Flavone content and glucosidase activity were analyzed in various species of the genera Chamomilla, Matricaria, and Anthemis, especially during the development of the chamomile flower heads. The accumulation profile of flavonoids and the increase in enzyme activity were similar during ontogenesis. The accumulation of apigenin derivatives in closely related species was always linked to the occurrence of a catabolic beta-glucosidase in the respective plant organ. The flavone-glucoside-cleaving beta-glucosidase (FGG) from the ligulate florets of chamomile was purified to electrophoretic homogeneity by the following procedure: ammonium sulphate fractionation, anion exchange on Mono Q, hydrophobic interaction chromatography on Bio-Gel TSK Phenyl-5-PW, and gel filtration 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 with an M (r) of 60 kDa could be detected after SDS-PAGE. The isoelectric point as determined by chromatofocussing on Mono P was at pH 4.6. During the purification procedure only one glucosidase activity appeared. A partially purified enzyme was used for characterization. The temperature optimum was at 37 degrees C and the pH-optimum 5.6. Energy of activation 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 hydrolyzed. Transglucosylation to an acceptor other than water was observed. Reagents interacting with sulfhydryl-groups strongly inhibited the enzyme.

mation of these coloured products can probably attributed to phenol oxidase reactions.The apparent Km for apigenin 7-glucoside was 0.25 mM and that for luteolin 7-glucoside was 0.29 mM. Acylated apigenin derivatives such as apigenin 7-(O-acetyl)glucoside were not cleaved in a direct way. However, an increase in the deacetylated glucoside was observed after incubation with acylated apigenin derivatives. This finding suggests the presence of an acetylesterase in the crude extract. Prior to glucoside degradation to apigenm, the acetyl function is cleaved. When apigenin 7-glucoside was used as a substrate the enzyme showed substrate inhibition at concentrations higher than 1.25 mM. Therefore 1 mM apigenin 7-glucoside was used in the standard assay.The formation of free apigenin in flower heads of Chamomilla recutita (L.) Rauschert (Asteraceae) has been explained as a result of the enzymic degradation of apigenin 7-glucoside and other apigenin derivatives and depends on certain post-harvest conditions (1). In this paper, the presence of an apigenin-7-glucoside-hydrolyzing glucosidase in protein extracts from chamomile flower heads is reported. Furthermore the activity of the enzyme is characterized.Alter the incubation of protein extracts prepared from fresh, dried, frozen and freeze-dried flower heads with apigenin 7-glucoside under standardized conditions, an increase of apigenin could be determined, which was correlated with a decrease in the amount of its glucoside. Control incubation without protein or heat-denatured protein showed no cleavage of the glucoside. For the preliminary characterization of the enzyme, acetone dry powders were used, which had been prepared from the flower heads and also showed high glucosidase activity.Mcllvaine's citrate-phosphate buffer pH 5.6 was used for extraction and incubation, but high glucosidase activity was found in extracts with only phosphate or only citrate buffers as well. The addition of detergents like Triton X-100 to the buffer, which is necessary when enzymes are particle-or membrane-bound, did not improve the enzyme activity of the extracts.Optimal glucosidase activity was found at pH 5.6 with half maximal activities at pH 4.3 and 6.7. The temperature optimum was at 36°C and 50% activity at 26°C and 45.5°C. The reaction velocity was linear with time over 10 minutes with protein adjusted to 0.06 mg/ml.In the presence of reducing agents the glucosidase activity was enhanced. Mercaptoethanol, tested in concentrations of 5, 10, and 20mM, caused increases in the relative activity (control without stabilizer = 100 percent) of 19, 35, and 9 percent. Dithiothreitol (2, 5, 10, and 20 mM) and ascorbate (5, 10, and 20 mM) resulted in increases of 26, 41, 56, and 74 percent and of 24, 36, and 49 percent, respectively. In the optimized enzyme assay mercaptoethanol was used in a concentration of 10mM. The absence of reducing agents led to coloured products when luteolin 7-glucoside was used as a substrate. The for-o-Gluconic acid-1,5-lactone is a specific inhibitor for fi-glucosid...