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

Abstract

Despite the unsurpassed selectivity that enzymesu sually offer, biocatalytic transformations repeatedlyf all shorto ft he robustness andp rocess efficiency demanded for production-scalec hemical 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 potentialo ft he GT reaction, it was vital to combine af ocused, kinetic characteristics-based enzyme selection with ar eaction design properly aligned to thermodynamic constraints.T he equilibrium constant (K eq )f or the conversion of sucrose and uridine 5'-diphosphate (UDP) into the target product UDP-a-d-glucose and d-fructose decreasedw ith increasing pH due to deprotonation of the b-phosphate group of UDP above the pK a of 6.0. Proton uptakec oupled to the glucosyl transfer made it essential that the pH was carefully con-trolledt hroughout the reaction. Comparing two SuSys from Acidithiobacillus caldus and Glycine max (soybean), substrate inhibition by UDPs uperseded catalytic efficiency as the prior selection criterion, demanding choice of the bacterial GT for use at high UDP concentrations.R eaction at the operational pH optimum, determined as 5.0, gave 255 mM (144 gL À1 )o fU DP-glucosei n8 5% yield from UDP. Using an enzyme concentration of only 0.1 gL À1 , as pace-time yield of 25 gL À1 h À1 was obtained. The mass-based turnovern umber (g product formed per ge nzyme added) reachedavalue of 1440f rom as ingle batch conversion. Therefore,t hese parameters of the UDP-glucose synthesiss how that the reaction of aG Tc an be pushed to ap rocess efficiency typicallyr equiredf or implementation in fine chemicals production.

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

“…8 and Tables S3 to S6). A similar pH dependence has been reported for UDP-dependent sucrose synthase (62). The application of eQuilibrator, version 2.0, to determine thermodynamic equilibria has successfully been implemented with sucrose synthase by other investigators (62).…”

“…Equilibrium constants of 23 and 225 were calculated for ADP and UDP, respectively, which reasonably matched the values that were observed experimentally (K eq values of 30 and 157, respectively). Based on this, our calculations predict a shift of the equilibrium constant toward the starting materials under increasingly acidic conditions due to the protonation of UDP (pKa 5.5 to 6.5 [61,62]) ( Fig. 8 and Tables S3 to S6).…”

Glycosyltransferase cascades for natural product glycosylation: Use of plant instead of bacterial sucrose synthases improves the UDP‐glucose recycling from sucrose and UDP

“…Before comparing UDP‐glucose recycling by the different SuSys we had to define useful reaction conditions for the cascade conversions. Unlike nothofagin synthesis by Os CGT (pH optimum ≈8.5), UDP‐glucose formation by SuSy was favored under acidic conditions (≈pH 5–6) . We previously recognized that both enzymatic conversions were well combinable when buffered to pH 7.5 with 50 mM HEPES containing 50 mM KCl and 13 mM MgCl 2 .…”

“…Therefore, UMP besides UDP and UDP-glucose had to be quantified in addition to phloretin and nothofagin. We noted that (sugar) nucleotides like UMP, UDP and UDP-glucose could also be analyzed by reversed-phase HPLC if an TBAB based ion-pairing protocol was used [11]. By applying an isocratic flow at 12.5% acetonitrile (sugar) nucleotides were separated in just 1.5 min (Supporting information, Fig.…”

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP‐glucose production with sucrose synthase

“…It was known from studies using the free enzyme (Gutmann and Nidetzky, ), that pH control at 5.0 is crucial for high conversion. The reaction was performed in a total volume of 400 mL using 84.1 mmol UDP disodium salt (38 g), 600 mmol sucrose (205 g), and 4 mmol MgCl 2 (0.38 g) dissolved in water.…”