R. Maier et al., PURIFICATION AND CHARACTERIZATION OF A FLAVONE 7-O-GLUCOSIDE-SPECIFICGLUCOSIDASE FROM LIGULATE FLORETS OF CHAMOMILLA-RECUTITA, Planta medica, 59(5), 1993, pp. 436-441
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.