Binding Kinetics and Fraction of Immobile Enzymes Bound to Cellulose Fibrils Studied Through Confocal Laser Scanning Fluorescence Microscopy and FRAP

Biofuels and bioproducts derived from cellulosic biomass represent great potential renewable and environmentally friendly technologies. Key to converting cellulosic biomass into soluble sugars is the depolymerization of the cellulose macromolecules by enzymes called cellulases. These enzymes depolymerize the cellulose chain by binding to the exposed cellulose surface and cleaving β-glucosidic bonds. Although much work has been done studying the dynamics of these enzymes in bulk solution, little is known about how these enzymes operate at the micron to nanoscale. To this end, our lab has fluorescently labeled three of these enzymes (Thermobifida fusca Cel9A, Cel5A and Cel6B) to study their binding and catalytic behavior through a variety of spectroscopic techniques. The work presented aims at quantifying the binding and unbinding kinetics, and the fraction of immobile enzyme bound to the cellulose substrate through scanning confocal microscopy and FRAP (fluorescence recovery after photobleaching). Sonicated BMCC (bacterial microcrystalline cellulose) was patterned on glass surfaces through "molecular combing" to produce oriented cellulose bundles and mats. The patterned cellulose was incubated with fluorescent cellulases at saturating conditions (2nM) for approximately three hours. Cellulose aggregates were imaged with a confocal laser scanning microscope. FRAP experiments were performed on both mats and fibril bundles at various temperatures to elucidate the kinetics of binding/unbinding, and to estimate the immobile fraction of cellulases on the cellulose surface. Results from this study showed that the binding/unbinding kinetics and the immobile fraction for each enzyme differ according to the cellulase mode of hydrolysis (random versus processive) and varied significantly with temperature. This study helps to further the understanding of the molecular basis of cellulose hydrolysis and could potentially aid in the development of more efficient enzymes suitable for industrial applications.