Investigating the enzymatic hydrolysis of crystalline cellulose using fluorescence based assays---Implications of synergism, binding and product inhibition

The hydrolysis kinetics of bacterial microcrystalline cellulose (BMCC) by the cellulases of Thermobifida fusca was studied using fluorescence based assays in three ways. First, the binding of fluorescence-labeled Cel5A, Cel6B and Cel9A in ternary synergistic mixtures was assessed. A rapid high-throughput binding assay using microwell plates was developed to measure the bound fractions of the three cellulases at varying mole ratios of Cel6B and Cel9A, with Cel5A fixed at 10% of the total cellulase loading. This study revealed that the bound fractions of cellulases in ternary mixtures were additive, unlike the hydrolytic activity which was synergistic. Second, an experimental system was developed for the application of high resolution fluorescence microscopy to examine the binding of individual Cel5A, Cel6B and Cel9A to immobilized cellulose with varying morphologies. The immobilization technique allowed deposition of cellulose morphologies ranging from nanoscale cellulose fibers, to microscale cellulose fibril mats to sub-millimeter scale cellulose particles. Fluorescently labeled Cel5A, Cel6B and Cel9A were successfully integrated with fluorescently labeled cellulose to obtain a miniaturized reaction system which retained the intrinsic binding and hydrolytic capabilities of cellulases. Direct visualization of the binding behavior of individual cellulases on cellulose with different morphologies was achieved using this system which showed that the binding behavior depended strongly on the morphology and complexity of cellulose aggregates. Third, the significance of product inhibition by cellobiose as a rate-retarding factor in the hydrolysis of BMCC by Cel9A and Cel9A-68, its construct lacking the family 2 cellulose binding module, was investigated. Fluorescently labeled BMCC was used as the substrate for an analysis of initial rates in the presence of exogenous cellobiose. Increasing cellobiose concentrations ranging from 1--5mM were found to decrease the initial rate by 10--30% but increasing cellobiose concentrations from 5 to 60 mM did not cause a further decline in initial rates, clearly ruling out classical competitive inhibition as a possible mechanism. No definitive correlation was observed between binding and cellobiose concentrations for both enzymes indicating that the presence of cellobiose does not lead to significant enhancement or inhibition of binding.