(292b) A Single-Molecule Analysis Reveals Morphological Targets for Cellulase Synergy

The mechanisms of enzyme activity on solid substrates are not well understood. Unlike enzyme catalysis in aqueous solutions, enzyme activity on surfaces is complicated by adsorption steps and structural heterogeneities that make enzyme-substrate interactions difficult to characterize.  We developed a single-molecule approach for quantifying organizational differences in enzyme binding targets on morphologically complex substrates, and we used this approach to explain the cooperative action of cellulase enzymes that target different cellulose structures.  Cellulases exhibit binding specificities for many cellulose morphologies, but the role of these specificities within multi-enzyme mixtures has remained unclear.  Using a new metric to quantify binding target arrangements determined by photoactivated localization microscopy (PALM), we show that combinations of cellulase enzymes designed to bind within similar but non-identical cellulose morphologies can exhibit synergistic activity. Our results reveal a strategy for improving the activity of cellulolytic mixtures and demonstrate a versatile method for investigating protein organization on heterogeneous surfaces.