PURIFICATION AND CHARACTERIZATION OF A FLAVONE 7-O-GLUCOSIDE-SPECIFICGLUCOSIDASE FROM LIGULATE FLORETS OF CHAMOMILLA-RECUTITA

Flavone content and glucosidase activity were analyzed in various spec ies of the genera Chamomilla, Matricaria, and Anthemis, especially dur ing the development of the chamomile flower heads. The accumulation pr ofile of flavonoids and the increase in enzyme activity were similar d uring ontogenesis. The accumulation of apigenin derivatives in closely related species was always linked to the occurrence of a catabolic be ta-glucosidase in the respective plant organ. The flavone-glucoside-cl eaving beta-glucosidase (FGG) from the ligulate florets of chamomile w as purified to electrophoretic homogeneity by the following procedure: ammonium sulphate fractionation, anion exchange on Mono Q, hydrophobi c interaction chromatography on Bio-Gel TSK Phenyl-5-PW, and gel filtr ation on Superose 12. The M(r) of the native enzyme was determined by gel filtration (500 kDa) and native PAGE (334 kDa). Only one subunit w ith an M(r) of 60 kDa could be detected after SDS-PAGE. The isoelectri c point as determined by chromatofocussing on Mono P was at pH 4.6. Du ring the purification procedure only one glucosidase activity appeared . A partially purified enzyme was used for characterization. The tempe rature optimum was at 37-degrees-C and the pH-optimum 5.6. Energy of a ctivation was 32.9 kJ/mol. The determination of the kinetic constants with various aryl glycosides proved a high affinity of the FGG towards flavone 7-O-glucosides. Alpha-Glycosides and disaccharides were not h ydrolyzed. Transglucosylation to an acceptor other than water was obse rved. Reagents interacting with sulfhydryl-groups strongly inhibited t he enzyme.