Hsieh M C, Graham T L
Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA.
Phytochemistry. 2001 Dec;58(7):995-1005. doi: 10.1016/s0031-9422(01)00380-6.
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.
通过对低速离心沉淀进行高盐提取以及随后的阴离子和阳离子交换色谱法，从大豆[Glycine max]根中纯化出了一种对异黄酮共轭物具有高比活性的β-葡萄糖苷酶。在分级分离过程中，需要在10%甘油中进行稳定化处理以实现纯化。该酶很可能是一种二聚体（约M(r) 165 kDa），潜在亚基的M(r)为80 kDa和/或75 kDa。最适pH和温度分别为pH 6和30℃。该酶具有高度的热稳定性。在所检测的各种潜在效应物中，银离子和汞离子的抑制作用最强。在存在250 microMβ-巯基乙醇的情况下，银离子的IC(50)从140 microM增加到14 mM。葡萄糖酸-δ-内酯的抑制作用不强（IC(50) 24 mM）。该酶对异黄酮共轭物具有高活性，其对异黄酮共轭物的特异性常数比对通用显色底物对硝基苯基β-葡萄糖苷高160 - 1000倍。该酶对所测试的黄酮醇糖苷无活性。部分纯化的酶对7 - O - 葡萄糖基 - 和7 - O - 葡萄糖基 - 6'' - 丙二酰 - 异黄酮具有相似的K(m)和k(cat)，表明它可能能够裂解酯化的葡萄糖基共轭物。我们推测该酶参与了大豆苷元和染料木黄酮的释放，这两种物质在大豆防御中都起着核心作用。
