酵母醇脱氢酶在双水相系统中的亲和分配行为

Afinity Partition of Yeast Alcohol Dehydrogenase in the Aqueous Two-phase Systems

将均三嗪型活性染料—活性嫩黄M—7G共价偶于聚乙二醇(PEG6000)分子上,构建双水相系统,能使醇母脱氢酶(以下简称ADH)分配系数增值11倍。观察了影响ADH分配系数的各种因素,设计了借液—液萃取方式快速部分纯化ADN的方案,经部分纯化后,ADH的比活力按每毫克制剂含单位数计算提高至700倍;按每毫克蛋白质含单位数计算提高至16倍,酶活力回收率可达86％。

Enzymes are increasingly employed as reagents in clinical practice and in industrial processes. Simple, rapid and efficient large scale procedures suitable for enzyme recovery and purification are needed urgently. It is well kwown that aqueous two-phase systems suit the purpose,and they have been used for enzyme extraction and partial purification in this laborato- ry. The method has recently been refined by coupling a low cost affinity ligand, a triazine dye (Reactive Light Yellow M-7G) to PEG6000 covalently. It re- sulted in a 11-rold increase in the partition coefficient of the enzyme ADH. The present work was carried out to study a rapid partial purification method for Baker’s Yeast ADH with affinity liquid-liquid extraction procedure. The puri- fication protocol involved two steps, resulted in the production of a rather highly purified ADH with good yield. By using the procedure,the specific activity (units/mg protein) and the practical purity (units/mg preparation) of the puri- fied ADH were 16-fold and 700-fold higher than that of the crude preparations respectively, and the recovery of the ADH activity was up to 86%.