莲子多酚氧化酶的分离与纯化
Purification and isolation of polyphenol oxidase from lotus seed
-
摘要: 由多酚氧化酶（polyphenol oxidase,PPO）导致的酶促褐变严重影响莲子的外观。本研究旨在探讨莲子PPO的提取纯化方法,为研究其蛋白结构、进一步探究莲子褐变机理提供理论依据。以酶活力和酶比活力为指标,对比了提取PPO的四种方法,发现匀浆吸附后丙酮沉淀法提取莲子PPO可获得较高的酶比活力,达289.04 U/mg;采用单因素实验对该提取方法进行优化,得最佳提取参数为:料液比1∶5、提取时间2 h、p H=7、含2%PVPP和0.8%Triton X-100提取溶液,优化后PPO粗酶液酶比活力为294.40 U/mg;采用DEAE Sepharose Fast FLow阴离子交换层析、Sephadex G-75凝胶柱层析对粗酶液进行纯化,得到分子量大约为58 ku莲子PPO,酶比活力为7926.68 U/mg,纯化倍数为26.92,回收率为38.48%。Abstract: The enzyme browning induced by polyphenol oxidase( PPO) affects nutrition,flavor and economic value of lotus seed.Insight into the mechanism of the enzyme browning is based on the structure and characteristic of PPO.Therefore,to obtain a fairly pure PPO is the purpose of this study.The comparation between four methods of extracting lotus seed PPO was made,homogenization post- acetone extraction method was better in PPO specific activity and maintaining the total activity.The results of optimizing homogenization post- acetone extraction were as follows: the ratio of solid- to- solvent 1 ∶ 5,extract time 2 h,phosphate buffer containing 0.8% Triton X- 100 and 2%PVPP at p H7,and the specific activity was 294.40 U / mg.The rude extraction was purified by DEAE Sepharose Fast Flow ion exchange column chromatography and Sephadex G-75 gel filtration chromatography.The specific activity of purified PPO was 7926.68 U / mg,a purification factor of 26.92,yield was 38.48%.The molecular weight of this enzyme was about 58 ku.
下载: