| Project/Area Number |
16360411
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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 |
Biofunction/Bioprocess
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| Research Institution | Nagoya University |
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Principal Investigator |
NAKANO Hideo Nagoya University, Graduate School of Bioagricultural Sciences, Professor, 大学院生命農学研究科, 教授 (00237348)
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| Co-Investigator(Kenkyū-buntansha) |
IWASAKI Yugo Nagoya University, Graduate School of Bioagricultural Sciences, Associate Professor, 大学院生命農学研究科, 助教授 (50273214)
加藤 且也 独立行政法人)産業技術総合研究所中部センター, 主任研究員 (70356781)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 2006: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2004: ¥7,500,000 (Direct Cost: ¥7,500,000)
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| Keywords | lipase / cell-free protein synthesis / single-molecule PCR / enantioselectivity / high-throughput screening / emulsion / ハイスループットスクリーニン / ファジーニューラルネットワーク |
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| Research Abstract |
In this research, it has aimed to create the lipase group that has a variety of substrate specificities by introducing the combinatorial mutation only into the substrate binding pocket of an industrially useful lipase as a structural frame. The outline of the result is shown as follows.
1. The combinatorial library where four amino acid in the substrate binding pocket had been made a target based on the computer model of the enzyme complex was made by the SIMPLEX method. Acquired data in the first screening was analyzed by fuzzy neural net work (FNN), and some fuzzy rules were derived from the analysis. Finally, the obtained rules were proven to be useful in obtaining new mutant enzymes.
2. The Burkholderia cepacia KWI-56 lipase shows only a low optical selectivity to 2-phenylbutyrate. Then, the structural model of the reaction intermediate between the substrate and the enzyme was constructed on the computer to give possible residues for introducing mutations. Four residues were selected in the pocket, and combinatorial mutation limited to the hydrophobic amino acids (seven kinds) was introduced to the selected site by the SIMPLEX method. The screening of the SIMPLEX library gave some clones showing a higher enantioselectivity than the wild type.
3.To make the SIMPLEX method more effective, a new technology amplifying single DNA molecules on microbeads in emulsion was developed. After one bead PCR on a 384-well plate, DNA library could be constructed on the plate. The mutation library of the lipase was constructed as above, and screened against the above-mentioned substrate, resulting that a high enantioselecitve mutant was obtained.
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