Enzymatic Synthesis Of Rare Sugars With Dihydroxyacetone Phosphate-dependent Aldolases And Glycerol Kinase

Rare sugars are monosaccharides and their derivatives, which are particularly uncommon in nature. Rare sugars possess many potential applications in the food, pharmaceutical and nutrition industries. But the synthetic routes of most rare sugars are limited. This thesis mainly use L-rhamnulose-1-phosphate aldolase from the thermophilic strain Thermotoga maritima MSB8 and glycerol kinase from Escherichia coli to synthesize rare sugars via one-pot multienzyme system.In order to investigate the application of RhaDT.mari in rare sugar synthesis, the gene rhaDT.mari encoding RhaDT.mari was cloned into the plasmid pET-43 a to express Nus-RhaDT.mari in E. coli Rosetta(DE3). Nus-RhaDT.mari protein with a molecular weight of 90 kDa was purified. When Nus tag was cleaved with thrombin, RhaDT.mari protein without Nus tag was obtained with a molecular weight of 27 kDa. The study on the stereoselectivity of RhaDT.mari demonstrated that the stereoselectivity of RhaDT.mari was different in contrast to that of RhaDE.coli when accepting D/L-glyceraldehyde as receptors. Interestingly, Nus tag exerted some influence on the stereoselectivity of RhaDT.mari. In addition, the effect of temperature on the activity of RhaDT.mari was studied and the result showed that the optimum temperature of RhaDT.mari was 50℃. At last, Nus-RhaDT.mari was employed to synthesize rare sugars through a “one-pot four-enzyme” approach with DL-glycerol 3-phosphate as the precursor and D/L-glyceraldehyde as receptors. The products and ratios were detected by HPLC. The four rare sugars(D-psicose, D-sorbose, L-fructose and L-tagatose) after purification were identified by 1H NMR.As the starting material DL-glycerol 3-phosphate in “one-pot four-enzyme” method is relatively expensive, this method was optimized using glycerol kinase. Fisrtly, the gene glpK encoding GK was amplified and cloned into the expression plasmid pET-28 a. The recombinant plasmid was transformed into E. coli to express GK. GK protein with a molecular weight of 56 kDa was purified. On the one hand, L-glycerol 3-phosphate could replace DL-glycerol 3-phosphate produced by GK using cheap substrate glycerol, which was further converted into DHAP by glycerol phosphate oxidase(GPO). On the other hand, DHAP could be directly produced from DHA catalyzed by GK. The generated DHAP could be coupled with the acceptor D-glyceraldehyde catalyzed by different DHAP-dependent aldolases to produce ketose-1-phosphates, which could be dephosphorylated with acid phosphatase(AP) to get ketoses. The products, yields and ratios could be detected by TLC and HPLC. The results demonstrated that ketoses including D-psicose, D-sorbose and D-fructose could be successfully synthesized using one-pot multienzymatic reaction with the cheap substrate glycerol or DHA as the precursor. This approach could provide the theory basis for the synthesis of more other rare sugars and their derivatives.