| Project/Area Number |
07555252
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
生物・生体工学
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| Research Institution | Tohoku University |
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Principal Investigator |
KOYAMA Taketoshi Tohoku University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (20089808)
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| Co-Investigator(Kenkyū-buntansha) |
KOIKE Ayumi Toyota Motor Corp., Division of the 1st.FP,Researcher, 第一FP部, 研究員
OGURA Kyozo Tohoku University, lnst.for Chem. Reac.Sci., Professor, 反応化学研究所, 教授 (80006303)
NISHINO Takuzo Tohoku University, Faculty of Engineering, Professor, 工学部, 教授 (90005827)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1996: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | farnesyl diphosphate / prenyltransferase / thermostable enzyme / polyprenyl diphosphate / isoprenoid chain elongation / isoprenoid biosynthesis / overexpression / prenyl diphosphate synthase. / ファルネシルニリン酸 / ポリプレニルニリン酸 / 合成酵素 / プレニル二リン酸 / ポリプレノール / 人口基質ホモログ |
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| Research Abstract |
1. In order to construct new enzymes catalyzing novel types of C-C bond formation that is useful for application to organic synthesis of many kinds of biologically active compounds, we introduced several kinds of mutations in the structural gene of the thermostable famesyl dihosphate (FPP) synthase of Bacillus stearothermophilus and obtained the following results : (1) Identification of binding sites for an ally lic substrate such as dimethylallyl-or gernyl diphosphate and for the homoally lic substrate, isopentenyl diphosphate. (2) Identification of critical amino acid residues that reside in the catalytic site(s)of this preny ltransferase. (3) Conversion of cataly tic function synthesizing C-15 prenyl diphosphate (famesyl diphsphate) to catalyze the synthesis of longer prenyl chain length up to C-25 (farnesyl geranyl diphosphate). (4) Construction of several kinds of catalytically active FPP synthases comprising heteromeric subunits that contain amino acid replacements in the catalytically important amino acid residues in the catalytic site. (5) Construction of several kinds of chimeric enzymes consisting half of the farnesyl diphosphate synthase subunit and half of the heptaprenyl diphosphate synthase of B.Stearmophilus.
2. Molecular cloning the genes encoding several kinds of bacterial preny ltransferases was carried out. The cloned preny ltransferase genes were : (1) Heptaprenyl diphosphate [(all-E)-C_<35>PP] synthase of B.stearothermophilus. (2) Hexaprenyl diphosphate [(all-E)-C_<30>PP] synthase of Micrococcus luteus B-P 26. (3) Decaprenyl diphosphate [(all-E)-C_<50>PP] synthase of paracoccus denitrificans and (4) Undecaprenyl diphosphate [(Z,E-mixed)-C_<55>PP] synthase of M.luteus B-P 26. Construction of each of these clone expression system which enables overproduction of the encoded preny ltransferase in Escherichia coli cells has also been carried out.
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