Analysis of active center in hyperthermophilic cellulase from Pyrococcus horikoshii

J Microbiol Biotechnol. 2007 Aug;17(8):1249-53.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cellulases / chemistry
  • Cellulases / genetics*
  • Cellulases / metabolism*
  • Cellulose / metabolism
  • Enzyme Stability
  • Mutagenesis, Site-Directed
  • Pyrococcus horikoshii / enzymology*
  • Pyrococcus horikoshii / genetics
  • Sequence Deletion


  • Cellulose
  • Cellulases