Elucidating the Molecular Basis of Cellulase Synergism Through High Resolution Quantitative Fluorescence Microscopy

Abstract

Converting cellulose into fermentable sugars presents significant challenges to producing bioenergy from lignocellulose. Individual cellulases exhibit low rates and extents of hydrolysis. However, mixtures of cellulases and other cell wall degrading enzymes exhibit rates of hydrolysis that are much greater than would be predicted by summing individual rates. Thus, understanding the molecular mechanisms that give rise to synergistic behavior is essential for engineering more effective enzyme cocktails. Previous studies by the Walker Lab revealed mixtures of cellulases Cel9A, a processive endocellulase, and Cel6B, an exocellulase, exhibited higher extent of binding that would be predicted by summing the individual binding extents. A major question is whether this is driven by intrinsic cellulase binding kinetics or are changes in these two cellulases’ diffusion rates into the cellulose macrostrucutre yielding this behavior.In this study, bacterial microcrystalline cellulose fibrils were immobilized on a solid substrate using polymer lift-off. Cel9A and Cel6B were fluorescently labeled with either of two colors and purified into populations with known degree of labeling. These labeled cellulase populations were tested to validate the previous observation that labeling does not inhibit cellulose depolymerization. The binding of labeled cellulases on immobilized cellulose fibrils was observed using fluorescence microscopy for a period of 95 minutes, with images taken every minute for the first 10 minutes, every 2.5 minutes for the next 10 and every 5 minutes for the remainder. Individual binding curves were established for each enzyme in each color using different populations to characterize binding of enzymes with different numbers of labels. The effect of synergism was investigated by combining Cel9A and Cel6B, labeled in different colors, in varying molar ratios and observing effects of synergism and competition on diffusion and substrate binding in the system.