Dissecting and Reconstructing Synergism: IN SITU VISUALIZATION OF COOPERATIVITY AMONG CELLULASES* |
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Authors: | Thomas Ganner Patricia Bubner Manuel Eibinger Claudia Mayrhofer Harald Plank Bernd Nidetzky |
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Affiliation: | From the ‡Institute for Electron Microscopy and Fine Structure Research, Graz University of Technology, Steyrergasse 17, A-8010 Graz, Austria, ;the §Center for Electron Microscopy, Steyrergasse 17, A-8010 Graz, Austria, and ;the ¶Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria |
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Abstract: | Cellulose is the most abundant biopolymer and a major reservoir of fixed carbon on earth. Comprehension of the elusive mechanism of its enzymatic degradation represents a fundamental problem at the interface of biology, biotechnology, and materials science. The interdependence of cellulose disintegration and hydrolysis and the synergistic interplay among cellulases is yet poorly understood. Here we report evidence from in situ atomic force microscopy (AFM) that delineates degradation of a polymorphic cellulose substrate as a dynamic cycle of alternating exposure and removal of crystalline fibers. Direct observation shows that chain-end-cleaving cellobiohydrolases (CBH I, CBH II) and an internally chain-cleaving endoglucanase (EG), the major components of cellulase systems, take on distinct roles: EG and CBH II make the cellulose surface accessible for CBH I by removing amorphous-unordered substrate areas, thus exposing otherwise embedded crystalline-ordered nanofibrils of the cellulose. Subsequently, these fibrils are degraded efficiently by CBH I, thereby uncovering new amorphous areas. Without prior action of EG and CBH II, CBH I was poorly active on the cellulosic substrate. This leads to the conclusion that synergism among cellulases is morphology-dependent and governed by the cooperativity between enzymes degrading amorphous regions and those targeting primarily crystalline regions. The surface-disrupting activity of cellulases therefore strongly depends on mesoscopic structural features of the substrate: size and packing of crystalline fibers are key determinants of the overall efficiency of cellulose degradation. |
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Keywords: | Atomic Force Microscopy Biotechnology Carbohydrate-binding Protein Carbohydrate Processing Cellulase Enzyme Mechanisms Cellobiohydrolase Cellulose Polymorphism Endoglucanase Synergism |
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