The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66→Asn and Asp-66→Glu mutation remarkably decreased kinetic parameters such as Vmax and kcat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49→Ser/Tyr and Cys-72→Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211→Ser/Tyr mutant enzyme was less than 10 min at 80°C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211→Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.
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