| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2006: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Since chitin, the substrate for chitinases, is a rigid, insoluble and crystalline polysaccharide, understanding of mechanism for crystalline chitin hydrolysis is a critical issue for chitinase study. Most studies aimed at understanding mechanism for crystalline chitin hydrolysis carried out so far are focused on the role of amino acid residues in the chitinase domains. In this study, we newly focused on the local structure of the chitinase molecules in addition to the amino acid residues, to get further insight into crystalline chitin hydrolysis.
1)Importance of over hung-loop structure in crystalline hydrolysis
Chitinase A1 from Bacillus circulans WL-12 has an overhung-loop structure on the catalytic cleft and it has been suggested to be important for crystalline chitin hydrolysis. Deletion mutagenesis of this loop structure was carried out and the effect was analyzed. The mutant chitinase decreased the hydrolytic activity against crystalline chitin significantly and, thus, the loop str
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ucture was proved to be important for crystalline chitin hydrolysis.
2)The mechanism for chitin binding of ChBD specific for crytalline chitin
ChBD in B.circulans chitinase A1 is a unique chitin-binding domain, since it is specific for crystalline chitin. Site-directed mutagenesis for screening amino acid residues involved in chitin binding activity of this ChBD revealed Gln679 as a new residue important for chitin binding, in addition to W687.
3)The role of aromatic amino acid residues exposed on the surface of Serratia chitinase B
To clarify the roles of the four aromatic amino acid residues (Y240,W252,W479 and Y481) aligned to the catalytic cleft, site-directed mutagenesis to replace Y with W and W with Y was carried out. Y to W mutation increased and W to Y mutation decreased binding activity, while all mutations decreased hydrolytic activity significantly. From these results, it was concluded that proper balance between mobility of enzyme and binding activity is important for crystalline chitin hydrolysis. Less
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