Unlocking the Potential of Leloir Glycosyltransferases for Applied Biocatalysis: Efficient Synthesis of Uridine 5 '-Diphosphate-Glucose by Sucrose Synthase

摘要

Despite the unsurpassed selectivity that enzymes usually offer, biocatalytic transformations repeatedly fall short of the robustness and process efficiency demanded for production-scale chemical synthesis. Nucleotide sugar-dependent "Leloir" glycosyltransferases (GTs) are fine catalysts of glycosylation but there is concern as to whether reactions from this enzyme class are fit for industrial process development. We demonstrate in this study of sucrose synthase (SuSy; EC 2.4.1.13) that, in order to unlock the synthetic potential of the GT reaction, it was vital to combine a focused, kinetic characteristics-based enzyme selection with a reaction design properly aligned to thermodynamic constraints. The equilibrium constant (K-eq) for the conversion of sucrose and uridine 5'-diphosphate (UDP) into the target product UDP-alpha-D-glucose and D-fructose decreased with increasing pH due to deprotonation of the beta-phosphate group of UDP above the pK(a) of similar to 6.0. Proton uptake coupled to the glucosyl transfer made it essential that the pH was carefully controlled throughout the reaction. Comparing two SuSys from Acidithiobacillus caldus and Glycine max (soybean), substrate inhibition by UDP superseded catalytic efficiency as the prior selection criterion, demanding choice of the bacterial GT for use at high UDP concentrations. Reaction at the operational pH optimum, determined as 5.0, gave 255 mM (144 gL(-1)) of UDP-glucose in 85% yield from UDP. Using an enzyme concentration of only 0.1 gL(-1), a space-time yield of 25 gL(-1)h(-1) was obtained. The mass-based turnover number (g product formed per g enzyme added) reached a value of 1440 from a single batch conversion. Therefore, these parameters of the UDP-glucose synthesis show that the reaction of a GT can be pushed to a process efficiency typically required for implementation in fine chemicals production.