The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei (TrCel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual TrCel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of TrCel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of TrCel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of TrCel7A binding provides microscopic insight into interactions between TrCel7A and the cellulose substrate.
Keywords: Bioenergy; Biofuel; Cellulase; Cellulose; Fluorescence; Molecular Imaging; Single Molecule.