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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2017, Vol. 43 Issue (5): 579-588    DOI: 10.3785/j.issn.1008-9209.2017.02.121
园艺学     
勐库大叶种茶树多酚氧化酶粗酶的酶学性质
伍梦瑶，黄莹捷，姚燕妮，黄友谊*
园艺植物生物学教育部重点实验室/华中农业大学园艺林学学院茶学系，武汉 430000
Characterization of crude polyphenol oxidase isolated from Camellia sinensis var. assamica cv. Mengku
WU Mengyao, HUANG Yingjie, YAO Yanni, HUANG Youyi*
(Ministry of Education Key Laboratory of Horticultural Plant Biology/Department of Tea Science, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430000, China)
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摘要：

以云南大叶种代表性品种勐库大叶种茶树鲜叶为原料，对以硫酸铵沉淀制备的多酚氧化酶（polyphenol oxidase, PPO）粗酶进行部分酶学性质研究。结果表明，勐库大叶种PPO最适反应pH为4.4，最适反应温度为37 ℃，对焦性没食子酸、邻苯二酚有催化活性，而对间苯二酚、对苯二酚基本无催化活性。以焦性没食子酸、邻苯二酚为底物时，勐库大叶种PPO的米氏常数（Km）分别为4.2 mmol/L和36.5 mmol/L，最大单位酶活力（Vmax）值分别为2 000 U/mL和5 000 U/mL，且对焦性没食子酸的催化效果优于邻苯二酚。抑制剂乙二胺四乙酸二钠（EDTA-2Na）、亚硫酸钠、抗坏血酸和L-半胱氨酸对勐库大叶种PPO的抑制效果均随抑制剂浓度的增大而增强，其中亚硫酸钠的抑制作用最强。在终浓度为0～10.00 mmol/L范围内，除Na+对勐库大叶种PPO活性有一定的抑制作用外，K+、Cu2+和Mg2+对勐库大叶种PPO活性均有不同程度的促进作用。不同来源的茶树PPO酶学性质有着明显的区别，可能与品种适制性有直接的联系。
Abstract: Polyphenol oxidase (PPO, EC 1.10.3.1) has great significance to the growth and development of Camellia sinensis, for instance it participates in adjusting and maintaining the normal physiology and metabolism, plays an essential role in the course of the quality formation of tea products, and contributes to the oxidation reaction of tea polyphenols. The oxidation products, such as quinines, theaflavins and thearubigins, have a remarkable effect on the quality of black tea. The existing researches of C. sinensis PPO involve in various varieties and multiple aspects, including separation and purification, enzymatic property analysis, genetic structure, etc. Nevertheless, there is no research on C. sinensis var. assamica cv. Mengku, even though it carries relative original genetic information and has a great suitability for black tea manufacture.
In this study, the fresh leaves of Camellia sinensis var. assamica cv. Mengku were used as materials. The crude polyphenol oxidase (PPO) from tea leaves was extracted by using homogenate extraction technology and purified through (NH4)2SO4 precipitation. The characteristics including optimum reaction pH, optimum reaction temperature, substrate specificity, Michaelis-Menten constants and the effect of inhibitors and metal ions on its activity were studied. It aims to lay the foundation for regulating PPO activity and illuminating the suitability for black tea manufacture of C. sinensis var. assamica.
The results showed that the optimum pH of crude PPO was 4.4, and its optimum temperature was 37 ℃. It had the catalytic activity on catechol and pyrogallic acid, but not on hydroquinone and resorcinol. When pyrogallic acid and catechol as its substrate, their Km values were 4.2 and 36.5 mmol/L, respectively, and their Vmax values were 2 000 and 5 000 U/mL, respectively, indicating that the catalytic efficiency of pyrogallic acid was better. The inhibition of EDTA-2Na, sodium sulfite, ascorbic acid and L-cysteine on crude PPO was enhanced with the increasing concentration, and sodium sulfite had the strongest inhibitory effect on it, while EDTA-2Na showed the lowest inhibitory rate, with 3.64% in 1.00 mmol/L. In this experimental condition, citric acid showed no inhibitory effect on crude PPO, on the contrary its activity was slightly increased (P＞0.05). Na+ had inhibition action on the crude PPO, whereas both K+ and Mg2+ showed a promotion effect on the crude PPO. Cu2+ of 10 mmol/L increased the enzymatic activity of crude PPO to 172.85%, which shows that Cu2+ has a strong promotion effect on the crude PPO.
In conclusion, the relative lower optimal pH and optimum reaction temperature of the crude PPO are advantageous to the formation of main quality components in black tea, particularly the formation of theaflavins, which may account for that C. sinensis var. assamica cv. Mengku is fit to manufacture black tea. The characteristics of the crude PPO, which was extracted and purified from C. sinensis var. assamica cv. Mengku, show visible differences with other varieties. This may be caused by discrepancy of protein structure, and lead to suitability for manufacture of tea varieties.
收稿日期: 2017-02-12 出版日期: 2017-05-03
CLC:  S 571.1  
基金资助: 国家自然科学基金（31270731）
通讯作者: 黄友谊（http://orcid.org/0000-0001-5735-2830）     E-mail: youyi@mail.hzau.edu.cn
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引用本文:

伍梦瑶，黄莹捷，姚燕妮，黄友谊. 勐库大叶种茶树多酚氧化酶粗酶的酶学性质[J]. 浙江大学学报(农业与生命科学版), 2017, 43(5): 579-588.

WU Mengyao, HUANG Yingjie, YAO Yanni, HUANG Youyi. Characterization of crude polyphenol oxidase isolated from Camellia sinensis var. assamica cv. Mengku. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(5): 579-588.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2017.02.121    或    http://www.zjujournals.com/agr/CN/Y2017/V43/I5/579

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