Evaluation of Cellulose Reactivity with CBH1 and/or EG2 During Hydrolysis

In this study, we applied a restart protocol to explore how cellulose reactivity changed with conversion, the interaction major cellulase components from wild type Trichoderma reesei with cellulose and synergism over the course of enzymatic hydrolysis. For the first time, our results revealed dynamic profiles of adsorption, sugar release, and oligosaccharide yields over time for interrupted enzymatic hydrolysis of Avicel. Although interruption of Avicel hydrolysis over 0-15 h showed negligible changes in crystalinity and reducing end content as cellulose hydrolysis proceeded, CBH1 and EG2 adsorption capacities decreased rapidly and then gradually when these enzymes were applied individually. The synergistic adsorption capacities of both enzymes were much lower when applied together than when they were applied alone while the hydrolysis rate was much higher when the two enzymes worked together. As hydrolysis proceeded, cellulose provided more binding sites for EG2 than CBH1. Initial (1 hour) sugar release patterns indicated a sharp drop in rate during later stages of interrupted Avicel hydrolysis, consistent with absorption capacities, likely due to “depleting” of binding sites. However, the 24 hr sugar release pattern (equilibrium) suggested that DP>3 oligosaccharides may further contribute to the drop in hydrolysis rates, especially at lower enzyme loadings or with unbalanced enzyme mixtures. A new model of enzymatic hydrolysis of cellulose that indicates cellulose reactivity, oligomer distribution, effective enzyme binding capacity, and equilibration of depolymerization limiting factors was developed as well.