| The hydrolytic conversion of cellulosic waste materials to glucose can provide a valuable feedstock for our global fuel and chemical needs. Although cellulose is quite recalcitrant to hydrolysis, degradation by cellulase systems may offer an environmentally benign solution to the problem.; The complex structure of cellulose means that the cellulases must navigate a complicated matrix when accessing the substrate. Hydrolysis is a surface reaction and the formation of the enzyme-substrate complex is dependent on proximity. Additionally, it is theorized that synergism between cellulases is optimized when there is equal accessibility for all system components within the matrix, and pore-sieving effects are minimized.; A packed column size-exclusion chromatography technique was developed in order to characterize the cellulose matrix of Avicel PH 102 and observe the movement of cellulase catalytic domains within the matrix. Highly reproducible elution profiles were observed using a series of inert poly-ethylene glycol (PEG) probes to characterize the pore size distribution of the Avicel. The PEGs exhibited concise size-based trends with regard to accessible volume, probe velocity, system dispersion, and accessible surface area. From the elution data, mathematical analysis determined the surface area accessible to a 5.1 nm molecule (the approximate size of a cellulase) be 31.0 m2/g cellulose.; The elution profiles of three cellulase catalytic domains also resulted in highly reproducible elution profiles and were used to estimate several governing transport parameters of flow through porous media. The effects of the binding of the catalytic domains to the cellulose on molecule velocity and dispersion were investigated. Analysis revealed that the both the PEGs and enzymes followed size based trends but binding of the enzymes resulted in retarded velocity through the column and decreased system dispersion. |
