Identification and Enzymatic Characterization of a Sugar Phosphatase
[Objective]High value-added monosaccharides can be synthesized through multi-enzyme cascade reactions using starch or dextrin as substrates.In the reaction systems,sugar phosphatase catalyzes the irreversible dephosphorylation reaction of the phosphorylated sugar intermediates to generate the corresponding sugars.This work is aimed to mine the new sugar phosphatase and to characterize this enzyme.[Method]In this study,a gene with unknown function,named as TsPase derived from the thermophilic bacterium Thermoproteus sp.CIS_19,was obtained from gene mining and screening.TsPase was found to have sugar phosphatase activity after its heterologous soluble expression with Escherichia coli BL21(DE3),purification and enzymatic characterization.[Result]TsPase has a Tm value of(80.3±1.3)℃,while the optimal reaction temperature of 70℃and pH of 4.Our study indicated that metal ion Mg2+has a strong promoting effect on TsPase.With the substrate tagatose 6 phosphate kinetic constant Km is(2.40±0.98)mmol/L,and the catalytic constant kcat is(102.50±8.60)min-1.TsPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin.The production of D-tagatose in the reaction equilibrium system was(4.26±0.03)g/L and the conversion rate reach 42.6%±0.3%.[Conclusion]Overall,TsPase possesses fine thermal stability and activity,as well as high conversation rate in vitro synthetic enzymatic biosystem.These characteristics make it great value in future theoretical research and industrial production.
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