Protein Engineering, Vol. 16, No. 1, 19-25,
January 2003
© 2003 Oxford University Press
Protein engineering to improve the thermostability of glucoamylase from Aspergillus awamori based on molecular dynamics simulations
Department of Chemical Engineering, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao E. Road, Taipei 106, Taiwan
* To whom correspondence should be addressed. E-mail: f10894{at}ntut.edu.tw
Twelve mutations were constructed to improve the thermostability of glucoamylase from Aspergillus awamori based on the results of molecular dynamics simulations. The thermal unfolding of the catalytic domain followed a putative hierarchical behavior. In addition, the unfolding of the 13 
-helices obeyed the random ordered mechanism, in which the -helices 8, 1 and 11 unfolded more rapidly than the others. The catalytic center was well protected by the (/)6-barrel at simulation temperatures up to 600 K, whereas the catalytic base, E400, migrated from its original interior pocket to the surface of the catalytic domain by surmounting the hydrophobic barrier provided by -helices 12 and 13 at 800 K. The disulfide bonds engineered to ‘lock’ the -helix 11 on the surface of the catalytic domain dramatically increased the thermostability. Substituting G396 and G407 with Ala residues slightly increased the thermostability, whereas their specific activity and catalytic efficiency were reduced. This indicates that the introduced residues with higher hydrophobicity were favorable in the loop between -helices 12 and 13, whereas they partially destroyed the hydrogen bond and salt linkage network in the catalytic center. -Helices 12 and 13 can be stabilized by introducing residues with higher hydrophobicity, except for the H391M mutation.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Purmonen, J. Valjakka, K. Takkinen, T. Laitinen, and J. Rouvinen Molecular dynamics studies on the thermostability of family 11 xylanases Protein Eng. Des. Sel., November 1, 2007; 20(11): 551 - 559. [Abstract] [Full Text] [PDF]
|
|