title: Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis.
abstract: 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 degrees 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.
authors.affiliation: Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University, Seoul.
authors.forename: H G
authors.initials: HG
authors.lastname: Yoon
authors.forename: H Y
authors.initials: HY
authors.lastname: Kim
authors.forename: Y H
authors.initials: YH
authors.lastname: Lim
authors.forename: H K
authors.initials: HK
authors.lastname: Kim
authors.forename: D H
authors.initials: DH
authors.lastname: Shin
authors.forename: B S
authors.initials: BS
authors.lastname: Hong
authors.forename: H Y
authors.initials: HY
authors.lastname: Cho
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D006026:Glycoside Hydrolases; C019868:chitosanase