Beta-Glucosidases from Cicer arietinum L. Purification and Properties of isoflavone-7-O-glucoside-specific beta-glucosidases.

Beta-Glucosidases specific for isoflavone 7-O-glucosides have been isolated from garbanzo plants, Cicer arietinum L. These aryl-beta-glucohydrolases occur in the different organs of the plant as multiple molecular forms. The major isoenzymes of the roots, the leaves and the hypocotyl were purified to electrophoretic homogeneity. When subjected to isoelectric focussing in polyac rylamide gels the electrophoretically homogeneous glucohydrolases were found to consist of one or two major and several minor enzymically active molecular species. In roots the beta-glucohydrolase isoenzymes constitute a considerable portion of the extractable protein, so that purification to an electrophoretically homogeneous form is easily attainable. All beta-glucosidases analyzed possess molecular weights in the range of 125 000 (ultracentrifugation) to 135 000 (Sephadex G-200) and contain two subunits of molecular weight near 68 000. The pH optimum for enzymic activity is 7--7.5 with a second optimum of 4.5--5. The isoelectric points of the various species range between pH 5.9 AND 7.1. Staining for glycoprotein was positive. Kinetic analysis demonstrated a pronounced specificity of the enzymes for aromatic substrates with glucose as the sugar moiety. alpha-Glucosides as well as disaccharides were not hydrolyzed at all. Isoflavone 7-O-glucosides are the most favoured substrates with a Km of 2 x 10(-5) M, while the Km with aromatic glucosides (i.e. salicin, 4-nitrophenyl glucoside) are 100 times larger. In addition the beta-glucosidases show a pronounced specificity for glucose in the 7-position of the flavonoid nucleus. Using isoflavone aglycones as substrates glucose transferase activity was also demonstrable. The beta-glucohydrolase activity is strongly inhibited by Hg2plus. This inhibition is partially reversible and preferentially influences the Km values of the enzymes compared to V. Agplus, glucono-1,5-lactone, ethyleneglycol monomethyl ether and glycerol are only weakly inhibitory, while glucose, p-chloromercuribenzoate and Cu2plus are without effect.