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

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Abstract

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 mm), 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.

Original languageEnglish
Pages (from-to)1387-1390
Number of pages4
JournalChemBioChem
Volume18
Issue number14
DOIs
Publication statusPublished - 18 Jul 2017

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Keywords

  • biocatalysis
  • carbohydrates
  • glycosylation
  • phosphorylation
  • reaction mechanisms

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

Cooperations

  • NAWI Graz

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