| Project/Area Number |
18608002
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
極限環境生物学
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| Research Institution | Kyoto University |
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Principal Investigator |
MATSUMI Rie Kyoto University, Graduate School of Engineering, Researcher (90397597)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥4,240,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥540,000)
Fiscal Year 2007: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | secretion / gene disruption / genetics / signal peptide / signal peptide peptidase / transporter / archaea / hyperthermophiles / 超好熱始原菌 / 膜透過 / 分泌タンパク質 / 抗生物質耐性 / simvastatin / sugar transporter / amylopullulanase |
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| Research Abstract |
We identified and characterized the signal peptide peptidase (SppA) from the Thermococcus kodakaraensis. In a search for additional enzymes involved in signal peptide degradation, a gene encoding a putative membrane-bound peptidase with similarity to SppA was found. Western blot analysis indicated that the protein (SppB) is membrane-associated. In contrast to SppA, SppB prefers basic amino acids at the P-2 site and hydrophobic residues at the P-1 position. As archaeal signal peptides are comprised from an n-domain (with basic residues) and an h-domain (with hydrophobic residues), the substrate specificities of SppA and SppB can be expected to efficiently complement one another in the initial breakdown of signal peptides at the cytoplasmic membrane. A detailed site-directed mutagenesis study indicated that the peptidase activity of SppB_<Tk> is dependent on a Ser130-His226-Asp154 catalytic triad, distinct to the Ser-Lys catalytic dyad of SppA.
We developed a gene disruption system in T. kodakaraensis based on antibiotic resistance. With this system, we disrupted the amylopullulanase gene (apu) or a gene cluster which includes apu and genes of a putative sugar transporter. Cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Δapu strain could not grow on pullulan and displayed only low levels of growth on amylase, suggesting that Apu is a major polysaccharide-degrading enzyme in T. kodakaraensis.
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