Purification and characterization of a flavonol 3- O-β-heterodisaccharidase from the dried herb of Fagopyrum esculentum Moench

• Published in: Phytochemistry (Oxford) Vol. 64; no. 2; pp. 411 - 418
• Main Authors: Baumgertel, Andreas, Grimm, Rudi, Eisenbeiß, Wilhelm, Kreis, Wolfgang
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 01.09.2003; Elsevier
• Subjects: Amino Acid Sequence; Animals; Antibodies - immunology; Biological and medical sciences; Chromatography, Gel; Chromatography, Ion Exchange - methods; Enzymes; Fagopyrum - enzymology; Fagopyrum esculentum; Flavonoids; Flavonols - chemistry; Flavonols - metabolism; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - physiology; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Metabolism; Mice; Molecular Weight; Peptide Fragments - genetics; Plant physiology and development; Polygonaceae; Purification; Rutin; Rutin - metabolism; Rutinoside; Sequence Analysis, Protein; Substrate Specificity; Thermodynamics; β-Glycosidase; Rutinoside; Purification; Fagopyrum esculentum; Rutin; Polygonaceae; β-Glycosidase; Enzyme; Characterization; Enzymatic activity; Dicotyledones; Glycosidases; Substrate specificity; Angiospermae; Quercetin; Hydrolases; Isolation; Above ground plant part; Spermatophyta; Kinetics
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/S0031-9422(03)00418-7

A flavonol-3- O-β-heterodisaccharide glycosidase (FHG I) was isolated from dried overground parts of Fagopyrum esculentum Moench. FHG I is a monomeric glycoprotein with a carbohydrate content of 23% and a molecular mass of 74.5 kDa. A flavonol-3- O-β-heterodisaccharide glycosidase (FHG I) was isolated from dried aerial tissues of Fagopyrum esculentum Moench (Fagopyri herba). It has a specific enzyme activity of ca. 3.5 nkat mg −1 protein in buffered extracts when rutin (quercetin-3- O-rutinoside) was used as substrate and an optimal enzyme activity was seen at around pH 4.8 and 30 °C. FHG I was purified about 156-fold to apparent homogeneity by hydrophobic interaction, anion exchange and size exclusion chromatographic steps. The apparent molecular mass of FHG I was 74.5±2 kDa as determined by SDS-PAGE and it is a monomeric glycoprotein with a carbohydrate content of 23%. The isoelectric point as determined by isoelectric focusing was 5.7 and the energy of activation was 32 kJ mol −1. FHG I exhibits a high substrate specificity, preferring flavonol 3-O-glycosides comprising the disaccharide rutinose. The K m and V max values for the natural substrate rutin were calculated to be 0.561 μM and 745 nkat mg  −1 protein, respectively. Two oligopeptide fragments obtained after enzymatic digestion of FHG I were sequenced and showed similarities to sequences of β-glucohydrolases from other plant species. Polyclonal antibodies were raised and their specificities determined. Another flavonol 3- O-β-heterodisaccharide glycosidase (FHG II) could also be detected in buckwheat herb, having a molecular mass of 85.3±2 kDa and an isoelectric point between pH 6.0 and 6.5.