An improved method to directly estimate cellulase adsorption on biomass solids

Based on nitrogen factors (NF) determined for two commercial cellulases produced by the fungus Trichoderma reesei, a nitrogen analyzer was sed to directly estimate the amount of enzyme adsorbed on biomass solids, rather than relying on the difference between initial and final dissolved rotein as typically practiced. For adsorption of cellulase at 4 ◦C where reactions are minimal, the results for this method agreed closely with those ndirectly determined by the usual BCA measurements of dissolved protein. But for hydrolysis at 50 ◦C, the BCA method indicated that much less rotein was adsorbed on the solids compared to the NF method. Because the reagents used in conventional methods are very sensitive to reducing ugars, reagents, chemicals, and especially lignin components released during hydrolysis of lignocellulosic biomass, the result was a high amount f free protein in solution which indirectly affected the amount of protein adsorbed on the solid. Another important advantage of this approach ompared to conventional methods is that material balances on protein can be closed. Application of the NF method revealed that sugars and specially cellobiose strongly inhibit cellulase binding, and cellulase binding was completely shut down at cellobiose concentrations greater than 50 g/L. However, this binding inhibition can be reversed using a higher ratio of substrate concentration to inhibitors. 2008 Elsevier Inc. All rights reserved.