| Although there have been several attempts at improving cellulase stability, the thermodynamics of unfolding of these enzymes remains largely undetermined. Furthermore, the fundamental conditions for synergistic enhancement of enzymatic cellulose degradation are also poorly understood. Here, I aim at determining some of the underlying mechanisms for cellulase stability and I also sought to improve cellulase stability and study the effects on synergistic enhancement in activity, of a specific registry of scaffolded enzymes. In chapter two, as a case study, I start by inserting myoglobin globular domain into ankyrin repeats scaffolds. I determine the best insertion sites and find that both scaffold and inserted myoglobin remain folded and stable. In chapter three, I use the consensus ankyrin repeats array, as means of stabilizing and improving the activity of singly inserted cellulases, as seen by both thermal denaturations and cellulolytic assays. In chapter four, I determine the conditions for reversible unfolding and refolding of CA catalytic domain and the scaffolded CA. I found that the consensus ankyrin scaffold stabilized CA catalytic domain at low denaturant concentrations and that these constructs unfold through a series of equilibrium intermediates. In chapter five, I use the consensus ankyrin domain to tether catalytic domains in a linear array, bringing the cellulases in close proximity, and aiming at determining the role of spacing, order and enzyme identity in synergism enhancement, for enzymes with distinct and complementary reactivities. To facilitate these studies, I have improved upon the traditional assay for cellulase activity (the DNS assay), adapting it to a high-throughput format using a 96-well plate reader. |
