Joint Studies on Dextran-Producing and -Degrading Enzymes

葡聚糖蔗糖酶是葡聚糖酶的一种，可以催化蔗糖生成葡聚糖。最近，金博士建立了一种新的突变技术——真空紫外线辐射。突变菌株获得高产葡聚糖蔗糖酶组成，提供了生产大量葡聚糖的可能性。右旋糖酐及其低聚糖已被发现对人体具有多种有益功能。在国际项目中，我们试图阐明右旋糖酐生成酶和降解酶的结构和功能之间的关系，并通过这两种酶制备右旋糖酐和低聚异麦芽糖。(1)成功分离了葡聚糖蔗糖酶高产基因并在大肠杆菌中表达。在启动子区域发现了突变位置。(2)克隆了异麦芽糖三葡聚糖酶基因，并在大肠杆菌中表达。α-葡萄糖苷酶基因位于该基因的上游区域。这两个基因形成了一个由一个启动子控制的簇状结构。(3)过量生产葡聚糖蔗糖酶制备了大量葡聚糖。研究了利用异麦芽糖三葡聚糖酶从葡聚糖中提取纯异麦芽糖的有效方法。(4)从右旋糖酐中纯化出一种生成四糖和五糖的酶，并对其性质进行了研究。(5)设计了基于机理的葡聚糖酶灭活剂。失活动力学研究表明，合成的化合物是新的葡聚糖酶自杀底物。

Dextransucrase, one of glucansucrases, catalyzes the formation of dextran from sucrose. Recently Dr. Kim has established a novel mutation technique, Vacuum UV radiation. The mutant strains obtained hyper-produced dextransucrase constitutively, giving a possibility to produce the large amount of dextran. Dextran and its oligosaccharides have been found to have several useful functions to human. In the international project we tried to elucidate the relationship between structures and functions of dextran-producing and -degrading enzymes, and prepared the dextran and isomaltooligosaccharides by both enzymes.(1) We succeeded in isolation of a hyper-produced dextransucrase gene and its expression in Escherichia coli. The mutated position was found in the promoter region. (2) A gene of isomaltotrio-dextranase was cloned and expressed in E. coli. The α-glucosidase gene was found in the upstream region of this gene. Both genes made a cluster structure, which was controlled by one promoter. (3) The large amount of dextran was prepared by hyper-produced dextransucrase. We examined the effective production method of pure isomaltotriose from dextran using isomaltotrio-dextranase. (4) An enzyme giving tetra- and penta-saccharides from dextran was purified and the properties were investigated. (5) Mechanism-based inactivator for dextranase was designed. Kinetic studies on inactivation indicated that compounds synthesized were found to be novel suicide substrate for dextranase.