose to synthesize D-allulose.Various DPEases have been reported,but most of them have poor thermal stability and optimal reaction environment is alkaline.DPEase from Dorea sp.CAG:317 has strong acid and alkali resistance,and DPEase from Clostridium cellulolyticum H10 has better thermal stability.In this study,the above two kinds of DPEase were expressed separately in Bacillus subtilis,and their enzymatic properties were analyzed.Then the coexpression strain was constructed by concatenating the two enzymes,and the co-expression level of DPEase was improved by optimizing the promoter combination.Titanium dioxide,sodium alginate and glutaraldehyde were used as immobilized cell materials.The conditions of immobilized cells were optimized,and their properties were analyzed,which provided a theoretical basis for the application of DPEase in industrial production.(1)The DPEase gene fragments of Dorea sp.CAG:317 and C.cellulolyticum H10 were cloned by designed primers,and the recombinant strains B.subtilis 168/pMA5-Ds-dpe and B.subtilis 168/pMA5-Rc-dpe were constructed.The target protein was successfully expressed after 24 h of shaking flask culture.The cells were collected,and then the total enzyme was extracted and purified to study the enzymatic properties of DPEase.The optimal reaction temperature of DSDPEase is 60?,which is not resistant to high temperatures and has a half-life of less than 1 h at 45?.The optimal reaction temperature of RCDPEase is 70?,the relative activity of the enzyme at 65?75? is greater than 90%,the half-life at 45? is more than 4 hours,and the thermal stability is good.The optimal reaction pH of the two DPEases is 8.5,and the acid and alkali resistance of DSDPEase is better than RCDPEase.The results show that the reaction solution of enzyme-catalyzed monosaccharide is not easy to browning at pH 6.5,OD₄₂₀ is 0.114,and the reaction solution at pH 8.5 has deeper browning,OD₄₂₀ is0.261 and 0.259.(2)Firstly,the bicistronic recombinant strains B.subtilis 168/pMA5-Ds-Rc-dpe and B.subtilis 168/pMA5-Rc-Ds-dpe were constructed to co-express DSDPEase and RCDPEase.The specific enzyme activities of the total enzyme solution of the above recombinant strains at pH 6.5 were 18.9 U·mL^-1 and 17.5 U·mL^-1,respectively,which exceeded the specific enzyme activities of single enzyme expression.Through the comparison of specific enzyme activity,the optimal sequence of DSDPEase and RCDPEase genes is determined to be5'-Ds-Rc-dpe-3'.(3)To construct a dual-promoter recombinant expression vector,first use the P_{Hpa II}carried by pMA5 itself as the first promoter to select P_{Hpa II},P₄₃,P_srfA,and P_spoVG as the second promoters to construct recombinant bacteria B.subtilis 168/pMA5-Ds-dpe-P_{Hpa II}-Rc-dpe,B.subtilis 168/pMA5-Ds-dpe-P₄₃-Rc-dpe,B.subtilis 168/pMA5-DS-dpe-P_srfA-Rc-dpe,B.subtilis168/pMA5-Ds-dpe-P_spoVG-Rc-dpe,after shaking flask fermentation,DPEase can be successfully expressed,The specific enzyme activity of the crude enzyme solution at pH 6.5was determined.The specific enzyme activity of B.subtilis 168/pMA5-DS-dpe-P_srfA-Rc-dpe was the highest,reaching 154.43 U·mL^-1,which was 9 times the specific enzyme activity of single enzyme expression.Construct a double-promoter recombinant expression vector.First,pMA5 itself carries P_{Hpa II} as the first promoter,and P_{Hpa II},P₄₃,P_srfA,and P_spoVG are used as the second promoters to construct recombinant bacteria B.subtilis168/pMA5-Ds-dpe-P_{Hpa II}-Rc-dpe,B.subtilis 168/pMA5-Ds-dpe-P₄₃-Rc-dpe,B.subtilis168/pMA5-Ds-dpe-P_srfA-Rc-dpe,B.subtilis 168/pMA5-Ds-dpe-P_spoVG-Rc-dpe,after shaking flask fermentation,DPEase can be successfully expressed.The specific enzyme activity of the total enzyme solution under pH 6.5 was determined.Among them,the specific enzyme activity of B.subtilis 168/pMA5-Ds-dpe-P_srfA-Rc-dpe was the highest,reaching 154.43U·mL^-1,which was 9 times the specific enzyme activity of single enzyme expression.Then use P_srfA as the second promoter,and replace the first promoter with P₄₃ and P_spoVG to construct recombinant bacteria B.subtilis 168/pMA5-P_spoVG-Ds-dpe-P_srfA-Rc-dpe,B.subtilis168/pMA5-P₄₃-Ds-dpe-P_srfA-Rc-dpe,SDS-PAGE analysis results showed that B.subtilis168/pMA5-P_spoVG-Ds-dpe-P_srfA-Rc-dpe can successfully express DPEase,but at 33 k Da B.subtilis 168/pMA5-P₄₃-Ds-dpe-P_srfA-Rc-dpe lanes did not appear obvious specific bands.Under the condition of pH 6.5,the specific enzyme activity of B.subtilis168/pMA5-P_spoVG-Ds-dpe-P_srfA-Rc-dpe is 228.5 U·mL^-1,which is 13.7 times higher than the total specific enzyme activity of single enzyme expression.(4)Adding appropriate amount of titanium dioxide to the traditional immobilized material sodium alginate can significantly improve the enzyme activity recovery and mechanical strength of the immobilized cells.Experimental results showed that 2%SA,2%CaCl₂,0.03%glutaraldehyde solution and,the ratio of titanium dioxide(TiO₂)to sodium alginate of 1:4 were optimal for immobilization.Under these conditions,the recovery rate of immobilized cells activity was as high as 82%.Compared with free cells,the optimal reaction temperature of immobilized cells remained unchanged at 80? and the thermal stability was improved.After 10 consecutive cycles,the enzyme activity recovery rate still maintained at58%,with the mechanical strength of 100%,the conversion rate of 28.8%,and the residual enzyme activity of approximately 70.5%.