双水相纯化淀粉糖化酶的影响因素研究

Study on influencing factors of salt/PEG aqueous two-phase systems for the purification of glucamylase

以四种不同的淀粉糖化酶发酵液为研究对象,用纯化倍数作为参考指标。在单因素实验基础上,选取四种分子量的聚乙二醇(PEG600、PEG800、PEG1000、PEG2000)、三种盐(MgSO4、(NH4)2SO4、KH2PO4)和四种发酵的淀粉糖化酶酶液(米根霉、根霉、酒精酵母、酯化酵母)为自变量。得到最佳的提取条件为:米根霉在PEG1000浓度为15%(均为质量浓度),MgSO4浓度为22.86%,纯化倍数可达3.35;其酒精酵母和酯化酵母在PEG1000浓度均为15%,(NH4)2SO4浓度分别为15.73%和13.33%,得到纯化倍数为1.82、1.50;根霉在PEG2000浓度为15%,盐(NH4)2SO4添加14.37%,纯化倍数达2.07。该方法在淀粉糖化酶含量有差异的情况下,能保存酶活去除杂蛋白,分离出淀粉糖化酶。

Extraction and separation of glucamylase by two-phase aqueous systems was investigated. The effects of the molecular weight （600,800,1000,2000） and concentration of polyethylene glycol （PEG）, the concentrations of salt（MgSO4, （NH4）SO4, KH2PO4） and the four crude enzymes of glucamylase（Rhizopusoryzae, Rhizopus,Distillery yeast,Esterification yeast） on the purification factor were studied. The results showed that Rhizopusoryzae under the PEG1000＇s mass concentration of 15%, MgSO4 with the concentration of 22.86% got the purification factor were 3.35. Distillery yeast and Esterification yeast at the PEG1000＇s concentration with 15%, the （NH4） SO4＇ s concentration were 15.73% and 13.33%, respectively, got the purification fold were 1.82,1.50. Rhizopus at the molecular weight of PEG with 2000,concentration was 15% ,the （NH4）2SO4＇s concentration was 14.37% ,The purification fold were 2.07. This method could keep biological activity and simplicity of operator, had good application prospects.