杨桃多酚氧化酶的部分纯化及其特性研究

Partial Purification and Characterization of Polyphenol Oxidase from Carambola

杨桃多酚氧化酶粗酶液经过DEAE-Toyopearl650M离子交换柱层析,Butyl-Toyopearl650M疏水柱层析后,被纯化了21.6倍,回收率为45.2%。该酶能迅速地催化焦性没食子酸的酶促氧化反应,而对邻苯二酚、间苯二酚、对苯二酚和绿原酸则完全无催化活性。该酶对焦性没食子酸的Km值为7.92mmol/L,其最适pH为8.0,pH稳定性范围在pH4.0-11.0,最适温度为60℃,热稳定性相对较高,在≥90℃加热30min后仍残留约9%的酶活性。该酶的最佳抑制剂是抗坏血酸,其次是NaHSO3和盐酸-L-半胱氨酸,Cu2+、Zn2+、Ca2+等金属离子对该酶也有一定的抑制作用。

Polyphenol oxidase in carambola was partially purified to 21.6-fold with a recovery of 45.2% through chromatographies on DEAE-Toyopearl 650M and Butyl-Toyopearl 650M columns. The purified enzyme quickly oxidized pyrogallic acid, and had no activity towards catechol, resoreinol, hydroquinone and chlorogenic acid. The Km value of the enzyme for pyrogallic acid was 7.92mmol/L. The optimum pH was 8.0, and the enzyme activity was stable with pH in the range of 4.0 -11.0. The enzyme had an optimum temperature of 60℃ and was relatively stable at higher temperature： 9% of the PPO activity remained after a heat treatment at 90℃ for 30 min. The strongest inhibitors of the enzyme activity were ascorbic acid, followed by NaHSO3 and hydrochloride-L-cysteine. The enzyme was also inhibited by metal ion such as Cu^2＋ , Zn^2＋ and Ca^2＋.