Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis

Nonlinear kinetics are commonly observed in the enzymatic hydrolysis of cel lulose. This nonlinearity could be explained by any or all of the following three factors: enzyme inactivation, product inhibition, or substrate heter ogeneity. 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 o ut the possibility of product inhibition as a cause of the observed nonline arity. The results from the other three approaches all provide strong evide nce against enzyme inactivation and strong evidence for substrate heterogen eity as the cause of the nonlinear kinetics. The most direct evidence for s ubstrate heterogeneity is that pretreatment of swollen cellulose with eithe r E2cd or E5cd gave a product that was hydrolyzed at a much (3-to 4-fold) s lower rate than untreated swollen cellulose even though the initial treatme nt degraded only 15-18% of the substrate. Furthermore, the activation energ y of E2 catalyzed hydrolysis of swollen cellulose increased from 10 kcal/mo l for the initial rate to 29 kcal/mol for hydrolysis after 24% digestion. ( C) 1999 John Wiley & Sons, Inc.