贵州豆豉纤溶酶发酵液中活性酶的盐析分离纯化

Separation and Purification of Active Enzyme from Fibrinolytic Enzyme Fermentation Broth of Guizhou Lobster Sauce with Salting Fractionation Method

目的:对贵州豆豉发酵液中纤溶酶分离纯化。方法:贵州特色药食两用的豆豉中提取得到的纤溶酶菌株(DCK-B),用液体发酵法将其发酵,采用硫酸铵盐析法进行粗提纯,并对0～80%浓度的一级硫酸铵梯度盐析与0～30%、30%～60%两段硫酸铵梯度盐析的结果进行比较。结果:硫酸铵梯度盐析在30%以前出现的沉淀为杂蛋白,且较复杂;30%梯度以后的目标蛋白大量沉淀出来,到50%时活性蛋白几乎沉淀完全;经两段盐析,当0～30%盐析时有少量活性蛋白盐析出来,绝大部分蛋白在30%～60%这个盐析梯度沉降下来。结论:确定了贵州豆豉纤溶酶发酵液中活性酶的两段硫酸铵盐析分离纯化方法。

Objective： To culture fibrinolytic enzyme strains （DCK-B）extracted from Guizhou characteristic lobster sauce with liquid ferment method, and to develop a method for primary purifica- tion of active fibrinolytie enzyme from the fermentation products. Methods： Ammonium sulphate （AS） salting out method was used for primary purification, and the effects of one step salting out method with 0% -80% AS and two step salting out method with AS of 0% -30% and 30% -60% were com- pared. Results： The precipitate was impure protein when AS concentration was lower than 30%. A lot of target protein precipitated when AS concentration was higher than 30%. When the concentration was up to 50%, almost all the active protein precipitated. It indicated that a few active protein was salted out when AS concentration was between 0 -30% and most of active protein salted out when AS con- centration was between 30% - 60%. Conclusions： The two step AS salting out method for separation and purification of active enzyme from fibrinolytic enzyme fermentation broth of Guizhou lobster sauce is established.