Yang Liu, Ning Zhou S, Shi Chen Z, Chang Zhong Y, Huan Liu Y
The State Key Laboratory for Biocontrol, Sun Yet-Sen University, Guangzhou, 510275, PR China.
J Agric Food Chem. 2004 Apr 7;52(7):1940-4. doi: 10.1021/jf030476c.
An isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) from endophytic bacterium, Pseudomonas ZD-8 was purified to homogeneity by successive ammonium sulfate precipitation, gel filtration on SephadexG-100, DEAE-sephrose CL-6B and DEAE-Sephacel chromatography. The enzyme was a monomeric protein with an apparent molecular mass of 33 kDa as determined by SDS-PAGE and gel filtration. It was optimally active at pH 6.0 and 40 degrees C and had a specific activity of 1485 U mg of protein(-1) against genistin. The ICHG readily hydrolyzed rho-nitrophenyl-beta-glucoside, rho-nitrophenyl-beta-galactoside, genistin, daidzin, with Km values of 1.64, 1.87, 0.012, 0.014 mM, respectively. The ICHG showed a pronounced specificity for glucose in the 7-position of isoflavone and flavone conjugates and hydrolyzed effectively malonyl isoflavone glucosides as well as isoflavone glucosides with similar kinetics. Glucose and glucono-delta-lactone inhibited the enzyme competitively with Ki values of 84 mM and 23 mM, respectively. The enzyme did not require divalent cations for activity, and its activity was strongly inhibited by Hg2+, Ag+, rho-chloromercuribenzoate, iodoacetic acid, and N-ethylmaleimide while reducing agents such as beta-mercaptoethanol, dithiothreitol, dithioerythritol, glutathione slightly activated the enzyme.
从内生细菌假单胞菌ZD - 8中分离得到一种异黄酮共轭水解β - 葡萄糖苷酶（ICHG），通过连续的硫酸铵沉淀、SephadexG - 100凝胶过滤、DEAE - 琼脂糖CL - 6B和DEAE - 琼脂糖凝胶柱层析将其纯化至同质。经SDS - PAGE和凝胶过滤测定，该酶为单体蛋白，表观分子量为33 kDa。其最适pH为6.0，最适温度为40℃，对染料木苷的比活性为1485 U mg蛋白-1。ICHG能轻易水解对硝基苯基 - β - 葡萄糖苷、对硝基苯基 - β - 半乳糖苷、染料木苷、大豆苷，其Km值分别为1.64、1.87、0.012、0.014 mM。ICHG对异黄酮和黄酮共轭物7位上的葡萄糖具有显著特异性，能有效水解丙二酰异黄酮葡萄糖苷以及异黄酮葡萄糖苷，且动力学相似。葡萄糖和葡萄糖酸 - δ - 内酯对该酶有竞争性抑制作用，Ki值分别为84 mM和23 mM。该酶的活性不需要二价阳离子，Hg2 +、Ag +、对氯汞苯甲酸、碘乙酸和N - 乙基马来酰亚胺能强烈抑制其活性，而β - 巯基乙醇、二硫苏糖醇、二硫赤藓糖醇、谷胱甘肽等还原剂能轻微激活该酶。
