Walking a Fine Line with Sucrose Phosphorylase: Efficient Single-Step Biocatalytic Production of L-Ascorbic Acid 2-Glucoside from Sucrose

The 2-O-alpha-d-glucoside of L-ascorbic acid (AA-2G) is a highly stabilized form of vitamin C, with important industrial applications in cosmetics, food, and pharmaceuticals. AA-2G is currently produced through biocatalytic glucosylation of L-ascorbic acid from starch-derived oligosaccharides. Sucrose would be an ideal substrate for AA-2G synthesis, but it lacks a suitable transglycosidase. We show here that in a narrow pH window (pH 4.8-6.0, with sharp optimum at pH 5.2), sucrose phosphorylases catalyzed the 2-O-alpha-glucosylation of L-ascorbic acid from sucrose with high efficiency and perfect site-selectivity. Optimized synthesis with the enzyme from Bifidobacterium longum at 40 degrees C gave a concentrated product (155 gL(-1); 460 mm), from which pure AA-2G was readily recovered in similar to 50% overall yield, thus providing the basis for advanced production. The peculiar pH dependence is suggested to arise from a reverse-protonation mechanism in which the catalytic base Glu232 on the glucosyl-enzyme intermediate must be protonated for attack on the anomeric carbon from the 2-hydroxyl of the ionized L-ascorbate substrate.