title: Partial purification and characterization of a soybean beta-glucosidase with high specific activity towards isoflavone conjugates.
abstract: A beta-glucosidase with high specific activity towards isoflavone conjugates was purified from soybean [Glycine max] roots by high salt extraction from a low speed centrifugal pellet and subsequent anion and cation exchange chromatography. Purification required stabilization throughout fractionation in 10% glycerol. The enzyme is most likely a dimer (approximate M(r) 165 kDa) with potential subunits of M(r) 80 and/or 75 kDa. The pH and temperature optima are pH 6 and 30 degrees C, respectively. The enzyme was highly heat-stable. Of the various potential effectors examined, silver and mercury ions were the most inhibitory. The IC(50) of silver ions was increased from 140 microM to 14 mM in the presence of 250 microM beta-mercaptoethanol. Glucono-delta-lactone was not strongly inhibitory (IC(50) 24 mM). The activity was highly active against isoflavone conjugates, with a specificity constant 160-1000 fold higher for isoflavone conjugates over the generic chromogenic substrate, p-nitrophenyl beta-glucoside. The enzyme was inactive against the flavonol glycosides tested. The partially purified enzyme had similar K(m) and k(cat) towards 7-O-glucosyl- and 7-O-glucosyl-6"-malonyl-isoflavones, suggesting that it may be able to cleave the esterified glucosyl conjugate. We hypothesize that the enzyme is involved in the release of daidzein and genistein, both of which play central roles in soybean defense.
authors.affiliation: Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Hsieh
authors.forename: T L
authors.initials: TL
authors.lastname: Graham
mesh_terms: D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007529:Isoflavones / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007529:Isoflavones; D001617:beta-Glucosidase