茶叶多酚氧化酶同工酶PPO1合成茶黄素TF作用机理研究

Theaflavins have a variety of pharmacological effects closely related to human health, and it is also important in quality components of black tea. Polyphenol oxidase is a key enzyme that catalyzes the oxidation of catechins to the synthesis of theaflavins, which has been studied in detail at home and abroad, but it is unclear whether its isozyme synthesize a single theaflavin component. Based on our previous experiments, it was found that a tea polyphenol oxidase isozyme (PPO1) can enzymatically synthesize theaflavin TF monomer from catechin mixture. This study intends to separate and prepare the polyphenol oxidase isozyme from fresh tea leaves by protein purification system. The purified tea polyphenol oxidase isozyme N-terminal and endopeptide amino acid sequences will be analyzed by Edman degradation method and MALDI-TOF MS/MS tandem mass spectrometry. According to the obtained partial amino acid sequence, the full-length cDNA sequence of polyphenol oxidase isozyme gene will be obtained by PCR and RACE technology, and the prokaryotic expression vector will be constructed and the recombinant enzyme will be obtained. The polyphenol oxidase isozyme and the recombinase will be analyzed and identified on the enzymatic properties, protein structure and function. The research results of this project have important theoretical and practical significance on understanding the mechanism of the synthesis of theaflavin compound from tea polyphenol oxidase isozyme and exploring the tea quality and the large-scale development of theaflavin monomer.

茶黄素类具有多种与人体健康密切相关的药理功效，也是构成红茶的重要品质成分。多酚氧化酶是催化儿茶素合成茶黄素的关键酶，国内外对其催化合成茶黄素进行了详尽研究，但对其同工酶合成茶黄素研究极少，现发现茶叶多酚氧化酶同工酶PPO1能够将儿茶素混合物合成茶黄素TF单体。本项目拟用蛋白质纯化系统分离制备出茶鲜叶多酚氧化酶同工酶，利用Edman降解法和MALDI-TOF MS/MS串联质谱对纯化的茶叶多酚氧化酶同工酶N-末端和内肽氨基酸序列进行分析；根据获得的部分氨基酸序列，通过PCR和RACE技术得到多酚氧化酶同工酶基因全长cDNA序列，构建原核表达载体并得到重组酶；将多酚氧化酶同工酶和重组酶在酶学性质、蛋白质结构、功能特征上进行分析鉴定。本项目研究结果，对于深入了解茶叶多酚氧化酶同工酶催化合成单一茶黄素化合物TF的作用机理和探讨茶叶品质调控、茶黄素单体规模开发均具有重要的理论和实践意义。

茶叶多酚氧化酶（PPO）是催化儿茶素类形成茶黄素混合物的关键酶，影响茶叶的风味品质和营养价值。现阶段研究多以PPO整体为研究对象，但对其同工酶特性及催化合成茶黄素研究极少。本项目通过匀浆浸提、色谱法、膜法等技术成功提取纯化出电泳纯PPO同工酶。围绕酶动力学、氨基酸序列、酶学性质、功能结构、酶促合成茶黄素情况进行系统研究，结果发现获得一个约58 KDa的PPO同工酶，经高效液相色谱（HPLC）和液质联用（UHPLC-MS）验证能够体外酶促合成单一茶黄素组分TF，且不受反应底物儿茶素组分的配比组成、反应温度、pH值、反应时间等因素影响，即产物类型单一，仅生成量变化。对电泳纯PPO同工酶蛋白进行MALDI-TOF/MS鉴定，发现其具体蛋白分子量为58.369 kDa，等电点pI为6.4，该蛋白在Uniport数据库中鉴定为山茶属（Camellia sinensis）的多酚氧化酶。基于茶树基因组数据库对PPO基因家族进行了生物信息学分析，发现除CsPPO4外，CsPPO1、CsPPO2、CsPPO3的基因序列差别不大，且均为亲水性蛋白；系统进化树分析显示CsPPO1、CsPPO2、CsPPO3的亲缘关系较近，其中CsPPO1和CsPPO3的亲缘关系最近，qPCR荧光定量显示在同一茶树品中具有表达差异性。研究建立了三相分离纯化法提取PPO，对获得酶蛋白进行酶学特性分析，证实该法具有稳定有效、简单便捷的优势。利用上述建立的三相分离纯化法分离游离态PPO（sPPO）和膜结合态PPO（mPPO），对酶学性质和酶促合成茶黄素进行比较研究，结果发现两种形态的酶蛋白均对二酚表现出高活性以及对单酚表现出低活性，最适pH均为5.5。mPPO的最适温度为25 ℃，显著低于sPPO，EDTA表现出抑制sPPO和激活mPPO作用，同时mPPO在酸性和高酯型儿茶素为底物的条件下具有高稳定性。本项目研究结果的获得对于深入了解茶叶PPO形态及其同工酶、酶促合成茶黄素、茶叶加工品质调控具有重要的现实意义。