一种具有纤溶活性的蛋白酶的分离纯化及性质

Purification and characterization of a fibrinolytic enzyme from Bacillus subtilis Jin2-1

目的分离纯化BacillussubtilisJin2 1发酵产生的具有纤溶活性的蛋白酶 ,并对其性质进行研究。方法通过硫酸铵分级盐析 ,SerineSepharose 2B和SephacrylS— 2 0 0色谱柱进行分离纯化 ,用SDS PAGE测定纯度 ,SDS PAGE和凝胶层析测定相对分子质量 ,纤维蛋白平板法测定酶活力。结果分离得到了电泳纯的酶 ,该酶的相对分子质量为 2 8kD ,等电点约为 8 6 ,最适 pH为 7 5 ,最适温度为 4 0℃ ,在 4 0℃以下较稳定 ,超过 5 0℃时酶活力开始下降 ;金属离子Mn2 + 对酶有明显的激活作用 ,而Zn2 + 对酶有抑制作用 ,二异丙基磷酰氟 (DFP)和苯甲基磺酰氟 (PMSF)完全抑制酶活性。结论该酶是一种丝氨酸蛋白酶 ,与纳豆激酶相似 。

Objective To purify the fibrinolytic enzyme from the Bacillus subtilis Jin2 1 and analyze its properties.Methods Purifying the enzyme by the ammonium sulfate fractionation,Serine Sepharose 2B,Sephacryl S-200 chromatography and so on.Results The molecular weight was detected by the SDS PAGE and gel chromatography.The enzymatic activity was estimated by the fibrin plate method.The purified enzyme showed a single protein band in the SDS PAGE.Its relative molecular weight was 28 kD,and the isoelectric point was 8 6.The optimum pH and temperature were 7 5 and 40 ℃ .The enzyme was stable at pH 7 ～9 and less than 40 ℃ .The Mn 2+ could activate the enzymatic activity and Zn 2+ could inhibit the activity.The DFP and PMSF completely inhibited the enzymatic activity.Conclusions The enzyme is a serine protease and it is similar to nattokinase.It is expected to be a new thrombolytic medicine.