Journal of Microbiology and Biotechnology

  • 제17권8호
  • /
  • Pages.1249-1253
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  • 2007
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Analysis of Active Center in Hyperthermophilic Cellulase from Pyrococcus horikoshii

  • Kang, Hee-Jin (National Institute of Advanced Industrial Science and Technology (AIST, Kansai)) ;
  • Ishikawa, Kazuhiko (National Institute of Advanced Industrial Science and Technology (AIST, Kansai))
  • 발행 : 2007.08.30
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초록

A hyperthermostable endoglucanase from Pyrococcus horikoshii with the capability of hydrolyzing crystalline cellulose was analyzed. A protein engineering study was carried out to obtain a reduced-size mutant. Five amino acid residues at both the N- and C-terminus were found to be removable without any loss of activity or thermal stability. Site-directed mutagenesis was also performed on R102, N200, E201, H297, Y299, E342, and W377, residues possibly involved in the active center or in the recognition and binding of a cellulose substrate. The activity of the resulting mutants was considerably decreased, confirming that the mutated residues were all important for activity. A reduced-size enzyme, as active as the wild-type endoglucanase, was successfully obtained, plus the residues critical for its activity and specificity were confirmed. Consequently, an engineered enzyme with a reduced size was obtained, and the amino acids essential for activity were confirmed by site-directed mutagenesis and comparison with a known three-dimensional structure.

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참고문헌

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