云南大叶种多酚氧化酶高活性机理研究

Although China is the largest tea-producing country in the world, the export of black tea has been in a serious recession for its poor quality, resulting in waste of a large number of tea resources and low efficiency of the tea industry. However, black tea sale in the international tea market trade is always about more than 80 percent, and the quality of Chinese black tea urgently needs to be improved. In spite of playing a key role on the quality formation of black tea and having a lot of reports on it, there is a lack of research to a single isoenzyme of polyphenol oxidase (PPO), and the high enzymatic activity mechanism of PPO is unclear..Based on the cloned tea ppo genes and the heterologously expressed PPO in our group, the differently expressed proteins from the samples of Yunnan-Dayezhong (Camellia sinensis) with high enzymatic activity of PPO, and Longjing 43 (Camellia sinensis) with low enzymatic activity of PPO, would be isolated and identified by non-denatured two-dimensional electrophoresis and mass spectrometry. And their encoding genes and their modification method of translated peptide chains would be determined by gene level validation. PPO isoenzymes with high enzymatic activity would be screened out, be heterologously expressed, and be characterized. It will be to understand the formation mechanism of tea PPO isoenzymes, to clarify the high enzymatic activity mechanism of Yunnan-Dayezhong PPO, which will be as a base for black tea processing, the production of highly active PPO enzyme, and black tea varieties breeding, in order to improve China's black tea quality and its export.

我国是世界上最大产茶国，但我国红茶却因品质差而出口严重萎缩，导致全国茶资源大量浪费、茶产业效益低下。然而国际茶叶市场贸易量约80％以上是红茶，因此亟待提高我国红茶品质。茶叶多酚氧化酶（Polyphenol oxidase，PPO）对红茶品质形成具有重要作用，尽管对PPO研究很多，但缺乏单一同工酶的研究，尚不清楚PPO高活性的机制。.在我们已克隆茶树ppo基因并成功异源表达的基础上，以云南大叶种（PPO高活性）和龙井43号（PPO低活性）为材料，利用非变性蛋白质二维电泳和质谱技术分离鉴定PPO表达差异蛋白，通过基因水平的验证，确定其编码基因及译后肽链修饰方式；筛选获得高活性PPO同工酶，并进行异源表达和酶性质分析。本课题将了解茶树PPO同工酶形成的机制，阐明云南大叶种PPO高活性机理，为红茶加工、高活性PPO酶制剂生产和适制红茶品种选育提供理论基础，以提高我国红茶品质并促进出口。

多酚氧化酶(Polyphenol oxidase，PPO，EC 1.10.3.1)对茶叶加工以及茶树生理代谢都有着重要意义。作为茶叶中最受关注的酶类，人们对PPO的研究从未间断，但多是以粗酶为研究对象，对茶树PPO同工酶研究不足。本课题以PPO高活性的云南大叶种（勐库大叶种）和低活性的龙井43号为材料，分离纯化获得8条PPO单一同工酶，对部分PPO差异单一同工酶进行了鉴定，分析了酶学性质、茶黄素合成条件等。主要研究结果如下：.龙井43号、勐库大叶种鲜叶分别以料液比1∶2、1∶3，加入内含5% PVP(w/v)预冷的0.1 mol/L柠檬酸-磷酸盐缓冲液(pH7.2)，冰浴研磨，4 ℃浸提12 h，于4 ℃以9000 r/min离心35 min，上清液过滤制得粗酶液。通过30%-80%硫酸铵的分级沉淀、DEAE-Sepharose CL-6B离子交换层析、Sephadex G-150凝胶过滤层析，从龙井43号和勐库大叶种中分离纯化获得分子量为35、45、50、60、80 ku的茶树PPO单一同工酶。.对部分龙井43号PPO差异单一同工酶进行了质谱和氨基酸N端序列测定，质谱鉴定检测到其中3个PPO差异同工酶与4种不同的蛋白相似，而通过氨基酸N端测序成功检测到8个氨基酸，其余7个可能由于甲基化修饰而未能检出。.分别对龙井43号、勐库大叶种的PPO粗酶和PPO差异单一同工酶PPO I-1进行酶学性质比较分析，龙井43号和勐库大叶种PPO在底物特异性和亲和性方面差异显著，前者作用的底物范围广，而勐库大叶种PPO仅对邻苯二酚和焦没食子酸具有催化作用。最适pH、最适温度、热稳定性、抑制剂以及金属离子影响方面，二者的PPO粗酶及PPO I-1也表现出明显差异。与龙井43号相比，勐库大叶种PPO的最适pH低、低温(30 ℃)条件下具有更强的热稳定性，这可能是导致其适制性差异的重要原因。.分析了不同因素对龙井43号、勐库大叶种的PPO粗酶和差异PPO I-1合成茶黄素的影响，发现它们催化合成茶黄素的最适pH、最适温度接近，但最适底物浓度以及茶黄素产量方面勐库大叶种PPO远高于龙井43号，这可能是造成两个茶树品种适制性差异的又一重要原因。