从花椰菜（甘蓝型油菜）中纯化和表征多酚氧化酶。

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.).

机构信息

Laboratory of Food Science, Faculty of Agriculture, Saga University, Saga, Japan.

出版信息

J Agric Food Chem. 2012 Apr 11;60(14):3673-8. doi: 10.1021/jf300380b. Epub 2012 Apr 3.

Abstract

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).

摘要

采用间苯三酚作为底物，从花椰菜中纯化得到多酚氧化酶（PPO），比活力达到 282 倍，回收率为 8.1%。SDS-PAGE 显示该酶为单一条带。SDS-PAGE 和凝胶过滤分别估计该酶的分子量为 60 和 54 kDa。纯化后的酶被称为间苯三酚氧化酶（PhO），可氧化间苯三酚（K(m) = 3.3 mM）和间苯三酚羧酸。该酶还具有过氧化物酶（POD）活性。最后一步，纯化的花椰菜 POD 的活性提高了 110 倍，回收率为 3.2%。PhO 和 POD 的最适 pH 值分别为 8.0 和 4.0，在 5 °C 下，pH 值在 3.0-11.0 范围内稳定 20 h，在 5.0-8.0 范围内稳定 20 h，分别。PhO 的最适温度为 55 °C，POD 的最适温度为 20 °C。PhO 的最有效抑制剂是 10 mM 的二乙基二硫代氨基甲酸钠（IC(50) = 0.64 和 K(i) = 0.15 mM），POD 的最有效抑制剂是 1.0 mM 的氰化钾（IC(50) = 0.03 和 K(i) = 29 μM）。