Tuesday, May 3, 2011
Two common approaches to improve the conversion of cellulose to glucose are: reducing cellulose crystallinity by thermochemical treatments or improving cellulase activity by protein engineering. However, we demonstrate here a third approach, based on reorganizing the hydrogen bonding network within crystalline cellulose to produce cellulose III. Crystalline cellulose can exist in various allomorphic states (e.g., I, II, III), however, there has never been a detailed kinetic study characterizing the formation of cellulose III and its influence on cellulase hydrolytic activity. We have carried out a detailed kinetic study to determine the effect of pretreatment parameters (e.g., ammonia/water loading, total residence time, reaction temperature) on the conversion of cellulose I to III using various cellulosic substrates. Cellulose crystallinity and extent of conversion between I and III allomorphic states was determined using X-ray diffraction (spectral deconvolution approach). The substrates were then subjected to hydrolysis by various combinations of purified Trichoderma reesei cellulases (e.g., Cel7A, Cel6A, Cel7B, Cel5A, Cel12A, Cel61). Based on protein structural analysis and activity assay data, it appears that a cellulase such as Cel7B with a more open and unrestricted active site cleft may further enhance the degradation of cellulose III. Molecular dynamics simulations also revealed that cellulose III fibrils are readily hydrated, explaining its reduced hydrophobically-driven binding to cellulases and that their surface has structural and dynamical features similar to the ones found in amorphous cellulose. Glucan chains within cellulose III are more readily accessible by cellulases due to reduced intra-chain and intra-sheet hydrogen bonding compared to native cellulose.