Purification and characterization of a flavonol 3-O-beta-heterodisaccharidase from the dried herb of Fagopyrum esculentum Moench.
A flavonol-3-O-beta-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 degrees 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 microM 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 beta-glucohydrolases from other plant species. Polyclonal antibodies were raised and their specificities determined. Another flavonol 3-O-beta-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.
Baumgertel
Andreas
A
Lehrstuhl für Pharmazeutische Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Grimm
Rudi
R
Eisenbeiss
Wilhelm
W
Kreis
Wolfgang
W
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods; D019612:Fagopyrum / Q000201:enzymology*; D005419:Flavonoids; D044948:Flavonols / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics; D012431:Rutin / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013816:Thermodynamics
D000906:Antibodies; D005419:Flavonoids; D044948:Flavonols; D010446:Peptide Fragments; D012431:Rutin; D006026:Glycoside Hydrolases