底物亲和法分离纯化蕲蛇胃蛋白酶

Separation and Purification of the Pepsin from Snake Stomach by it's Affinity with Substrate

利用酶(胃蛋白酶)与其底物(酪蛋白)的亲和性,从蕲蛇胃中提取得到一种胃蛋白酶。让底物和酶在pH 2.5的乳酸缓冲液中充分结合,然后调pH至4.0(底物的等电点)沉淀底物和酶的结合物,随后让沉淀物再溶解于乳酸缓冲液中,添加低浓度的SDS将底物和酶分离,最后用DEAE离子交换色谱柱进行纯化,经SDS—聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,分离得到的胃蛋白酶为电泳纯,其分子量为33KD。酶的最适pH值小于3.0,最适温度为50℃。该结果证明胃蛋白酶能利用其与酪蛋白的亲合性及SDS沉淀作用有效地分离,这种胃蛋白酶能在食品生产的加热、强酸处理过程中应用。

A kind of pepsin from snake stomach was separated and purified via affinity between pepsin and its unique substrate, casein. The pepsin extracted from the stomach of Agkistrodon Acutus was dissolved together with casein in lactic acid buffer (pH2.5). After completely interaction, the pH was adjusted to 4.0 isoelectric point of the substrate to precipitate the substrate-enzyme complex. The precipitated complex was re-dissolved in the same lactic acid buffer. Sequentially casein was removed from the complex by centrifugation after adding low concentration SDS to the buffer. The supernatant was applied to an ion-exchange chromatographic column to obtain a fraction with highest proteolytic activity. The fraction showed a single band purity of electra phoretic grade in SDS-PAGE. The molecule weight was 33 KD. The optimal pH of this purified fraction was less than 3. These results demonstrated that pepsin could be efficiently separated by its affinity with casein and SDS precipitation. This pepsin is possible to be used in thermal and strong acidic process of food production.