Abstract
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.
Highlights
Low cost and readily available lignocellulosic materials, such as wheat straw, switchgrass, or poplar, are composed of cellulose and other polysaccharides that can be converted to ethanol
Analysis of enzyme activity and protein concentration Cellulase activity was measured following the NREL filter paper assay (Adney & Baker, 2008) and reported in filter-paper units (FPU) per milliliter of solution. β-glucosidase activity was measured using the method described by Wood & Bhat (1988) and reported in cellobiase units (CBU)
Stability of commercial enzyme preparations The enzyme system commonly used for enzymatic hydrolysis is a mixture of cellulases and β-glucosidases
Summary
Low cost and readily available lignocellulosic materials, such as wheat straw, switchgrass, or poplar, are composed of cellulose and other polysaccharides that can be converted to ethanol. As lignocellulose contains non-hydrolysable lignin to which cellulases adsorb irreversibly, some cellulases are lost during hydrolysis (Farinas et al, 2010; Jørgensen, Kristensen & Felby, 2007; Zhou et al, 2009). These factors necessitate a high enzyme load, which increases hydrolysis costs; one of the key economic factors and impediments to lignocellulosic ethanol production and commercialization is the amount and cost of enzyme needed for enzymatic hydrolysis (Lynd et al, 2008; Qi et al, 2011). The recycling of cellulases bound to the residual substrate as well as enzymes in the reaction suspension is relatively new technology that has been gaining popularity because of its potential to decrease enzyme requirements during ethanol production (Qi et al, 2011)
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