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Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis

Authors
 H.-G. Yoon  ;  H.-Y. Kim  ;  Y.-H. Lim  ;  H.-K. Kim  ;  D.-H. Shin  ;  B.-S. Hong  ;  H.-Y. Cho 
Citation
 APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, Vol.56(1~2) : 173-180, 2001 
Journal Title
 APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 
ISSN
 0175-7598 
Issue Date
2001
MeSH
Amino Acid Sequence ; Catalysis ; Circular Dichroism ; Enzyme Stability ; Glycoside Hydrolases/chemistry* ; Glycoside Hydrolases/metabolism ; Hot Temperature ; Kinetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Protein Conformation
Keywords
Enzyme ; Catalytic Activity ; Bacillus ; Amino Acid Residue ; Residual Activity
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 V max and k cat 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.
Full Text
http://link.springer.com/article/10.1007/s002530000571
DOI
10.1007/s002530000571
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Biochemistry and Molecular Biology (생화학-분자생물학교실) > 1. Journal Papers
Yonsei Authors
Yoon, Ho Geun(윤호근) ORCID logo https://orcid.org/0000-0003-2718-3372
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/141961
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