Quinoprotein-dependent periplasmic oxidase system in aerobic bacteria

与来自德国和荷兰的三个不同研究所合作了一年，取得了以下成果：(1)利用富集法从土壤中分离到一株新的细菌菌株恶臭假单胞菌HK5。当生长在不同的酒精上时，表达三种不同的藜麦蛋白酒精脱氢酶。它们是一种藜麦蛋白和两种藜麦血色素蛋白，在周质氧化酶的基础上，它们在生化、生理学和应用酶学方面表现出了有趣的方面。(2)葡萄糖脱氢酶是一种典型的藜麦蛋白，根据它们的定位可分为膜结合酶和可溶性酶。这两种酶被高度纯化并转化为脱辅酶。研究了PQQ与辅酶的结合过程，发现这两种酶在很多方面是完全不同的。(3)从亚羟基葡糖杆菌膜组分中分离纯化了一种乙醇脱氢酶失活酶。非常有趣的是，在消耗生物能量的情况下，失活的酶可以转化为有活性的酶。(4)亚氧葡萄糖杆菌的乙醇脱氢酶由三个不同的亚基组成。第二个亚基含有三个摩尔的细胞色素c，在乙醇氧化的催化活性中表现出一个有趣的现象。首次提出了酶活性过程中电子在分子内流动的几个证据。

One year collaboration with three different institutes from Germany and the Netherlands has yielded the following results.(1) A novel bacterial strain, Pseudomonas putida HK5, was isolated from soil by enrichment technique. Three distinct quinoprotein alcohol dehydrogenases are expressed when grown on different alcohols. They are one quinoprotein and two quinohemoproteins and they showed interesting aspects in biochemistry, physiology and applied enzymology, on the basis of periplasmic oxidase.(2) Glucose dehydrogenase, a typical quinoprotein, is divided into two species depending on their localization, membrane-bound and soluble enzymes. The two enzymes were highly purified and converted to the apo-enzymes. The binding process of PQQ to the apo-enzymes was investigated revealing that the two enzymes are completely different each other in many aspects.(3) An inactive enzyme of alcohol dehydrogenase has been purified from the membrane fraction of Gluconobacter suboxydans. It is interesting very much to know that the inactive enzyme can be converted to the active one by the expenses of biological energy.(4) Alcohol dehydrogenase from Gluconobacter suboxydans is consisted of three different subunits. The second subunit contains three moles of cytochromes c and shows an interesting phenomena during the catalytic activity of alcohol oxidation. Several evidence for intramolecular flow of electron during enzyme activity were presented for the first time.