Monday, April 19, 2010
LL Conference Facility (Hilton Clearwater Beach)
Cellobiohydrolase (CBH I) secreted by Trichoderma reesei, known as processive exoglucanase is one of the key enzymes used currently in a commercial cellulase mixture for processing biomass to biofuels. CBH I enzyme contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highly-glycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain, cleaves the β-1,4-glycosidic bond, and releases cellobiose as the end product. The objective of this study is to directly image the CBH I enzyme hydrolysis and CBM binding to cellulose. Atomic force microscopy (AFM) is employed to investigate the cellulose structure and morphological changes made by CBH I and CBMs that are derived from fungi and bacterial cellulase, respectively, decreasing cellulose size as well as increasing surface roughness after CBH I hydrolysis were observed. Optical techniques (Total Internal Reflection Fluorescence microscopy or TIRF-M, and Defocused Orientation and Position Imaging or DOPI) are employed to analyze at the single-molecular scale the binding orientations and molecular motion of CBMs that are tagged with green fluorescence protein (GFP). The preliminary results have revealed a confined orientation and nanometer-scale movement of a family 1 CBM (TrCBM1 from fungus T. reesei CBH I) and a family 2 CBM (AcCBM2 from bacterium Acidothermus cellulolyticus E1) bound to cellulose.