Application Of L-rhamnulose Kinase On Production Of D-psicose

As one of the most attractive rare sugars,D-psicose has a wide application prospect in food,medicine and other fields due to its bioactivities.D-psicose can be produced from D-fructose using D-psicose-3-epimerase?DPE?.However,the reaction catalyzed by DPE is reversible,which leads to a low conversion rate.This research mainly focuses on D-psicose synthesis using DPE,L-rhamnulose kinase?RhaB?and polyphosphate kinase?PPK?,co-expressed in E.coli by whole-cell transformation.Thus,an increased production of D-psicose can be expected.Meanwhile,the substrate specificity of RhaB and its application for rare sugar synthesis are investigated.In this paper,the gene rhaB encoding RhaB was amplified and cloned into the expression plasmid pET-28a.The recombinant plasmid was transformed into the E.coli Rosetta?DE3?to overexpress the protein.Then enzymatic properties of Rha B and its optimum conditions were investigated with D-psicose as the substrate.Our expression results revealed Rha B protein with a molecular weight of 53 kDa was purified,and its optimum p H and temperature were determined as 8.0 and 35?,respectively.Mg²⁺was the optimum metal ion for the reaction.In the cascade reaction combined with DPE from Clostridium cellulolyticum H10,the optimum conditions with D-fructose as the substrate were also tested and determined as follows:pH,temperature and metal ion were 8.5,35?and Mg²⁺,respectively;The optimum ratio for the combination of DPE and RhaB was 1:2.In the co-expression system based on DPE and RhaB in E.coli,the optimum cell concentration was 20 g?L^-1 and the yield of D-psicose could reach 70%with 20 g?L^-11 D-fructose as the substrate.The whole-cell cascade reaction system based on DPE and Rha B was successfully established.However,the amount of ATP would be reduced due to the ATP price.Thus,PPK from E.coli was overexpressed in the cascade reaction to establish the ATP-regeneration system for the overall whole-cell biotransformation.The results demonstrated that the optimum temperature,pH and metal ion by whole-cell biotransformation were 37?,8.5 and Mg²⁺,respectively.The activity of whole cells was very stable within pH 6.0-10.0 and the bioconversion yield from D-fructose to D-psicose was 70%under the optimum conditions.Thus,the whole-cell cascade reaction system significantly improves the yield of D-psicose,which has practical application significance.Eventually,the substrate specificities of RhaB were analyzed and indicated as follows:RhaB prosessed broad substrate specificities and catalyzed the phosphorylation of C₃-R ketoses in an irreversible way,such as D-psicose,L-fructose,L-tagatose,D-sorbose and so on.Like the production of L-tagatose,in the cascade reaction based on RhaB and D-tagatose-3-epimerase?DTE?which was expressed in E.coli Rosetta?DE3?and purified,the yield of L-tagatose could reach 68%with L-sorbose as the substrate.Our results provide efficient method for the production of rare sugars.