| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1995: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the chitosanase gene from Streptomyces sp.N174, and purified by S-Sepharose and Bio-Gel A column chromatography. The chitosanase thus obtained produced alpha-anomer from beta-1,4-linked oligosaccharide substrate, indicating that the enzyme is an inverter. When glucosamine hexasaccharide was used as the substrate, the product distribution was trimer>>dimer>tetramer. Time-courses of oligosaccharide hydrolysis showed a decrease in rate of substrate degradation from hexamer to pentamer to tetramer. It is most likely that the substrate binding cleft of the chitosanase can accommodate at least six glucosamine residues, and that the cleavage point is located at the midpoint of the binding cleft.
For identification of the amino acid residues essential for catalysis, site-directed mutagenesis of the chitosanase was carried out, and the activity and stability were determined for each of the mutant chitosanases. Substitutions of Glu22 or Asp40 reduced drastically specific activity and kappacat, while KAPPAmwas only slightly changed. These results suggested that Glu22 and Asp40 are directly involved in the catalytic center of the chitosanase. The mutation of Asp37 considerably reduced the enzymatic activity, and significantly decreased the midpoint temperature of thermal unfolding transition (Tm). In the crystal structure, the carboxyl side chain of Asp37 points away from the binding cleft, but makes a close interaction with the imidazole nitrogen of His90. It is likely that Asp37 stabilize the structure of substrate binding cleft by interacting with His90 and its mutation destabilize the structure resulting in the lower affinity to the substrate.
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