Purification of Polyphenol Oxidase from Tea Leaf by Three Phase Partitioning and Enzymatic Properties

In order to study a fast and efficient method for obtain polyphenol oxidase (PPO) from tea leaf, three phase partitioning (TPP) were used to purified PPO and then its enzymatic properties were studied. The results showed that PPO from tea leaf was purified with 15.76-fold enrichment in the specific activity of 210.46 U/mg and 8.04% recovery by TPP purification method for the second time. The strongest affinity substrate was pyrogallol (K_m=6.82 mmol/L, V_max=5.36×10⁻² OD/min) for purified tea PPO. The optimum temperature and pH of the enzyme were 45 ℃ and 5.5, respectively. The enzyme had great stability under condition of 25 ℃ and pH4.5~7.0. The most significant inhibitory effect of different compounds on purified tea PPO was ascorbic acid (IC₅₀=2.42±0.18 mmol/L), followed by oxalic acid and EDTA. The inhibitory effect of citric acid was the weakest, while halides had no significant inhibitory effect (P>0.05). This work proves that the TPP method can better retain the tea PPO activity, and easy to operate, time-consuming and low-cost for the purification of tea PPO, which is beneficial to industrial applications.