| Project/Area Number |
13460049
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Bioproduction chemistry/Bioorganic chemistry
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
SAKATA Kanzo Kyoto University, Institute for Chemical Research, Professor, 化学研究所, 教授 (20087563)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
HIRATAKE Jun Kyoto University, Institute for Chemical Research, Associate Professor, 化学研究所, 助教授 (80199075)
MIZUTANI Masaharu Kyoto University, Institute for Chemical Research, Instructor, 化学研究所, 助手 (60303898)
SHIMIZU Bunichi Kyoto University, Institute for Chemical Research, Instructor, 化学研究所, 助手 (50324695)
KATO Hiroaki Harima Institute, RIKEN, Membrane Dynamic Research Group, Cheam Leader, 速度論的結晶学研究チーム, チームリーダー (90204487)
SAKATA Kanzo Kyoto University, Institute for Chemical Research, Professor (20087563)
|
|---|
| Project Period (FY) |
2001 – 2003
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥18,600,000 (Direct Cost: ¥18,600,000)
Fiscal Year 2003: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2002: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2001: ¥10,000,000 (Direct Cost: ¥10,000,000)
|
|---|
| Keywords | β-primeverosidase / diglycosidase / disaccharide glycoside specific hydrolase / glycosyl hydrolase family 1 / furcatin hydrolase / vicianin hydrolase / glycosylamidine / affinity adsorbent / カラスノエンドウ / グリコシダーゼファミリー / フルカチン / ムシカリノキ / 生体防御 / 二糖配糖体 / 二糖配糖体加水分解酵素 / コンビナトリアル / 基質特異性 |
|---|
| Research Abstract |
The following research results have been obtained by this research project.
(A)Clarification of catalytic mechanisms of the diglycosidases and their stereostructures
1)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 roles
The 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.
|
