Integration of Computer Modeling and Initial Studies of Site-Directed Mutagenesis to Improve Cellulase Activity on Cel9A from Thermobifida fusca

摘要

Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material.Unfortunately,cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases,which has led to a strong interest in improving their activities.Thermobifida fusca secretes six cellulose degrading enzymes:two exo-and three endocellulases and an endo/exocellulase Cel9A (formerly called E4).Cel9A shows unique properties because of its endo-and exocellulase characteristics,strong activity on crystalline cellulose,and good synergistic properties.Therefore,it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation.In this article,we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling.A computer model of Cel9A was created using the program CH ARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model.Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD).The interaction between this cellulose chain and conserved CBD residues was determined in the model,and mutations likely to improve the binding properties of the CBD were selected.Site-directed mutations were carried out using the pET vector pET26b,Escherichia coll DH5-alpha,and the QuickChange mutagenesis method.