茶叶多酚氧化酶同工酶PPO1合成茶黄素TF作用机理研究

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。

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.