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Walking a Fine Line with Sucrose Phosphorylase: Efficient Single‐Step Biocatalytic Production of l ‐Ascorbic Acid 2‐Glucoside from Sucrose
Author(s) -
Gudiminchi Rama Krishna,
Nidetzky Bernd
Publication year - 2017
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700215
Subject(s) - ascorbic acid , chemistry , sucrose , substrate (aquarium) , anomer , protonation , glucoside , catalysis , yield (engineering) , stereochemistry , organic chemistry , food science , medicine , ion , oceanography , alternative medicine , pathology , geology , materials science , metallurgy
The 2‐ O ‐α‐ 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 ‐α‐glucosylation of l ‐ascorbic acid from sucrose with high efficiency and perfect site‐selectivity. Optimized synthesis with the enzyme from Bifidobacterium longum at 40 °C gave a concentrated product (155 g L −1 ; 460 m m ), from which pure AA‐2G was readily recovered in ∼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.