Recycling of enzymes during biomass conversion is one potential strategy to reduce the cost of the hydrolysis step of cellulosic ethanol production. Devising an efficient enzyme recycling strategy requires a good understanding of how the enzymes adsorb, distribute, and interact with the substrate during hydrolysis. We investigated the interaction of individual Trichoderma reesei enzymes present in a commercial cellulase mixture during the hydrolysis of steam-pretreated corn stover (SPCS). The enzyme profiles were followed using zymograms. gel electrophoresis. enzyme activity assays and mass spectrometry. The adsorption and activity profiles of 6 specific enzymes Cel7A (CBH I), Cel7B (EG I), Cel5A (EG II), Xyn 10 (endo-1,4-beta-xylanase III), Xyn 11 (endo-xylanase II), and beta-glucosidase were characterized. Initially, each of the enzymes rapidly adsorbed onto the SPCS. However, this was followed by partial desorption to an adsorption equilibrium where the Cel7A, Cel7B, Xyn 10, and beta-glucosidase were partially adsorbed to the SPCS and also found free in solution throughout the course of hydrolysis. In contrast, the Cel5A and Xyn 11 components remained primarily free in the supernatant. The Cel7A component also exhibited a partial desorption when the rate of hydrolysis leveled off as evidenced by MUC zymogram and SDS-PAGE. Those cellulase components that did not bind to the substrate were generally less stable and lost their activities within the first 24 h when compared to enzymes that were distributed in both the liquid and solid phases. Therefore, to ensure maximum enzyme activity recovery, enzyme recycling seems to be most effective when short-term rounds of hydrolysis are combined with the recovery of enzymes from both the liquid and the solid phases and potentially enzyme supplementation to replenish lost activity. (C) 2011 Elsevier Inc. All rights reserved.