| Project/Area Number |
09460053
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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 |
応用微生物学・応用生物化学
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| Research Institution | Tokyo University of Agriculture and Technology (1998-1999)
The Institute of Physical and Chemical Research (1997) |
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Principal Investigator |
YOHDA Masafumi Tokyo University of Agriculture and Technology, Department of Life Science and Biotechnology, Associate Professor, 工学部, 助教授 (50250105)
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| Co-Investigator(Kenkyū-buntansha) |
EASKI Nobuyoshi Kyoto University, Institute for Chemical Research, Professor, 化学研究所, 教授 (50135597)
IMAI Kazuhiro The University of Tokyo, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学系研究科, 教授 (50012620)
KAGAWA Yasuo Kagawa Nutrition University, Department of Medical Chemistry, Professor, 医化学研究室, 教授 (30048962)
MARUYAMA Tadashi Marine Biotechnology Institute, Kamaishi Laboratories, Senior Researcher, 釡石研究所, 主任研究員
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥13,600,000 (Direct Cost: ¥13,600,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1998: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1997: ¥7,200,000 (Direct Cost: ¥7,200,000)
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| Keywords | Amino Acid Racemase / D-Amino Acid / Archaea / Hyperthermophile / Epimerase / Aspartic Acid / Diaminopimelate / PLP Independent / 超好熱性菌 |
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| Research Abstract |
We have revealed that D-amino acids exist ubiquitously in hyperthermophilic archaea, Pyrococcus and Thermococcus strains. Especially, aspartic acid was almost completely racemized. The full length gene of aspartate racemase was cloned from Thermococcus strain KS-8, and it was expressed in E.coli. The recombinant protein exhibited high aspartate racemase activity at 70 ℃ and trace glutamate racemase activity. Several aspartate racemase homologue genes were found in the total genomic sequences of hyperthermophilic archaea. Among them, two aspartate racemase homologues of Pyrococcus horikoshii (PH0670, PH 1733), and one homologue of Archaeoglobus fulgidus (AF l422) were expressed in E.coli. All of them were highly expressed, and easily purified by heat treatment and chromatography. Among them, only PH0670 exhibited asparate racemase activity. The Km and Kcat values were estimated to be 3.6 E-3 (M) and 4.2E-10 (mol/sec), respectively. On the contrary, PH 1733 and AF1422 showed no aspartate racemase activity. The fact that they are highly homologous to 0rfX of E.coli suggested that they have diaminopimelate epimerase activity. However, they could not epimerize an enantiomer of diaminopimelate. We could have make crystals of PH0670, PH1733, AF1422 and Archaeoglobus diaminopimelate epimerase. In addition, we have found that aspartic acid in Themoplasma acidophilum was also highly racemized.
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