| Project/Area Number |
07405060
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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 | 一般 |
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| Research Field |
高分子合成
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KIMURA Shunsaku (1996-1997) Kyoto University, Graduate School of Engineering, Associate Professor, 工学研究科, 助教授 (80150324)
今西 幸男 (1995) 京都大学, 工学研究科, 教授 (00025991)
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| Co-Investigator(Kenkyū-buntansha) |
IMANISHI Yukio Nara Institute of Science and Technology, Department of Material Science, Profes, 物質創成科学研究科, 教授 (00025991)
伊藤 嘉浩 京都大学, 工学研究科, 助教授 (40192497)
木村 俊作 京都大学, 工学研究科, 助教授 (80150324)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1997: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1996: ¥3,800,000 (Direct Cost: ¥3,800,000)
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| Keywords | Mutants / Recombinant DNA Technique / Artifical Enzymes / Esterase / Subtilisin / Enzymatic Activity / Protein Structure / タンパク質工学 / 遺伝子工学 / リゾチーム / tRNA / ポリプロリン / 非天然アミノ酸 |
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| Research Abstract |
Hydrophobilized mutant enzymes of esterase from Pseudomonas sp.KWI-56 by connection of 10 or 20 tyrosine residues at the C terminus were synthesized by using the recombinant DNA technique. The mutant protein with a longer poly (tyrosine) chain became more hydrophobic than that with a shorter chain. The structure of the mutant proteins was analyzed by CD spectroscopy, which revealed the decrease of alpha-helicalconformation and the increase of beta-sheet conformation. The enzymatic activity of the mutant esterase was abolished due to the structure change induced by connection of a poly (tyrosine) chain, which should interact with hydrophobic region of the protein. On the other hand, a hydrophobilized mutant enzyme of subtilisin Carsberg by connection of poly (oxyethylene) was synthesized and the structure-activity relationship was investigated in organic solvents. The mutant enzyme showed 150-fold higher activity than the wild type in the ester-exchange reaction in benzen solution. The high activity of the mutant enzyme was also observed in dichloromethane solution, but the activity was diminished in water-miscible organic solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran. In these solvents, the molecular structure of the mutant protein was change significantly probably due to depletion of the essential water from the enzyme.
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