| Project/Area Number |
14360043
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KIMURA Atsuo Hokkaido Univ., Grad.School of Agr., Prof., 大学院・農学研究科, 教授 (90186312)
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| Co-Investigator(Kenkyū-buntansha) |
MORI Haruhide Hokkaido Univ., Grad.School of Agr., Asso.Prof., 大学院・農学研究科, 助教授 (80241363)
OKUYAMA Masayuki Hokkaido Univ., Grad.School of Agr., Assi.Prof., 大学院・農学研究科, 助手 (00344490)
玖村 朗人 北海道大学, 大学院・農学研究科, 助教授 (00241365)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2004: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2003: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 2002: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Keywords | transglycosidation / glycosylase / synthase |
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| Research Abstract |
Glycosylases are enzymes that hydrolyze the glycosidic linkage. These enzymes also catalyze the transglycosidation, in which the glycosyl residue is transferred to the acceptor substrate. The transglycosidation is an important reaction i)to produce oligosaccharides valuable for foods and ii)to synthesize bio-active sugar-chains. Transglycosidation and hydrolysis proceed in the same time, meaning that the substrate for transglycosidation as well as its product(s) is cleaved by hydrolysis even under conditions of transglycosidation. We have studied the reactions of glycosylase, and have found the phenomena that catalyzed the transglycosidation only. In this study, we analyze the mechanism of valuable phenomena and perform their application. The results are summarized as follows. 1)We have determined the catalytic residue of negatively charged by the method using suicide substrate. Mutant enzyme (synthase), of which catalytic residue was replaced, was produce and purified. The enzyme showed no hydrolytic reaction, only catalyzed the synthesis of oligosaccharide(s) from fluoride-substrate and acceptor. Acceptor of aryl glycoside is a good substrate, meaning that the hydrophobic interaction between aryl-group and subsite +2 is important. 2)Mutant enzyme, which recognized the plane-shaped substrate, a mimic compound of reaction intermediate, was constructed, and its ability of oligosaccharide-synthesis was studied. The low production was observed. We changed the substrate concentration, and succeeded in the improvement of yield. Addition of alcohol to reaction mixture was also effective, but the high concentration of alcohol decreased the production of oligosaccharide. We have found a glycosidase resistant for alcohol. Currently, the conversion of alcohol-stable enzyme to mutant enzyme of same type is trying.
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