食品科学

• 生物工程 • 上一篇    下一篇

丝瓜多酚氧化酶的分离纯化及酶学性质

吴海霞,曹雨舟   

  1. 1.运城学院生命科学系,山西 运城 044000;2.山西昭鑫电力科技有限公司,山西 运城 044000
  • 出版日期:2014-04-15 发布日期:2014-04-18

Isolation, Purification and Some Enzymatic Properties of Polyphenol Oxidase from Loofah

WU Hai-xia, CAO Yu-zhou   

  1. 1. Life Sciences Department, Yuncheng University, Yuncheng 044000, China;
    2. Shanxi Zhaoxin Electric Power Technology Co. Ltd., Yuncheng 044000, China
  • Online:2014-04-15 Published:2014-04-18

摘要:

从丝瓜中分离纯化出多酚氧化酶(polyphenol oxidase,PPO),并对其部分酶学性质进行研究。采用硫酸铵分级盐析、透析、DEAE-Cellulose DE-52离子交换层析和Sephadex G-75分子筛凝胶过滤层析分离纯化丝瓜PPO。纯化所得酶的比活力为957.9 U/mg,纯化倍数为28.3,酶活力回收率为18.5%;十二烷基磺酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)及非变性聚丙烯酰胺凝胶电泳(Native-PAGE)检测结果显示该酶蛋白呈单一条带,为单亚基蛋白,其分子质量约为67.8 kD,且无同工酶;该酶最适pH6.0,最适温度30 ℃,以左旋多巴(L-dopa)为底物,其米氏常数(Km)为1.32 mmol/L,最大反应速率(Vmax)为0.22 OD475 nm/min。

关键词: 丝瓜, 多酚氧化酶, 分离纯化, 酶学特性

Abstract:

Polyphenol oxidase (PPO) from loofah was extracted, and some of its kinetic properties were studied. ThePPO was purified by ammonium sulfate precipitation, dialysis, DEAE-Cellulose DE-52 ion-exchange and SephadexG-75 gel filtration. The specific activity of the purified PPO was 957.9 U/mg, which exhibited a purification fold of 28.3and an activity recovery of 18.5%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nativepolyacrylamidegel electrophoresis (Native-PAGE) showed that the enzyme was homogeneous as a monomeric protein. Itsmolecular weight was estimated to be 67.8 kD and it had no isozymes. The kinetic properties of the enzyme showed thatthe optimal pH and temperature were pH 6.0 and 30 ℃, respectively. The Km and Vmax towards the substrate L-dopa were1.32 mmol/L and 0.22 OD475 nm/min, respectively.

Key words: loofah, polyphenol oxidase (PPO), purification, enzymatic properties