高纯度木瓜蛋白酶的分离纯化和性质研究

Purification and characterization of high purity papain from Carica papaya

目的建立木瓜蛋白酶的分离纯化工艺路径。方法采用初步采集处理,20%,40%硫酸铵分级沉淀、SP-sephadex C50柱色谱、羟基磷灰石柱色谱,从番木瓜乳汁中分离纯化木瓜蛋白酶。结果获得比活为1 184 U/mg的纯酶,活力回收率为55.79%,纯化的酶经SDS-PAGE呈单一条带,高效液相色谱呈单一条带,纯度达到99.31%。该酶耐高温,最适pH值为7.2,对酪蛋白的动力学常数(Km)为3.5×10-5mol/L。结论应用此方法成功纯化了高纯度的木瓜蛋白酶。

Purpose To construct a simple way for purifying papain from Carica papaya. Methods Using means of collection, 20% and 40 % saturated ammonium sulfate fractionation, SP-sephadex C 50 column chromatography and hydroxyapatite chromatography to purify papain from Carica papaya. Results A 55.79 % yield of enzyme with a specific activity of 1 184 U/mg was obtained. The purified enzyme shows a single band on SDS-PAGE and a purity of 99.31% on HPLC. The enzyme is able to bear high temperature, and its optimum pH is 7.2, the Km with casein is 3.5 ×10^-5 mol/L. Conclusion Papain was successfully purificated by a simple method.