State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China; Department of Food Engineering (Bioengineering), Xuzhou Institute of Technology, Xuzhou 221008, China.
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Food Chem. 2011 Dec 1;129(3):1012-8. doi: 10.1016/j.foodchem.2011.05.062. Epub 2011 May 20.
A polyphenol oxidase was purified and characterised from leaves of the common spiderflower. Purification sequentially with ammonium sulphate, dialysis, DEAE-Sepharose ion-exchange chromatography and Sephadex G-75 gel filtration chromatography resulted in 37.8-fold enrichment in the specific activity and 44.3% recovery of the total activity. Purified PPO is a monomeric protein of 52.6kDa revealed by Coomassie and active staining and Western blot. It was optimally active at pH 8.0 and 60°C, and stable from pH 3.0 to 9.0 and below 60°C. It displayed enzymatic activity towards monophenols, diphenols and triphenols, especially towards diphenols, and substrate specificity towards methylated and methoxylated substrates. Its activity was slightly increased by 0.1% SDS, heavily inhibited by Hg(2+) and Pb(2+), and completely inhibited by 1.0mM of ascorbic acid, l-cysteine, β-mercaptoethanol, sodium diethyldithiocarbamate and thiourea, and by 10mM of dithioerythritol, sodium metabisulphite and sodium sulphite.
从普通蜘蛛花的叶片中纯化和表征了一种多酚氧化酶。经过硫酸铵沉淀、透析、DEAE-琼脂糖离子交换层析和 Sephadex G-75 凝胶过滤层析的连续纯化，比活力提高了 37.8 倍，总活性回收率为 44.3%。纯化的 PPO 通过考马斯亮蓝和活性染色及 Western blot 显示为 52.6kDa 的单体蛋白。它在 pH8.0 和 60°C 时具有最佳活性，在 pH3.0 至 9.0 和 60°C 以下稳定。它对单酚、二酚和三酚均具有酶活性，特别是对二酚，对甲基化和甲氧基化底物具有底物特异性。0.1%SDS 略微增加其活性，Hg(2+)和 Pb(2+)严重抑制其活性，1.0mM 抗坏血酸、l-半胱氨酸、β-巯基乙醇、二乙基二硫代氨基甲酸钠和硫脲以及 10mM 二硫苏糖醇、亚硫酸钠和连二亚硫酸钠完全抑制其活性。
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