| Project/Area Number |
12480184
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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 |
Structural biochemistry
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| Research Institution | Tokyo University of Pharmacy and Life Science |
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Principal Investigator |
OSHIMA Tairo Tokyo University of Pharmacy and Life Science, Faculty of Life Science, Professor, 生命科学部, 教授 (60167301)
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| Co-Investigator(Kenkyū-buntansha) |
TAMAKOSHI Masataka Tokyo University of Pharmacy and Life Science, Faculty of Life Science, Assistant Professor, 生命科学部, 助手 (10277254)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥10,200,000 (Direct Cost: ¥10,200,000)
Fiscal Year 2002: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥3,600,000 (Direct Cost: ¥3,600,000)
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| Keywords | Extreme thermophile / Evolutionary engineering / Temperature adaptation / Isopropylmalate dehydrogenase / Evolutionary biochemistry / 実験室内進化 / 進化分子工学 / 好熱性細菌 / Directed Evolution / 熱安定酵素 / 3-イソプロピルリンゴ酸デヒドロゲナーゼ / 反応速度論 / ランダム変異 |
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| Research Abstract |
The aim of this project is to understand which sites are necessary to raise the catalytic ability of an enzyme and how the stability to heat and the functional efficiency are related in an enzyme. As a model enzyme, isopropylmalate dehydrogenase, an enzyme in leucine biosynthesis, was selected and leuB gene which cods the enzyme, was coned into E.coli and expressed. Since the enzyme activity of isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus, is poor at 40℃, the E.coli transformants which harbor leuB gene from T.thermophilus could not grow well in the absence of leucine. Mutants which can grow faster were picked up and the enzyme produced by the isolated mutants was analyzed and characterized. The conclusions are; 1 It is not necessary to trade the stability to heat for improved activity at lower temperatures, and it is possible to create a robust enzyme with high catalytic activity at mesophilic temperatures, and 2 It is not necessary to replace amino acids residues at the active site for improvement of the catalytic properties, rather displace the position of the residues at the active center by replacing the residues located far from the center.
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