Surface-Based Assays for Enzyme Adsorption and Activity on Model Cellulose Films

Transportation fuels produced by harvesting and breaking down sturdy, fast-growing prairie grasses offer a renewable alternative to diminishing fossil-fuel supplies. The rate-limiting step in the production of renewable fuels from these lignocellulosic feedstocks is the enzymatic deconstruction of solid cellulose into glucose oligomers that are subsequently processed to form transportation fuels and fuel additives. Despite continuing research interest and significant subsidy of biofuel production, the mechanisms and kinetics governing this fundamental interaction remain largely unknown./nCellulose, the world's most abundant biopolymer, is comprised of long glucose chains organized in an extensive hydrogen-bonding network that makes cellulose insoluble in water and recalcitrant to enzymatic degradation. Complete deconstruction of cellulose into soluble glucose oligomers requires the concerted action of several enzymes, collectively known as cellulases, that adsorb to the cellulose surface from aqueous solution and complex with cellulose chains. Current assays of cellulase activity are performed in the bulk, and thus fail to characterize this important surface interaction. Recently, thin model films of solid cellulose adhered to metal supports have become available. These model films offer well-defined substrates of known surface area on which cellulase activity can be characterized./nThis work describes the development and application of surface-based assays for elucidating cellulase kinetics on model films of cellulose. The developed surface-based assays allow continuous, non-invasive, inhibition-free measurement of both enzyme adsorption and activity, and are, therefore, preferable to bulk assays. Ellipsometry, an optical technique that uses changes in the polarization of light to detect film thickness, is applied to prove the efficacy of surface-based assays for measuring the activity of a cellulase mixture on model cellulose films. Degradation rates measured by ellipsometry are identical to those measured by a traditional bulk glycan assay on Avicel, a laboratory-standard cellulose. Quartz crystal microgravimetry (QCM), an acoustic technique that uses changes in the resonance of a quartz crystal to detect adsorbed mass, is then used to measure the competitive adsorption and cooperative activity of two individual cellulases and their binary mixtures. Results obtained from both the optical and acoustic assays are commensurate./nUsing data from these assays, cellulase adsorption and activity are described according to a two-enzyme surface kinetic model incorporating both Langmuir adsorption to the cellulose surface and Michaelis-Menten activity of adsorbed enzyme. The model additionally quantifies observed irreversible binding of cellulases and the cooperative activity of two cellulases in creating and degrading cellulose chain ends. Cel7A, a processive cellobiohydrolase that complexes with cellulose chain ends and digests cellulose chains into glucose oligomers, is shown to have 14 times higher adsorption affinity for the cellulose surface than does Cel7B, a non-processive endoglucanase that disrupts the hydrogen-bonding structure of the cellulose surface and creates chain ends. Both enzymes rapidly bind irreversibly to the cellulose surface, with 75 - 85% irreversibly bound after 1 h contact between aqueous enzyme and solid cellulose. Nevertheless, irreversibly bound enzymes remain catalytically active. The cellulytic activity of Cel7A is maximized by increasing cellulose surface chain-end concentration without leaving a large quantity of Cel7B irreversibly bound. These findings underscore the importance of considering surface concentration, rather than bulk concentration, in the design of optimal cellulase mixtures for biofuel production./nThe kinetic constants governing adsorption and activity of Cel7A and Cel7B on the cellulose surface are obtained from single-enzyme experiments and used subsequently to predict the transient behavior of binary enzyme mixtures. In all cases, good agreement is shown between kinetic model and experiment, validating the surface-based assays. The ellipsometry and QCM techniques described in this thesis can be used further to measure the adsorption and complexation constants of other cellulases, to inform the design of cellulase cocktails, to quantify cellulase inhibition by aqueous glycans, to explore the role of substrate structure in cellulase activity, and to characterize loss of cellulase activity due to surface and thermal denaturation. Surface-based assays, therefore, represent an important new tool for addressing many outstanding problems in cellulose deconstruction.

Advisor: Radke, Clayton J