Purification of d'Anjou Pear (Pyrus communis L.) Polyphenol Oxidase.
Purification of d'Anjou Pear (Pyrus communis L.) Polyphenol Oxidase.
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安茹梨 (Pyrus communis L.) 多酚氧化酶的纯化。
DOI：
10.1104/pp.78.2.256
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发表时间：
1985
期刊：
影响因子：
7.4
通讯作者：
M. W. Montgomery
中科院分区：
文献类型：
--
作者：
K. W. Wissemann;M. W. Montgomery
采用多酚结合剂ag2 - x8和triton X-100的萃取方法，广泛纯化了安珠梨（Pyrus communis L.）中的多酚氧化酶（PPO）。用色谱法除去叶绿素色素，得到透明、无色的酶提取物。用Phenyl Sepharose CL-4B、DEAE-cellulose和羟基磷灰石色谱柱对梨PPO进行纯化。只有当色谱柱在室温下而不是在4℃下运行时，才能获得尖锐的峰和良好的分辨率。整个方案的重现性很好，疏水树脂层析是成功纯化的关键。聚丙烯酰胺平板凝胶电泳后，用银染色对梨PPO的三个分离组分进行蛋白染色，其相对迁移率分别为0.41、0.43和0.73。研究了二甲亚砜对酶活性的影响，发现纯化后的梨PPO活性明显高于对照。
Polyphenol oxidase (PPO) was extensively purified to homogeneity from d'Anjou pear (Pyrus communis L.) by extraction in the presence of the phenolic binder AG 2-X8 andTriton X-100. Chlorophyll pigment was removed by chromatography resulting in a clear, colorless enzyme extract. Purification of pear PPO was achieved after chromatography on Phenyl Sepharose CL-4B, DEAE-cellulose, and hydroxylapatite columns. Only after the columns were run at room temperature rather than at 4 degrees C were sharp peaks and good resolution obtained. Reproducibility of the entire scheme was excellent with chromatography on the hydrophobic resin as a key to successful purification. Three separate fractions of pear PPO were homogeneous when stained for protein with the silver stain after polyacrylamide slab gel electrophoresis and corresponded to relative mobilities of 0.41, 0.43, and 0.73. The effect of dimethylsulfoxide on enzyme activity was investigated and found to increase significantly the activity of purified pear PPO over the control.