Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus.

Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each of these residues, we identified several mutations that reduced cellobiose inhibition of E1, including W212A, W213A, Q247A, W249A and F250A. One of the mutations (W212A) resulted in a 47-fold decrease in binding affinity of cellobiose as well as a 5-fold increase in the kcat. The mutation further increased E1 activity on Avicel and dilute-acid treated corn stover and enhanced its productivity at high-substrate loadings. These findings were corroborated by funnel metadynamics, which showed that the W212A substitution led to reduced affinity for cellobiose in the +1 and +2 binding sites due to rearrangement of key cellobiose-binding residues.

Hydrolysis of model cellulose films by cellulosomes: Extension of quartz crystal microbalance technique to multienzymatic complexes.

Bacterial cellulosomes contain highly efficient complexed cellulases and have been studied extensively for the production of lignocellulosic biofuels and bioproducts. A surface measurement technique, quartz crystal microbalance with dissipation (QCM-D), was extended for the investigation of real-time binding and hydrolysis of model cellulose surfaces from free fungal cellulases to the cellulosomes of Clostridium thermocellum (Ruminiclostridium thermocellum). In differentiating the activities of cell-free and cell-bound cellulosomes, greater than 68% of the cellulosomes in the crude cell broth were found to exist unattached to the cell across multiple growth stages. The initial hydrolysis rate of crude cell broth measured by QCM was greater than that of cell-free cellulosomes, but the corresponding frequency drop (a direct measure of the mass of enzyme adsorbed to the film) of crude cell broth was less than that of the cell-free cellulosomes, consistent with the underestimation of the cell mass adsorbed using QCM. Inhibition of hydrolysis by cellobiose (0-10g/L), which is similar for crude cell broth and cell-free cellulosomes, demonstrates the sensitivity of the QCM to environmental perturbations of multienzymatic complexes. QCM measurements using multienzymatic complexes may be used to screen and optimize hydrolysis conditions and to develop mechanistic, surface-based models of enzymatic cellulose deconstruction.

Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei.

Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in "biomass to biofuels" process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellobiose dehydrogenase (CDH) and cellulase both in vitro and in vivo. The CDH from Phanerochaete chrysosporium was heterologously expressed in Pichia pastoris. Supplementation of the purified CDH in Trichoderma cellulase increased the cellulase activities. Especially ß-glucosidase activity was increased by 30-100% varying at different time points. On the other hand, the cdh gene was heterologously expressed in Trichoderma reesei to explore the synergism between CDH and cellulases in vivo. The analyses of gene expression and enzymatic profiles of filter paper activity, carboxymethylcellulase (CMCase) and ß-glucosidase show the increased cellulase activity and the enhanced cellulase production in the cdh-expressing strains. The results elucidate a possible mechanism for diminishing the cellobiose inhibition of cellulase by CDH. These findings provide a novel perspective to make more economic enzyme cocktails for commercial application or explore alternative strategies for generating cellulase-producing strains with higher efficiency.