盐析法联合离子液体双水相纯化木瓜蛋白酶

Extraction and Purification of Papain from Papaya Fruit by Salting-out and Ionic Liquid Aqueous Two-Phase System

本实验以海南产番木瓜为研究对象,采取盐析法联合离子液体双水相对其富含的木瓜蛋白酶进行提取纯化。以不同饱和度的(NH_4)_2SO_4对粗酶液进行盐析,通过实验结果分析,确定了以饱和度分别为20%和45%的两步盐析法的最佳条件。然后再进行双水相萃取,通过单因素试验结果设计3因素3水平的响应面试验,优化双水相萃取条件为离子液体[C_4mim]N(CN)_2质量分数29.76%、(NH_4)_2SO_4质量分数16.08%、pH 8.18,在此最优条件下木瓜蛋白酶萃取率为89.01%,酶活力回收率为86.72%,所得到木瓜蛋白酶比活力为1 056 U/mg,纯化倍数达到9.6倍。然后对实验产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,经纯化的酶中杂条带明显减少,验证了分离纯化的效果。

In this experiment,salting-out followed by aqueous two-phase extraction was applied to purify papain from Hainan-grown papaya fruit.The crude enzyme was precipitated by a two-step salt-outing method with optimized saturation degree of(NH4)2SO4of20%and45%,respectively.Using one-factor-at-a-time method and response surface methodology with a three-factor and three-level Box-Behnken design,the optimized conditions for aqueous two-phase extraction were determined as follows:[C4mim]N(CN)2concentration29.76%,(NH4)2SO4concentration16.08%and pH8.18.Under these conditions,the extraction efficiency of papain was89.01%and the specific activity of the purified enzyme was1056U/mg with86.72%recovery and10.6-fold purification.The purified enzyme contained a significantly reduced number of impurity bands,as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),suggesting good purification efficiency.