Methanol → beta-d-Glucopyranosylascorbic acid

Participants

Reaction Conditions

Reaction Steps

Using, as an index, a retention time of 5.2 minutes in LC system manufactured by GILSON (master pump 305 type, UV detector 116 type), column: INERTSIL® ODS-3 (manufactured by GL Science, 4.6×150 mm, 5 μm) , mobile phase: 20% MeOH-20 mM phosphoric acid-5 mM tetra-n-amylammonium bromide, flow rate: 0.5 ml/min, detection wavelength: 254 nm) of a chemically synthesized product of 2-O-(β-D-glucopyranosyl)ascorbic acid, commercially available cellulase, (β-glucosidase, and β-glucanase enzyme preparations were searched. An enzyme reaction system was obtained by being dissolved in a 10 mM acetate buffer (pH 5.0) to 1 ml so that cellobiose became to be 0.3 M and ascorbic acid became to be 0.2 M. After addition of 50 μl of an enzyme solution, the reaction was initiated at 37° C. The reaction was stopped by heating at 100° C. for 5 minutes, and β-D-glucopyranosylascorbic acid produced was analyzed by high performance liquid chromatography. As a result, it was found that cellulase (manufactured by Sigma), β-glucosidase (Toyobo Co., Ltd., Nakarai Tesque), CELLULOSIN T2 (Hankyu Kyoei Bussan), cellulase “ONOZUKA” RS, “ONOZUKA” FA and PANCELLASE BR (Yakult Pharmaceutical Ind. Co., Ltd.) have (β-glucosyltransferase activity. Free ascorbic acid appeared a position of 4.0 minutes, while peaks were recognized at positions of 3.6 minutes and 5.2 minutes before and after that position, and they were designated as substance X and substance Y. A conversion rate of the substance X indicated 15.7%, and a conversion rate of the substance Y indicated 0.8%. By cochromatography analysis of this substance X and substance Y with chemically synthesized products, a retention time of the substance X was consistent with that of 6-O-(β-D-glucopyranosyl)ascorbic acid, and a retention time of the substance Y was consistent with that of 2-O-(β-D-glucopyranosyl)ascorbic acid.

Provenance & Evidence