| Project/Area Number |
09660082
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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 |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
INOUYE Kuniyo Grad, Sch.Agric Kyoto Univ.Professor, 農学研究科, 教授 (10223249)
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| Co-Investigator(Kenkyū-buntansha) |
TAKITA Teisuke Grad, Sch.Agric Kyoto Univ.Instructor, 農学研究科, 助手 (70263126)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Halophilic Enzyme / Thermolysin / Metalloproteinase / chemical modification / Enzyme Activity / Proteinase / Salt / Protein Engineering / 金属プロテアーゼ / アスパルテーム / プロテアーゼ / 蛋白質分解酵素 / 好塩性 / 酵素構造 / 金属酵素 |
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| Research Abstract |
Thermolysin (TLN) is a thermophilic neutral metalloprpteinase, and we have reported that TLN is highly halophilic and its activity enhances 30-60 times in the presence of saturated concentration of salts in comparison with that in the absence.
In this study, the halophilicity was examined from the point of view of the enzyme structure and medium properties.
1. The salt activation was shown to be dependent considerably on pH and temperature and the addition of organic solvent, suggesting that the activation might be related with electrostatic interaction on the surface of TLN.
2. Temperature stability of TLN was enhanced 2-3 times by the addition of 0.5-1.5 M NaCl. The dependence on the salt concentration was different from that of the activation, suggesting that the stability and activity can be increased by indepenent mechanisms.
3. The solubility of TLN is as low as 1 mg/ml in the common buffer. It was found that it increased remarkably by the addition of salts up to 70 mg/ml. TLN was revealed to be cold-soluble.
4. Trp 115 is at the S2 suibsite. By the site-directed mutagenesis, an aromatic amino acid was shown to be needed strictly at this position for the activity.
5. Nitration and amination of tyrosyl reasidues of TLN showed that introduction of negative charges on the surface of TLN decreased the degree of the salt activation.
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