Thornthan Sawangwan

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.

Glucosylglycerol and glucosylglycerate as enzyme stabilizers.

Compatible solutes constitute a diverse class of low‐molecular‐mass organic molecules that are accumulated in high intracellular concentrations in response to the external stress of hyperosmolality or high temperature. Many of these compounds like α, α‐trehalose are well known for their stabilizing effect on protein structure and could lead to development of more stable protein formulations. Negatively charged solutes like mannosylglycerate (R‐2‐O‐α‐D‐mannopyranosyl‐glycerate) are widespread among (hyper)thermophilic microorganisms and are thought to be exceptionally potent stabilizers of proteins under high‐temperature denaturation conditions. To further inquire into the role of compound charge for protective function, we have compared two naturally occurring and structurally related solutes, glucosylglycerol (2‐O‐α‐D‐glucopyranosyl‐sn‐glycerol) and glucosylglycerate (R‐2‐O‐α‐D‐glucopyranosyl‐glycerate), as stabilizers of different enzymes undergoing inactivation through elevated temperature or freeze drying, and benchmarked their effects against that of α,α‐trehalose. Glucosylglycerate in concentrations of ≥0.1 M was the most effective in preventing thermally induced loss of enzyme activity of lactate dehydrogenase, mannitol dehydrogenase, starch phosphorylase, and xylose reductase. α,α‐Trehalose could usually be replaced by glucosylglycerol without compromising enzyme stability. Glucosylglycerol and glucosylglycerate afforded substantial (eightfold) protection to mannitol dehydrogenase during freeze drying.

Single-step enzymatic synthesis of (R)-2-O-α-D-glucopyranosyl glycerate, a compatible solute from micro-organisms that functions as a protein stabiliser

Regioselective glucosylation of R-glycerate catalysed by sucrose phosphorylase in the presence of sucrose as the donor substrate provided the natural compatible solute (R)-2-O-alpha-D-glucopyranosyl glycerate with complete regioselectivity in an optimised synthetic yield of 90% R-glycerate converted and a concentration of about 270 mM.

Production of glucosyl glycerol by immobilized sucrose phosphorylase: Options for enzyme fixation on a solid support and application in microscale flow format

2-O-(α-d-Glucopyranosyl)-sn-glycerol (αGG) is a natural osmolyte. αGG is produced industrially for application as an active cosmetic ingredient. The biocatalytic process involves a selective transglucosylation from sucrose to glycerol catalyzed by sucrose phosphorylase (SPase). Here we examined immobilization of SPase (from Leuconostoc mesenteroides) on solid support with the aim of enabling continuous production of αGG. By fusing SPase to the polycationic binding module Zbasic2 we demonstrated single-step noncovalent immobilization of the enzyme chimera to different porous supports offering an anionic surface. We showed that immobilization facilitated by Zbasic2 was similarly efficient as immobilization by multipoint covalent attachment on epoxy-activated supports in terms of production of αGG. Enzyme loadings of up to 90mg enzyme g-1 support were obtained and the immobilized SPase was about half as effective as the enzyme in solution. The high regio- and chemo-selectivity of soluble SPase in αGG synthesis was retained in the immobilized enzyme and product yields of >85% were obtained at titers of ∼800mM. The Zbasic2-SPase immobilizates were fully recyclable: besides reuse of the enzyme activity, easy recovery of the solid support for fresh immobilizations was facilitated by the reversible nature of the enzyme attachment. Application of immobilized Zbasic2-SPase for continuous production of αGG in a microstructured flow reactor was demonstrated. Space-time yields of 500mmol αGG L-1h-1 were obtained at product titers of ∼200mM. The continuous microreactor was operated for 16days and an operational half-life of about 10days was determined.