A Multi-Scale Investigation of Cellulases and Their Interaction with Cellulose Allomorphs

The enzymatic degradation of cellulose into monomeric sugars is a bottleneck in cellulosic biofuel production. This process is dependent not only on the individual enzymes themselves but also on the physical properties of the substrate. In this study, we use a multi-scale experimental approach to investigate the interaction of cellulolytic enzymes on both native and non-native cellulose allomorphs; specifically, reducing-end specific cellobiohydrolase (CBH I) from Trichoderma reesei and its carbohydrate-binding module (CBM). Both native cellulose I and its unnatural allomorph, cellulose III, produced using a next-generation extractive ammonia based pretreatment were used in this work. Bulk experimental measurements have revealed that CBMs have a reduced affinity for non-native cellulose allomorphs (cellulose III) compared to native cellulose (cellulose I), despite higher enzymatic hydrolysis rates. Optical tweezer based single-molecule assays are used to characterize enzyme behavior and provide a molecular-level understanding of the bulk assay results. Together, our multi-scale experiments provide insight into the cellulose degradation mechanism as a basis for engineering more efficient processes for the enzymatic degradation of cellulosic feedstocks.