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

植物界二糖苷特异性糖基水解酶家族1新家族的建立

基本信息

批准号：
13460049
负责人：
SAKATA Kanzo
金额：
$ 11.9万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

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.

本研究项目取得了以下研究成果。（A）阐明二糖苷酶的催化机制及其立体结构1）二糖苷酶的底物特异性，如茶叶β-樱草糖苷酶（PRD）、Viburnum furcatum中的furcatin水解酶（FH）、Vicia angustifolia var. 2）在昆虫细胞中高效表达了PRD和VH的全长cDNA，并在昆虫细胞中成功表达了两种二糖苷酶。大肠杆菌中的FH重组PRD是在约。30毫克/升的培养基。这使我们能够尝试结晶的蛋白质的X射线晶体学分析。3）我们已经开发了一种新型的糖苷酶抑制剂，glycosylamidines，具有有效的和选择性的活动。以β-樱草糖基为配基，制备了一种亲和吸附剂，对PRD及其它二糖苷酶的纯化有较好的效果。（B）植物界一个新的二糖苷酶家族的建立及其生理功能的阐明用重组PRD制备的抗PRD抗体具有高度的选择性和敏感性。利用该抗体可以了解PRD基因在茶树幼叶中的高水平表达以及PRD基因在细胞膜和细胞间隙中的定位。β-樱草糖苷酶活性、TLC检测二糖形成和抗PRD抗体免疫印迹分析。这些结果有力地说明了二糖苷酶在植物界的广泛分布。