Biomass Depolymerization projects
Surface Kinetic Mechanisms of Enzymatic Cellulose Deconstruction - Completed
Assessment of cellulose deconstruction kinetics is a critical step in the design of improved cellulases for efficient production of liquid fuels from biomass. The project elucidated molecular interactions between cellulases and cellulose at the aqueous/solid interface using surface-sensitive techniques to understand, quantitatively and mechanistically, how cellulase adsorption onto cellulose controls cleavage kinetics. Based on measured molecular-kinetic rate constants, they were able to provide molecular guides for protein engineering of improved cellulase constructs. They also developed a surface-based assay for improved enzyme development.
The team used their surface-based quartz crystal microbalance assay to determine Langmuir adsorption, desorption and irreversible-binding constants for cellobiohydrolase I and endoß- glucanase, demonstrating that while CBH I adsorbs to the surface much faster, the rate of irreversible binding is identical for both enzymes. This result suggests a binding-domain only interaction governing irreversibility. Radke’s group also explored the synergy between endoglucanase and exoglucanase on model cellulose films, finding that a high-bulk concentration of endoglucanase results in competition for surface sites and thereby slow, rather than enhanced, cellulose degradation.
Radke’s group developed a surface-based assay to elucidate the kinetics of cellulase binding, activity, and deactivation on a solid cellulose substrate. They assessed adsorption, desorption, and denaturation rate constants for isolated endoglucanases and exoglucanases. They then incorporated these rate constants into a novel, two-enzyme surface model for cellulase activity on a solid cellulose surface. Cellulases adsorbed irreversibly and retained activity on the cellulose surface for only several hours after initial adsorption.