番石榴多酚氧化酶的部分纯化及其特性研究

番石榴PPO的粗提取液经过80%硫酸铵盐析和DEAE-Toyopearl 650M、CM-Sephadex C-50离子交换柱层析分离后,被纯化了约126倍,回收率为16.13%。该酶迅速地催化焦性没食子酸的酶促氧化反应,而对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4.6 mmol/L,其最适pH为7.5,pH稳定性范围在pH4.0~11.0,最适温度为50℃,热稳定性相对较高,在≥90℃加热10 min后仍残留约15%的酶活性。该酶的最佳抑制剂是抗坏血酸和NaHSO3,其次是植酸、盐酸-L-半胱氨酸、柠檬酸,Ca2+、Mg2+等金属离子对该PPO也有较强的抑制作用。

Partial Purification and Characterization of Polyphenol Oxidase from Guava (Psidium guajava L.)

Polyphenol oxidase(EC.1.10.3.1,PPO) in guava(Psidium guajava L.) was partially purified to 126-fold with a recovery of 16.13% though ammonium sulfate fractionation and chromatographies on DEAE-Toyopearl 650M and CM-Sephadex C-50 columns.The purified enzyme quickly oxidized pyrogallic acid,and had no activity towards hydroquinone and chlorogenic acid.The Km value of the enzyme for pyrogallic acid was 4.6mmol/L.The optimum pH was at 7.5,and the enzyme activity was stable in the range of pH 4～11.The enzyme had an optimum temperature of 50℃ and was relatively stable at high temperatures: 15% of the PPO activity remained after a heat treatment at 90℃ for 10 min.The strongest inhibitors of the enzyme activity were L-ascorbic acid and NaHSO_3,followed by phytic acid,hydrochloric acid-L-cysteine,citric acid.The enzyme was also strongly inhibited by Ca~(2+) and Mg~(2+).