D-(+)-Cellobiose (CNP0226604.15) → 6-o-(beta-d-Glucopyranosyl)ascorbic acid

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反応手順

Twenty milligram of a cellulase preparation (Sigma) was dissolved in 1 ml of 20 mM acetate buffer (pH 5.0), and the solution was subjected to DOWEX® MARATHON WBA (resin 0.5 ml, manufactured by Dow Chemical) equilibrated with the same buffer to obtain a void fraction. This enzyme solution was added to 10 ml of 20 mM acetate buffer (pH 5.0) in which 0.35 g of ascorbic acid and 1 g of cellobiose were dissolved. The reaction at 37° C. for 2 days afforded a reaction solution of 11.8% of 6-O-(β-D-glucopyranosyl)ascorbic acid and 0.8% of 2-O-(β-D-glucopyranosyl)ascorbic acid. The solution was filtered with an UF membrane to recover and remove the enzyme, thereby to obtain a solution (pH 4.3, electrical conductivity 1.6 mS/cm). The solution was charged on a column of DOWEX® 1-X8 (acetate form, 1.5×12 cm) at SV=2.5. After the charge, washing with an about 10 column volume (200 mL) of distilled water, and elution with 0 to 0.1 M acetic acid linear gradient concentration (80 mL×2), and 0.1 to 1.0 M acetic acid linear gradient concentration (80 mL×2) were performed. A 6-O-(β-D-glucopyranosyl)ascorbic acid-enriched fraction (fractions 65 to 68), an unreacted ascorbic acid-enriched fraction, and a 2-O-(β-D-glucopyranosyl)ascorbic acid-enriched fraction (fractions 101 to 108) were fractionated in this order. The fractions 101 to 108 were collected as a 2-O-(β-D-glucopyranosyl)ascorbic acid-enriched fraction (2.4 mg, recovery rate 45%).

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