Molecular Level Investigations and Mathematical Modeling of Cellulase-catalyzed Hydrolysis of Cellulose
- Author(s): Fox, Jerome M.
- Advisor(s): Blanch, Harvey W
- Clark, Douglas S
- et al.
Cellulose-to-glucose conversion costs comprise one of the largest expenses in the production of lignocellulosic biofuels, a renewable alternative to traditional fossil fuels. Efforts to reduce these costs by improving the activity of cellulase enzymes, which act within multi-enzyme mixtures to catalyze the hydrolysis of cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In this work, we employed mechanistic models of enzymatic action alongside experimental studies of enzyme-enzyme synergy to investigate kinetic impediments encountered by cellulase enzymes as they catalyze the hydrolysis of cellulose. Using several mechanistic models of enzymatic hydrolysis, we show how hydrolysis kinetics and optimal cellulolytic mixture compositions are dependent on the nature of the cellulosic substrate (i.e., particle shape, surface area, degree of polymerization) and the conditions under which it is depolymerized (i.e., hydrolysis and fermentation process conditions). By developing a method to estimate catalysis-specific products within multi-enzyme reactions, we show that cellobiohydrolase enzymes, which catalyze processive hydrolysis from cellulose chain ends, encounter rate limitations that result, not from intrinsic kinetics, but from slow rates of chain complexation and from morphological obstacles to processivity. And using photoactivated localization microscopy (PALM) to produce pointillistic maps of carbohydrate-binding modules (CBMs) bound to cotton, we develop an order parameter to quantify the different spatial arrangements of adsorbed CBMs and use that order parameter to explain synergy between cellulase enzymes designed to target different surface structures. The results of this work reveal strategies for using morphological targeting to enhance enzyme-enzyme cooperativity within cellulolytic mixtures, thereby improving their overall activity and lowering the cost of cellulose hydrolysis.