| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1992: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1991: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Productions, purifications and gene manipulations of bacterial chitinases are generally easier than those of chitinases from other organisms. Therefore, bacterial chitinases are suitable for studying basic structures and properties of the enzymes which enable efficient hydrolysis of an insoluble and stable substrate, chitin. Bacillus circulans WL-12 is one of the bacteria which excrete chitinases into culture medium. Chitinase system of the bacterium include at least six distinct chitinase molecules, chitinase A1, A2, B1, B2, C and D. Amino acid sequence features and the studies using various deletion derivatives of chitinase A1 revealed that chitinase A1 and D are both consisted of chitin binding domains, fibronectin type III-like domains and catalytic domains. The chitin binding domain is necessary for the efficient and complete hydrolysis of insoluble chitin substrate. Fibronectin type III-like domains participate efficient hydrolysis of chitin by the catalytic domain of chitinase A
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1 bound to chitin. N-terminal large domain contains catalytic site of this enzyme. By comparing catalytic domains of chitinase A1 and D, we found the regions sharing a weak amino acid sequence similarity. Corresponding regions were also found in other bacterial chitinases, class III plant chitinases, endo-beta-N-acetylglucosaminidases and a yeast killer toxin. The regions are suggested to be important for catalytic activities of these enzymes. In order to verify this assumption, Ser-160, Asp-200 and Glu-204 in chitinase A1 were chosen based on the amino acid sequence alignment of the regions and replaced by site directed mutagenesis. Kinetic studies of the mutant chitinases revealed direct involvement of Glu-204 and Asp-200 residues in the catalytic events. Since Asp-200 and Glu-204 were chosen based on the sequence alignments, it is highly probable that Asp and Glu residues corresponding to Glu-204 and Asp-200 of chitinase A1 are also involved in catalytic mechanisms of other chitinases and related enzymes. Less
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