茶叶多酚氧化酶三相分离纯化及酶学性质研究

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

为研究一种快速高效的茶叶多酚氧化酶(polyphenol oxidase,PPO)制备方法,本试验采用三相分离法(three phase partitioning,TPP)获取茶叶PPO,并研究其酶学特性。结果表明,通过两次三相分离纯化后可获得酶比活力210.46 U/mg、纯化15.76倍、回收率8.04%的茶叶PPO,其最亲和底物为邻苯三酚(K_(m)=6.82 mmol/L,V_(max)=5.36×10^(−2) OD/min),最适反应温度和pH分别为45℃和pH5.5,在25℃和pH4.5~7.0时具有较好的稳定性。对纯化后茶叶PPO抑制作用最显著的化合物为抗坏血酸(IC_(50)=2.42±0.18 mmol/L),其次是草酸和EDTA,柠檬酸抑制效果最弱,而卤化物无显著影响(P>0.05)。表明三相分离法能够较好的保留茶叶PPO活性,且操作简便、耗时短、成本低,有利于工业化应用。

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(IC_(50)=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.