Establishment of A New Group of Glycosyl Hydrolase Family 1 Specific to Disaccharide Glycosides in Plant Kingdom

本研究取得了以下研究成果：(1)阐明了双糖苷酶的催化机理及其立体结构1)双糖苷酶的底物特异性，如茶叶β-Primeverosidase(PrD)、Vicianin水解酶(VH)和Vicianin水解酶(VH)。不同人工合成和天然底物的研究表明，这些二糖二糖酶对带有β-1，6键的二糖苷类化合物是非常特异的。2)PRD和VH的全长基因在昆虫细胞和昆虫细胞中过表达。大肠杆菌中的FH。当培养基用量约为30 mg/L时，可获得重组PRD。这使得我们可以尝试蛋白质的结晶用于X射线晶体分析。3)我们开发了一种新型的糖苷酶抑制剂，糖基酰胺，具有强大的和选择性的活性。本文报道了一种以β为配体的亲和吸附树脂的制备及其对二糖苷酶的纯化效果。(B)植物二糖苷酶家族的建立及其生理功能的研究用重组二糖苷酶制备的抗体具有高度的选择性和敏感性。应用该抗体可以了解PrD基因在茶树幼叶中的高水平表达，以及PrD基因在细胞膜或细胞间隙中的定位。β-Primeveroside酶活性，薄层层析检测二糖形成和抗PRD二糖抗体免疫印迹分析，其中大多数已知含有二糖苷类化合物。这一结果有力地表明了二糖苷酶在植物界的广泛分布。

The following research results have been obtained by this research project.(A)Clarification of catalytic mechanisms of the diglycosidases and their stereostructures1)Substrate specificities of diglycosidases such as tea leaf β-primeverosidase (PRD), furcatin hydrolase (FH) in Viburnum furcatum, Vicianin hydrolase (VH) in Vicia angustifolia var. segetalis have been studied with various kinds of synthetic and natural substrates to show that these diglycosdiases are very specific to disaccharide glycosides with β-1,6 linkage.2)The full-length cDNA of PRD and VH were overexpressed in insect cells and that of. FH in E.coli. The recombinant PRD was obtained at ca. 30 mg/liter of the medium. This allowed us to try crystallization of the protein for X-ray crystallographic analysis.3)We have developed a new type of glycosidase inhibitors, glycosylamidines, with potent and selective activities. An affinity adsorbent with a ligand of β-primeverosyl has been prepared and was found to be quite effective for purification of PRD as well as the other diglycosidases.(B)Establishment of a new family of diglycosidases in plant kingdom and clarification of their physiological rolesThe anti-PRD antibody prepared with the recombinant PRD was found to be highly selective and sensitive. Application of this- antibody allowed us to know the expression of the PRD gene at higher level in the younger tea leaves and the localization of the PRD in cell membrane or intercellular space.Distribution of diglycosidases was surveyed by using. β-primeverosidase activity, detection of disaccharide formation with TLC and immunoblotting analysis with the anti-PRD antibody among several kinds of plants most of which are known to contain some disaccharide glycosides. The results strongly suggest wide range of distribution of diglycosidases in plant kingdom.