So far, there is a lot controversy concerning the binding reversibility of cellulases to substrate.  While some researchers have noticed exchange of cellulases on the substrate surface in their experiments, which indicates that the binding is somehow reversible, others insist the binding of cellulases to substrate is irreversible from evidence in their binding isotherms. Due to the contradiction among these experiments, it is necessary to investigate whether the binding that occurs during the enzymatic hydrolysis process is reversible or irreversible
According to the experiments here, the enzyme’s reactivity has dropped during the enzymatic hydrolysis of biomass. To investigate if the reasons for the reactivity drop are related to reversible or irreversible binding, cellulases are first incubated with different substrates. It is observed that substrates with higher crystalline content would cause lower reactivity of enzymes. It is thus suspected the reactivity drop mainly comes from the irreversible binding of exoglucanases, which are very effective in digesting of crystalline cellulose. When enzyme pre-incubates in substrate-free buffer solution, less reactivity drop is noticed compared to enzyme that pre-incubates with substrate. By this, it is inferred that the reactivity drop is likely caused by inactivation of intact enzyme due to its interactions with the substrate. It is concluded here some exoglucanases are irreversibly bound to substrate.