| Project/Area Number |
07660094
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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 | CHIBA UNIVERSITY |
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Principal Investigator |
ANDO Akikazu FACULTY OF HORTICULTURE,CHIBA UNIVERSITY,ASSOCIATE PROF., 園芸学部, 助教授 (80125898)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1996: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1995: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | CHITOSANASE / CRYSTALLIZATION / X-RAY CRYSTALLOGRAPHY / SITE-DIRECTED MUTAGENESIS / CATALYTIC AMINO ACIDS |
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| Research Abstract |
1 Structure of chitosanase from Bacillus circulans MH-K1.
Chitosanase was crystallized by a vapor-diffusion procedure using ammonium sulfate as a precipitate. Rod-shaped crystals belong to the orthorhombic system and the space group P2_12_12 with unit-cell dimensions of a=43.3, b=57.7, c=128.0 A.MAD Diffraction pattern of the crystals containing Pt was analyzed. Electron density map of 3A resolution was obtained. Most of alpha-helices were localized. Localization of beta-sheets are on the way. Refinement of the map using the data of native crystals should be done.
2 Site-directed mutagenesits.
Amino acids of MH-K1 chitosanase, which were conserved and showed similarity to other chitosanases and lysozymes, were selected and changed by site-directed mutagenesis. The chitosanase activity of mutants were measured. Mutants of Glu on 37 or Asp on 55 were lost its activity. It indicated that these amino acids were catalytically importnat. Asp on 52 also suggested to participate in catalytic reaction.
3 Cloning of other chitosanase.
Part of chitosanase gene from Nocardioides sp K-01 was cloned. Primary structure of the chitosanase was determined using protein-sequencing data and the clone's sequence.
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