| Project/Area Number |
14560059
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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 | NIIGATA UNIVERSITY |
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Principal Investigator |
WATANABE Takeshi NIIGATA UNIVERSITY, Faculty of Agriculture, Professor, 農学部, 教授 (10201203)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | Chitinase / Bacillus circula / Serratia marcescens / Chitin binding domain / Crystalline chitin hydrolysis / Aromatic amino acid residue |
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| Research Abstract |
An ability to hydrolyze insoluble and crystalline chitin is the most intrinsic and interesting feature of chitinases, In this study, the mechanisms for crystalline chitin hydrolysis was studied on the basis of their 3D-structures by using chitinase A1(ChiA1) from Bacillus circulans WL-12 and chitinase A(ChiA) and chitinase B(ChiB) from Serratia marcescens 2170.
1)Roles of the aromatic amino acid residues within the catalytic cleft of B.circulans ChiA1. ChiA1 comprises a catalytic domain, two FnIII domains and a chitin-binding domain. Two aromatic amino acid residues were shown to be essential determinants only for crystalline chitin hydrolysis.
2)Solution structure of B.circulans ChiA1l. 3D-structure of each domain constructing ChiA1 has been determined previously but whole structure of this chitinases is unknown. By X-ray scattering, we succeeded to estimate solution structure of entire ChiA1 molecule.
3)Binding mechanism of the chitin-binding domain(ChBD) of B.circulans ChiA1. Trp687 in ChBD was revealed to be most important for chitin binding. In addition, possibility of change in loop structure along with binding was strongly suggested.
4)Roles of exposed aromatic amino acid residues of S.marcescens ChiB. Two Tyr and two Trp linearly aligned toward the catalytic cleft were shown to be essential for binding to and hydrolysis of crystalline β-chitin. Contribution of Trp residues to binding activity was larger than that of Tyr residues.
5)Crystalline chitin hydrolysis by ChiA and ChiB from S.marcescens. Hydrolysis of chitin chain in opposite direction has been suggested by 3D-structural analysis of ChiA and ChiB. This was experimentally proved by reducing end labeling technique and tilt diffraction of crystalline β-chitin microfibrils.
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