硫酸铵沉淀和层析法分离纯化纳豆激酶的研究

Separation and Purification of Nattokinase Produced by Bacillus Subtilis with Ammonium Sulfate Precipitation and Chromatography

利用生产纳豆激酶的Bacillus subtilis进行深层发酵。采用发酵液离心除菌，20％-60％饱和度的硫酸铵沉淀，Superdex 75凝胶过滤层析和SP Sepharose Fast Flow离子交换层析对活性组分进行分离提纯。用纤维平版法测定了活力，并用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法（SDS-PAGE）对分离纯化的效果进行了检验。结果表明在SDS-PAGE中观察到单一条带，分子量27．7kD，最终纯化倍数和酶活回收率分别为8．4和49％。

In order to obtain high purity nattokinase from the fermentation broth of Bacillus subtilis, some separation and purification techniques were explored. The optimized separation and purification process includes the following steps： removing cells by the centrifugation, 20%-60% saturation ammonium sulfate precipitation, gel filtration chromatography with Superdex 75 and ion-exchange chromatography with SP Sepharose Fast Flow. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） was used to examine the purification effect, and the results indicate that the final product obtained is homogeneous and has a molecular weight around 27.7 kD. The fibrinolytic activity of the nattokinase was measured by means of fibrin plate method. The purification factor and activity recovery of the nattokinase are 8.4 and 49%, respectively. The specific activities of purified nattokinase could reach 28530 IU·mg^-1. The results demonstrate that the combination of bioseparation process of ammonium sulfate precipitation with gel filtration and ion exchange chromatography is suitable for the separation and purification of nattokinase from culture broth of Bacillus subtilis. Especially for lab scale, the process mentioned above can be easily performed as an efficient way to prepare pure nattokinase with high yield.