Abstract:D-psicose can be produced from D-fructose using D-psicose-3-epimerase (DPE). However, the reaction catalyzed by DPE is reversible, which eventually reaches equilibrium between the D-fructose and D-psicose leading to a low conversion rate. L-rhamnulose kinase (RhaB) catalyzes the phosphorylation of ketoses in an irreversible way. In a cascade reaction based on the combination with DPE, RhaB can break the equilibrium by phosphorylating the D-psicose. Thus, an increased production of D-psicose can be expected. Polyphosphate kinase(PPK) is overexpressed in the cascade reaction to establish the ATP-regeneration system in order to reduce the industrial costs. DPE, RhaB and PPK were overexpressed in E. coli Rosetta(DE3) respectively to explore the optimum condition and the product yield. RhaB protein with a molecular weight of 5.3×104 was thus achievedat pH 8.0 under 35 ℃. When RhaB coupled with DPE, the consequent yield of D- psicose could reach 70% under optimum condition, i.e.,pH 8.5, 35 ℃, Mg2+ and DPE∶RhaB of 1∶2. In the cascade reaction of DPE and RhaB combined with PPK, the ATP concentration was reduced to 1/5 of D-fructose and the yield of D-psicose was 50%. The results provide a theoretical basis for the industrial production of D-psicose.
