Monday, April 19, 2010
2-18

Studying cellulase hydrolysis of cellulose with AFM and TIRF-M

Yu-San Liu1, Yonghua Luo1, John O. Baker1, Yining Zeng1, Michael E. Himmel1, Steve Smith2, and Shi-you Ding1. (1) Chemical and Biosciences Center, National Renewable Energy Laboratory, 1617 cole blvd, Golden, CO 80401, (2) Nano-Science & Engineering PhD Program, South Dakota School of Mines and Technology, 501 East St. Joseph Street, Rapid City, SD 57701

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.

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