(372d) Modeling of Cellulose Hydrolysis By Clostridium Thermocellum Monitored By Quartz Crystal Microbalance
With a goal of improving the production of fermentable sugar from lignocellulose for further processing into biofuels and chemicals, many efforts have been made to quantify and model the enzymatic hydrolysis kinetics of the cellulose degradation process as a function of environmental process variables, cellulose feedstock, and enzyme source. However, most studies use bulk assays to quantify cellulose decomposition, which are unable to capture the individual steps of enzyme adsorption and enzyme hydrolysis on solid substrate. The quartz crystal microbalance with dissipation (QCM-D), an interfacial technique for measuring mass change at a solid interface, allows monitoring in situ and in real time of the binding and catalytic activity of cellulases on model cellulose substrates. The application of QCM to cellulose degradation investigations presents the possibility for screening the effect of substrate properties (crystalinity, surface accessibility etc.), and reaction conditions (pH, temperature, enzyme concentration) on enzyme adsorption and enzyme hydrolysis. The application of QCM to the investigation of cellulose hydrolysis has been limited to fungal cellulases. For the first time, the QCM measurement will be extended to complexed cellulases of C. thermocellum, also known as cellulosomes, which is a highly active cellulase system. Existing kinetic models have not been applied to the measurements of cellulosome adsorption and degradation on solid substrate by this interfacial technique. A fundamental interfacial model that describes cellulosome adsorption and degradation of cellulose thin films provides a basis for interpreting interfacial measurement under various conditions.
In this work, the activity of cellulases of C. thermocellum in the form of free cellulosomes on amorphous cellulose surfaces was measured by QCM as a function of the concentration of an inhibitor, cellobiose. An interfacial kinetic model, incorporating the geometry of solid substrate and the generation of available interfacial sites during hydrolysis, was proposed to describe the surface phenomena of cellulosome binding and hydrolysis on amorphous cellulose thin film as measured by QCM. In this model, cellulose was assumed to exist as cylindrical fiber, which was made up by multilayers of cellulose chains. Consequently, only the top layer of cellulose substrate is accessible to enzyme while the underlying substrate becomes new interfacial sites during hydrolysis. The proposed interfacial model successfully describes the change in mass of the solid substrate during hydrolysis by cellulosomes and is consistent with uncompetitive inhibition by cellobiose.