Expanding the high-pH range of the sucrose synthase reaction by enzyme immobilization

Abstract

The glycosylation of an alcohol group from a sugar nucleotide substrate involves proton release, so the reaction is favored thermodynamically at high pH. Here, we explored expansion of the alkaline pH range of sucrose synthase (SuSy; EC 2.4.1.13) to facilitate enzymatic glycosylation from uridine 5’-diphosphate (UDP)-glucose. The apparent equilibrium constant of the SuSy reaction (UDP-glucose + fructose ↔ sucrose + UDP) at 30 °C increases by ∼4 orders of magnitude as the pH is raised from 5.5 to 9.0. However, the SuSy in solution loses ≥80 % of its maximum productivity at pH ∼7 when alkaline reaction conditions (pH 9.0) are used. We therefore immobilized the SuSy on nanocellulose-based biocomposite carriers (∼48 U/g carrier; ≥ 50 % effectiveness) and reveal in the carrier-bound enzyme a substantial broadening of the pH-productivity profile to high pH, with up to 80 % of maximum capacity retained at pH 9.5. Using reaction by the immobilized SuSy with automated pH control at pH ∼9.0, we demonstrate near-complete conversion (≥ 96 %) of UDP-glucose and fructose (each 100 mM) into sucrose, as expected from the equilibrium constant (K_eq = ∼7 × 10²) under these conditions. Collectively, our results support the idea of glycosyltransferase-catalyzed synthetic glycosylation from sugar nucleotide donor driven by high pH; and they showcase a marked adaptation to high pH of the operational activity of the soybean SuSy by immobilization.