蓝莓多酚氧化酶的部分纯化及其酶学特性研究

Partial purification and characterization of polyphenol oxidase from blueberry

目的探究蓝莓多酚氧化酶(polyphenol oxidase,PPO)的酶学特性。方法分别采用硫酸铵分级沉淀法和疏水层析法分离纯化蓝莓PPO,比较2者的纯化效果,并探究蓝莓PPO的酶促反应动力学、最适反应温度、pH及稳定性。结果硫酸铵分级沉淀法与疏水层析法的纯化倍数相近,但2者的得率分别为61%和84%。以绿原酸和邻苯二酚为底物时,蓝莓PPO的米氏常数(K_(m))分别为23.38 mmol/L和6.13 mmol/L。该酶的最适pH为6.0,最适反应温度为25℃。该酶在40℃以上不稳定,在70℃时,其酶活在60 s后残余酶活仅为7.23%。随着pH的不断降低,其稳定性不断下降。结论相比于硫酸铵分级沉淀法,疏水层析法对蓝莓PPO的分离纯化效果更好,蓝莓PPO对邻苯二酚的亲和力更高,该酶对温度和pH都较敏感。

Objective To investigate the properties of polyphenol oxidase(PPO)from blueberry.Methods Ammonium sulfate fractional precipitation and hydrophobic chromatography were used to separate and purify PPO from blueberry.The purification effects of the two methods were compared.Moreover,the enzymatic reaction kinetics,optimal reaction temperature,pH and stability of blueberry PPO were studied.Results The purification fold of ammonium sulfate precipitation and hydrophobic chromatography was similar,but the yields of the two methods were 61%and 84%,respectively.The Michaelis constants(K_(m))of PPO of blueberry were 23.38 mmol/L and 6.13 mmol/L respectively when chlorogenic acid and catechol were used as substrates.The optimum pH value of the enzyme was 6.0 and the optimum reaction temperature was 25℃.The enzyme was unstable at 40℃and only the residual activity was 7.23%after incubated at 70℃for 60 s.With the decreasing of pH value,the stability of the enzyme decreased.Conclusion Compared with ammonium sulfate fractionated precipitation method,hydrophobic chromatography has a better separation and purification effect on blueberry PPO.Blueberry PPO has a higher affinity for catechol,and the enzyme is sensitive to both temperature and pH.