Enzymatic Formation of aGlucosylthiamin

1. A high performance liquid chromatography (HPLC) analysis of thiamin and its derivatives: HPLC (Asahipack GS 320 column [7.6 x 500 mg] [Asahi Chemical Industries, Tokyo, Japan) is carried out using a Waters Associates chromatography system consisting of two pumps (model 6000A and 501G), an injector model U6K, a UV absorbance detector model 441, and a stainless steel mixing coil, together with a Shimadzu fluorescence HPLC monitor model RF 530, and two recorders (Shimadzu chromatopac model C-R1B). In addition, prepare the following reagents: thiamin hydrochloride (Nacalai Tesque); 0.04 M Sodium phosphate buffer, pH 6.0, and 0.01% K3 (Fe[CN]6)-15% NaOH solution.

2. For PPC of thiamin and its derivatives, prepare: filter paper no. 50 (40 x 40 cm); solvent A; and a blue fluorescence-producing reagent: A mixture of ethanol and an alkaline KsFe[CN]6) solution (1:1,v/v). The alkaline solution contains 200 mL of 1% K3(Fe[CN]6) aqueous solution, 26.4 mL of 30% NaOH and 13.6 mL of distilled water.

3. Enzymatic glycosylation reaction of thiamin: prepare the following enzymes and reagents: a-glucosyl donor: dextrin (Pine-Dex #1, DE 8); acceptor: thiamin hydrochloride; CGTase from B. stearothermophilus and its homogeneous preparation purified by the method of Kitahata and Okada (13); glucoamylase from Rhizopus sp. (grade I) (38.3 U/mg); a blue fluorescence-producing reagent; calcium chloride, anhydrous; and 0.1 M sodium acetate buffer, pH 5.5.

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4. Isolation of a-glucosylthiamin: prepare the following enzymes and reagents: dextrin (Pine-Dex #1); thiamin hydrochloride; CGTase from B. stearothermophilus; glucoamylase from Rhizopus sp. (grade I); calcium chloride, anhydrous; 0.1 M sodium acetate buffer (ph 5.5); a blue fluorescence-producing reagent; filter paper no. 50 (40 ¥ 40 cm); solvent A; activated Vitachange (Wako Pure Chemical Industries, Osaka, Japan); 25% (w/v) KCl aqueous solution; activated carbon; 85% ethanol; and phosphorus pentoxide.

5. For the characterization of a-glucosylthiamin, prepare the following enzymes and reagents: a-glucosidase from pig liver (see Subheading 2.1.6.); a-glucosidase from rice (see Subheading 2.1.6.); a-glucosidase from M. javanicus (see Subheading 2.1.6.); P-glucosidase from almond (20-40 U/mg) (Sigma); D2O (Euriso-top, CEA); a solvent system of 1-propanol-2% NH4OH (2:1,v/v); a solvent for HPLC: acetonitrile-water (9:1,v/v); kieselgel 600 plate for thin-layer chromatography (Merck); and Waters carbohydrate analysis column (4 x 300 mm).

Enzymatic Formation of 4G-a-d-Glucopyranosyl-Rutin

Assay of Rutin and its Derivatives

Rutin and its derivatives are assayed by Imai and Furuya's method (14) with a slight modification. A suitable amount of the reaction mixture containing rutin and its derivatives is applied as a band on filter paper no .50 (40 x 40 cm) and developed twice by ascent in solvent A. After drying, the chromatogram is sprayed with a yellow fluorescence-producing reagent. Rutin and its derivatives on its chromatogram are shown as yellowish fluorescent bands under UV lamp (3650A filter). Each yellow band is cut out and extracted with 20 mL of the mixed aqueous solution of AlCl3 and CH3COOK (1:1,v/v) at 25°C for 2 h. The amount of rutin compound in the extract is measured by the optical density at 420 nm.

Assay of CGTase Activity (10)

Two hundred ^L of the enzyme solution is incubated with 5 mL of 0.3% soluble starch in 20 mM sodium acetate buffer, pH 5.5, containing 1 mM CaCl2 at 40°C. After 10 min, 0.5 mL of the reaction mixture is pipeted out and added to 15 mL of 0.02 N H2SO4. To the mixture, 0.2 mL of 0.1 N I2 solution is added, and then the color developed is measured at 660 nm. One unit of the enzyme activity is defined as the amount of the enzyme that catalyzes a 10% decrease of the absorbance per min under these conditions.

Paper Chromatography

PPC is done on filter paper no. 50 with solvent A. After drying, the chromatogram is irradiated with UV lamp (2537A filter) where the zones of rutin

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and its derivatives are identified by their brownish yellow fluorescence. Also, rutin and its derivatives on the chromatogram are detected as yellow fluorescence under UV lamp (3650A filter) after spraying with a yellow fluorescence-producing reagent. PPC of sugars is done with solvents A and B. The silver nitrate dip method (15) is used for sugar detection. The solutions (A, B, and C) are poured into three different Pyrex baking dishes, and the chromatogram is dipped into solution A and allowed to dry. The chromatogram is next dipped into solution B until the characteristic black spots of sugar appear. After washing with water, it is placed in solution C until the background coloration disappears. A final washing with water gives a stable chromatogram.

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