龙井茶树多酚氧化酶蛋白提取方法的优化

摘要

目的：优化缓冲液匀浆提取茶树多酚氧化酶的方法。方法以龙井43号茶树鲜叶为材料,采用缓冲液匀浆浸提法提取茶树多酚氧化酶,分别进行不同单因素浸提优化试验,测定比较提取的粗酶活性,确定适合的浸提条件。结果以鲜叶标准为一芽二叶,料液比1：2,加入内含5% PVP(w：v)、经预冷的pH5.6柠檬酸-磷酸盐缓冲液(0.15 mol/L),海砂适量,冰浴研磨,4℃条件下隔夜浸提12 h,于4℃、9000 r/min离心35 min,取上清液,为龙井43号鲜叶中多酚氧化酶缓冲液成浆浸提的最优条件。结论采用优化的浸提条件,可以制备得到高活性多酚氧化酶粗酶液,为茶树多酚氧化酶同工酶分离与酶性质研究提供了基础。%Objective To optimize the extraction method of the tea polyphenol oxidase in buffer solution homogenate.MethodsPolyphenol oxidase from fresh leaves of Longjing 43 was extracted by buffer homogenate method. Different single extraction factors were tested, and the extracted crude enzyme activity was measured and compared to determine the appropriate extraction conditions.Results The fresh leaves were of a bud with two leaves, solid (tea leaf)-liquid (extraction buffer) ratio was 1:2, and the pre-cooling citrate-phosphate buffer (pH 5.6, 0.15 mol/L) containing 5% PVP (w:v) was added, a small amount of sea sand was added, and tea leaves were ground in ice bath and extracted overnight about 12 h at 4℃. The extracted buffer was centrifuged for 35 min with 9000 r/min at 4℃, and the supernatant was obtained.Conclusion High activity of crude polyphenol oxidase solution can be prepared using the optimized extraction conditions, which benefits to the isolation and purification and the analysis of enzymatic properties for isozyme of tea polyphenol oxidase.