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

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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.