| Project/Area Number |
62480028
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵工学
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| Research Institution | Research Center for Advanced Science and Technoogy, University of Tokyo (1988)
Tokyo Institute of Technology (1987) |
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Principal Investigator |
KARUBE Isao Res.Cent. for Adv.Sci. & Technol., Univ. Tokyo, Professor, 先端科学技術研究センター, 教授 (50089827)
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| Co-Investigator(Kenkyū-buntansha) |
SODE Koji Res.Cent. for Adv.Sci. & Technol., Univ. Tokyo, Research Associate, 先端科学技術研究センター, 助手 (10187883)
TAMIYA Eiichi Res.Cent. for Adv.Sci. & Technol., Univ. Tokyo, Assoc. Professor, 先端科学技術研究センター, 助教授 (60179893)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1987: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Keywords | Escherichia coli / Hydrogenase / Hydrogen production / Protein engineering / hydA / hydB / 大腸菌 |
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| Research Abstract |
This research aimed the improvement of the enzyme character of hydrogenase by protein engineering. This enzyme dominates the hydrogen metabolism in anaerobic bacteria, therefore, the improvement of this enzyme will enhance the microbial hydrogen production efficiency.
First, we investigated the roll and the products of both hydA and hydB. According to the gene product analyses, we assumed, 1)hydA product seems to control the expression level of hydrogenase in vivo, and 2)hydB product seems to be one of the subunit constructing hydrogenase.
We then attempted the improvement of hydrogenase activity in vivo, by modifing hydA by site directed mutagenesis using synthetic DNA, synthesized by DNA synthesizer (Biosearch; Cyclone). The alternation of one amino acid, cysteine to serine, resulted in the improvement of hydrogenase activity (133% of wild strain). We assumed that the alternation of amino acid sequence in hydA might affect and change the expression level of hydrogenase.
We have also found that hvdB mutant restored its hydrogenase activity by cultivating in a medium containing nickel ion. We also found that hydB mutant cultivated in media containing iron or vanadium ion restored only hydrogen uptake activity but not hydrogen evolution activity of hydrogenase. The cultivation in the presence such metals might result in incorporation of these metals in active center of hydrogenase instead of nickel, hence the alternation in its character might occur.
In conclusion, we have been suceeded in the modification of hydrogenase by means of proteinengineering. These results will contribute to the research of microbial hydrogen production.
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