层析法分离纳豆激酶的研究

选高产酶的纳豆杆菌发酵,发酵液离心除菌后加入饱和度为30%硫酸铵去杂,然后加入65%的硫酸铵析出纳豆激酶粗提物,然后将粗提物分别过阴阳离子交换柱和疏水层析柱进行提纯酶。比较其提纯倍数和回收率,得出较好得分离纯化方案为:样品依次经过DEAE-SepharoseFastFlow阴离子层析、CM-SepharoseFastFlow阳离子层析和Penpyl-SepharoseCl-4B疏水层析柱,纳豆激酶最终纯化倍数达到32.2,回收率为13.2%。

Separation and Purification of Nattokinase with Chromatography

In order to obtain high purity nattokinase from the fermentative broth of Bacillus subtilis, some separation and purification techniques were explored. The optimized separation and purification process includes the following steps: first removing cells by centrifugation, then removing the impurity protein by 30% ammonium sulfate, precipitating the crude extracts of nattokinase by 65% ammonium sulfate and finally purifying the crude extracts by DEAE-Sepharose fast flow ion-exchange chromatography, CM-Sepharose fast flow ion exchange chromatography and Phenyl-Sepharose Fast Flow chromatography, respectively. Besides, the fibrinolytic activity of the nattokinase was measured by means of fibrin plate method. The purification factor and activity recovery of the nattokinase are 32.2% and 13.2%, respectively. The above-mentioned processes can serve as an efficient way to prepare pure nattokinase with high yield especially for lab scale.