Monday, April 30, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
The anaerobic bacterium Clostridium thermocellum produces extracellular multi-enzyme macromolecules called cellulosomes which are capable of efficiently hydrolyzing diverse plant cell wall polysaccharides. In order to enzymatically depolymerize carbohydrates, various cellulases, hemicellulases, and other enzymes are bound together via their dockerin domains to one of the seven cohesion domains of the scaffoldin protein to form this multifunctional macromolecular machine. Carbohydrate binding domains on individual enzymes and scaffoldins enable cellulosomes to bind to substrates. This has functional benefits such as proximal synergy and consequences which may be non-specific binding to lignin. Cellulosomes have been observed bound to the microbe cell surface or free in solution and can be ~18 nm in length and >1MDa in molecular weight. We purified large molecular weight cellulosomes from C. thermocellum to characterize the biophysical properties using, small angle x-ray scattering, analytical ultracentrifugation, size-exclusion chromatography and native poly-acrylamide gel electrophoresis. Using these purified cellulosomes we have analyzed the enzymatic conversion of biomass and model cellulose substrates into fermentable sugars. We are ultimately interested in the mechanisms of cellulosome hydrolysis, we will also discuss results from immunolabeled transmission electron microscopy studies and how these images have broadened our understanding into the differences of enzymatic mechanisms between the cellulosome and free fungal enzymes. We will present the results to our enzymatic and biophysical assays and discuss how these systems can improve the efficiency of converting biomass to sugars for biofuel production.