The cost of enzymes makes enzymatic hydrolysis one of the most expensive steps in the production of lignocellulosic ethanol. Diverse studies have used commercial enzyme cocktails assuming that change in total protein concentration during hydrolysis was solely due to adsorption of endo- and exoglucanases onto the substrate. Given the sensitivity of enzymes and proteins to media conditions this assumption was tested by evaluating and modeling the protein concentration of commercial cocktails at hydrolysis conditions. In the absence of solid substrate, the total protein concentration of a mixture of Celluclast 1.5 L and Novozyme 188 decreased by as much as 45% at 50 °C after 4 days. The individual cocktails as well as a mixture of both were stable at 20 °C. At 50 °C, the protein concentration of Celluclast 1.5 was relatively constant but Novozyme 188 decreased by as much as 77%. It was hypothesized that Novozyme 188 proteins suffer a structural change at 50 °C which leads to protein aggregation and precipitation. Lyophilized β-glucosidase (P-β-glucosidase) at 50 °C exhibited an aggregation rate which was successfully modeled using first order kinetics (R2 = 0.97). By incorporating the possible presence of chaperone proteins in Novozyme 188, the protein aggregation observed for this cocktail was successfully modeled (R2 = 0.96). To accurately model the increasing protein stability observed at high cocktail loadings, the model was modified to include the presence of additives in the cocktail (R2 = 0.98). By combining the measurement of total protein concentration with the proposed Novozyme 188 protein aggregation model, the endo- and exoglucanases concentration in the solid and liquid phases during hydrolysis can be more accurately determined. This methodology can be applied to various systems leading to optimization of enzyme loading by minimizing the excess of endo- and exoglucanases. In addition, the monitoring of endo- and exoglucanases concentrations can be used to build mass balances of enzyme recycling processes and to techno-economically evaluate the viability of enzyme recycling.
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