Enzymatic hydrolysis of biomass undergoes a significant decrease in rate, which is often attributed to activity loss of enzyme during the incubation. Activity loss due to both interaction with substrate (for example inactivation of adsorbed enzyme) and all combined environmental mechanisms in a substrate free buffer solution were compared in this study. Enzyme-substrate interactions contributed more towards the overall activity loss than did the combined environmental sources as evidenced from three independent metrics. (1) Relative extents of inactivation were higher for enzyme-substrate interactions than for environmental mechanisms. (2) Apparent half-lives (1.37-11.01h) following interaction with substrate were relatively small compared to environmental inactivation, which was 21.5h. (3) The inactivation rate constant for enzyme-substrate interactions (0.56h<SUP>-1</SUP>) was 46 times higher than that of environmental inactivation (0.0123h<SUP>-1</SUP>). These results suggest enzyme-substrate interaction is the main cause of cellulase activity loss and contributes significantly to the slow rate of hydrolysis.