12-06: Single Molecule Tracking of Carbohydrate-Binding Modules Bound to Cellulose Crystals

To develop more cost-effective approaches to liberate fermentable sugars from recalcitrant biomass, the enzyme cocktail used for saccharification must be improved. We have developed a single-molecule technique based on fluorescence imaging to track the motion of cellulase components with spatial resolution at several nanometers.  We used single molecule spectroscopy to study the behavior of carbohydrate-binding modules (CBMs) labeled with quantum dots (QDs) while bound to cellulose crystals.  These bio-assembles were subjected to total internal reflection fluorescence (TIRF) microscopy.  The concentrations of the CBMs and QDs were optimized to achieve single molecule resolution. This technique revealed a confined nanometer-scale movement of the CBMs bound to cellulose. Although the mechanism of CBM motion is still unknown, the single molecule approach used here offers new opportunities to guide us toward a fundamental understanding of cellulase function, especially the mechanism of the “processivity” of exoglucanse.