Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis

Nonlinear kinetics are commonly observed in the enzymatic hydrolysis of cellulose. This nonlinearity could be explained by any or all of the following three factors: enzyme inactivation, product inhibition, or substrate heterogeneity. In this study, four different approaches were applied to test the above hypotheses using two Thermomonospora fusca endocellulases, E2 and E5. The lack of stimulation of cellulase activity by beta-glucosidase rules out the possibility of product inhibition as a cause of the observed nonlinearity. The results from the other three approaches all provide strong evidence against enzyme inactivation and strong evidence for substrate heterogeneity as the cause of the nonlinear kinetics. The most direct evidence for substrate heterogeneity is that pretreatment of swollen cellulose with either E2cd or E5cd gave a product that was hydrolyzed at a much (3- to 4-fold) slower rate than untreated swollen cellulose even though the initial treatment degraded only 15-18% of the substrate. Furthermore, the activation energy of E2 catalyzed hydrolysis of swollen cellulose increased from 10 kcal/mol for the initial rate to 29 kcal/mol for hydrolysis after 24% digestion.