Seminar

The consensus ankyrin domain as a scaffold for protein engineering: applications in cellulose degradation

Eva Cunha, PhD

Degradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remains a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyper-stable -helical consensus ankyrin domain scaffolds. 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. We aimed at determining some of the underlying mechanisms for cellulase stability and we also sought to improve cellulase stability and study the effects on synergistic enhancement in activity, of a specific registry of scaffolded enzymes. We used the consensus ankyrin repeats array, as means of stabilizing and improving the activity of singly inserted cellulases, determined by both thermal denaturations and cellulolytic assays. Furthermore, we determined the conditions for reversible unfolding and refolding of Clostridium thermocellum CelA (CA) catalytic domain and the scaffolded CA. We 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. Finally, we used 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. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.