Separation and Characterization of Polyphenol Oxidase from Unfermented and Fermented Liupao Tea

In order to explore the source and activity of polyphenol oxidase (PPO) in the fermentation process of Liupao tea, PPO from unfermented and fermented Liupao tea was extracted by using buffer solutions at different pHs and different types and amounts of protective agents at different liquid-to-solid ratios, and was purified through sequential (NH₄)₂SO₄ precipitation, dialysis, and Sephadex G-100 gel filtration chromatography. The results showed that two PPO isozymes with molecular masses of 59 (PPO1) and 70 kDa (PPO2) were purified from Liupao tea. PPO1 may be derived from tea leaves, and PPO2 may be produced by microbial fermentation. When catechol was used as the substrate, the optimum pHs of PPO1 and PPO2 were 6.5 and 6.0, and the optimum temperatures were 40 and 35 ℃, respectively. The thermal stability of PPO1 was better than that of PPO2, although both were almost completely inactivated after heating at 90 ℃ for 5 min. Our data indicated that new isozymes produced during the fermentation of Liubao tea can synergistically improve the quality of dark tea, which will provide a theoretical basis for the control of dark tea processing conditions and the study of polyphenol oxidase of dark tea.