A Molecular View of Cellulase-Catalyzed Hydrolysis of Cellulose: A Single Molecule Approach

The efficient conversion of biomass to biofuels such as ethanol 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 by fermentation. The hydrolysis is complex - it entails the cooperative (synergistic) action of multiple enzymes known collectively as cellulases: endoglucanase, exoglucanase and beta-glucosidase on both insoluble and soluble substrates. In contrast to homogeneous solution-phase catalysis, the overall efficiency of this heterogeneous catalysis process depends on factors in addition to the catalytic rates of the cellulases, including: cellulase absorption, desorption and diffusion rates on the insoluble cellulose substrate, endoglucanase-catalyzed conversion of cellulose from its crystalline to amorphous forms, and the processivity of exoglucanase-catalyzed hydrolysis of individual cellulose strands. To date, due in large part to limitations of the analysis methods used for its study, this heterogeneous reaction is poorly understood. Here we use single-molecule imaging to directly elucidate molecular-level details of cellulase activity that cannot be readily inferred from ensemble averages reported by conventional, bulk analyses.In our research, the activities of exo- and endoglucanases on cellulose microfibrils are investigated at the single-molecule level. Total internal reflection fluorescence microscopy (TIRFM) is used to monitor the activity of individual fluorescently-labeled cellulases interacting with insoluble cellulose substrates. Using wide field imaging, we are able to simultaneously record the motion of multiple, individual cellulases with nanometer spatial resolution. Time-resolved localization microscopy provides insights on: (i) binding and diffusion of enzyme to ‘active’ sites on cellulose; (ii) lifetime of enzyme activity for hydrolysis; (iii) enzyme processivity; and (iv) the nature of synergy between cellulase enzymes.