Expression Of Epimerase And Efficient Production Of D-allulose

The rare sugar D-allulose has important physiological functions,which is an ideal substitute for sucrose production from D-fructose and has been used as an ingredient in foods and dietary supplements.D-Allulose has been produced through the isomerization of D-fructose by D-allulose 3-epimerase(DAEase)in the industry with the high price because of the low conversion rate and high cost of product separation and purification.In this study,an efficient and low cost process with two-stage for D-allulose producing was proposed.In the first stage,E.coli BL21(DE3)with the gene of D-allulose 3-epimerase(DAEase)from Flavonifractor plautii was constructed to realize the synthesis of D-allulose.The reaction conditions by whole recombinant cells were optimal at p H 7.5,65 ~oC,and the cell was extremely thermostable with the half-life at 60~oC was 2.7 h without metal ions.Different buffer were compared on the conversation yield of the D-allulose,and Tris-HCl,PBS and pure water did not affect the conversion rate,which all remained about 33%.The production rate of D-allulose was 231 g/L/h with conversion rate 33%at 2.4 g/L E.coli in pure water.With boric acid added(boric acid to D-fructose was 0.4),the conversion rate was as high as 63%with 378 g/L/h.In the second stage,the Kluyveromyces marxianus was used to consume residual D-fructose to produce ethanol in the mixed system.D-Allulose accounts for 100%of the total sugar concentration without isolating after the ethanol fermentation.In order to improve the thermal stability of the enzyme,a semi-rational design method was used to find the suitable mutation sites and carry out site-directed mutation.In this study,the homology modeling,sequence alignment and proline effect were used to search for suitable mutation sites,and the following four sites were selected for corresponding mutation(G61P,L62P,V109P and G108P).The mutation results were evaluated.In summary,these results laid a good foundation for low cost industrial production of D-allulose.