The role of cellulose in the mechanism of its hydrolysis by cellulases

Lignocellulose is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction is still unsolved. Solving the reaction mechanism of cellulose hydrolysis is crucial for overcoming reaction rate limiting factors, assessing transport limitations (e.g. heat and mass transfer) during the reaction and effective reaction engineering. Cellulases must adsorb to the surface of cellulose, but only when it complexes with the substrate (i.e. engages an isolated molecule into the active site of the catalytic domain) can the enzyme hydrolyze glycosidic bonds of cellulose. Recent mechanistic models of cellobiohydrolase hydrolysis of cellulose demonstrated that the catalytic rate of productively bound cellulases is not limiting; rather, cellulose hydrolysis rates are sensitive to the rate of productive binding (complexation) and how quickly productively bound enzymes release from cellulose. Models that focus on elementary cellulase-cellulose interactions often treat cellulose as an inert/unchanging substrate and only capture hydrolysis trends over a short period from the start of the reaction. Models that attempt to capture cellulose changes during the reaction by modeling shrinking particles of various geometric complexity or varying distributions of cellulose degrees of polymerization have also failed to predict experimental cellulose hydrolysis curves, revealing that the rate limiting property of cellulose is neither specific surface area nor polydispersity. We present our hypothesis that the true limiting property of cellulose is its productive cellulase binding capacity, and discuss a cellulose hydrolysis mechanism that incorporates time dependent evolution of the productive cellulase binding capacity of cellulose with elementary cellulase-cellulose interactions.