可溶态和膜结合态双孢蘑菇多酚氧化酶的分离纯化及酶特性分析

Purification and Enzymatic Properties of Soluble and Membrane-Bound Polyphenol Oxidase from Agaricus bisporus

为开发一种双孢蘑菇两种不同形态多酚氧化酶(polyphenol oxidase,PPO)的提取纯化方法,通过温度诱导相分配、分步硫酸铵沉淀盐析、DEAE阴离子交换层析,对双孢蘑菇中的可溶态PPO(soluble PPO,sPPO)和膜结合态PPO(membrane-bound PPO,mPPO)进行分离纯化,并通过十二烷基硫酸钠-聚丙烯酰氨凝胶电泳和Native-聚丙烯酰氨凝胶电泳及米氏方程对纯化后两种形态PPO的纯度、分子质量及酶促反应动力学进行探究。双孢蘑菇sPPO和mPPO经纯化后对邻苯二酚的比活力分别达到6912.88 U/mg和19092.94 U/mg,相比于粗酶液,酶的比活力分别提高到12.20倍和10.86倍,且纯化后mPPO的比活力显著高于sPPO。酶促反应动力学结果显示,sPPO和mPPO对不同底物的催化活性差异较大,对底物邻苯二酚的催化活性较高,且mPPO比sPPO对底物邻苯二酚的亲和力更强,酶促反应速度更高。sPPO和mPPO对邻苯二酚的最适反应温度均为30℃,最适pH值均为6.8。本研究为双孢蘑菇两种状态PPO的质谱鉴定、酶学特性以及抑制酶促褐变的发生等研究奠定了基础。

An extraction and purification method for soluble polyphenol oxidase(sPPO)and membrane-bound polyphenol oxidases(mPPO)from Agaricus bisporus was developed involving temperature-induced phase partitioning,fractional precipitation with ammonium sulfate,and DEAE anion exchange chromatography.The purity,molecular mass and enzymatic reaction kinetics of purified PPO were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),Native-PAGE and Michaelis-Menten equation.The specific activities of sPPO and mPPO toward catechol were 6912.88 and 19092.94 U/mg,and increased by 12.20 and 10.86 times,respectively,compared with that of crude PPO.The results of enzymatic reaction kinetics showed that the catalytic activities of sPPO and mPPO toward different substrates were greatly different and were higher toward catechol.mPPO had a stronger affinity to the substrate catechol and a higher reaction rate than sPPO.The optimum pH and temperature for both PPO enzymes were 6.8 and 30℃,respectively.This study may lay a foundation for future research on mass spectrometry identification and enzymatic properties two forms of PPOs from A.bisporu and inhibition of enzymatic browning caused by PPO.