Mechanistic Studies Of Thermobifida Fusca Exocellulase Cel6B
Understanding the mechanism by which cellulases catalyze cellulose hydrolysis can greatly contribute to the development of biofuels. The thermophilic bacterium Thermobifida fusca, a major degrader of plant cell walls in certain environments, secretes seven different cellulases including exocellulase Cel6B. This cellulase acts by an inverting mechanism; however, its catalytic acid and base residues had not been identified. Biochemical approaches confirmed D274 to be the catalytic acid residue. A single catalytic base residue could not be determined, as sodium azide assays showed no activity rescue for any single mutations of candidate residues. However, a double mutation of D226A and S232A knocked out enzymatic activity and its activity was partially rescued by sodium azide. We therefore propose a novel hydrolysis mechanism for T. fusca Cel6B involving a proton-transferring network to carry out the catalytic base function. T. fusca exocellulase Cel6B was also engineered to gain knowledge on the relationship between processivity and synergism as these properties are important for hydrolyzing crystalline cellulose. Mutations of several residues in the active site tunnel of Cel6B gave higher processivity. This improvement was confirmed by two assays: the ratio of soluble/insoluble reducing sugars as well as the ratio of oligosaccharide products. Surprisingly, the mutant enzyme, which has the highest processivity, showed the least synergism in mixtures with endocellulases, suggesting that improving exocellulase processivity might not always be an effective strategy for producing improved cellulase mixtures for biomass conversion. The highly processive Cel6B mutant enzymes were successfully fluorescently labeled, so these species can be used to visualize binding and track their movement on cellulose. The catalytic domains of Cel6B was found to bind non-productively to other polysaccharides; therefore, the balance between specific binding and non-specific adsorption should be always considered when engineering cellulases for hydrolyzing complex substrates. Using immuno-precipitation, Cel6B was demonstrated to contribute greatly to the hydrolysis of crystalline cellulose by T. fusca.
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