The Study of Cellulose Structure and Depolymerization Through Single-Molecule Methods

Renewable energy has gained importance due to rising energy demands and diminishing fossil resources. Lignocellulosic biomass, with a core consisting of crystalline cellulose, has the potential to become a renewable source of fermentable sugars for energy production. However, to utilize this resource, biomass has to be broken down through physical, chemical, or enzymatic treatments. The biochemical hydrolysis of cellulose by cellulases offers an economical alternative to hazardous chemicals. Thus, complete knowledge of the molecular interactions between cellulases and cellulose would help to optimize the efficiency of industrial enzyme cocktails. Single-molecule (SM) methods study molecular events by visualizing individual molecules instead of measuring averages, thereby providing a detailed view of nanoscale processes with high spatial and temporal resolution. SM fluorescence microscopy utilizes enzyme and cellulose labeling, along with localization and tracking algorithms, to yield particle or fluorophore positions with nanoscale precision. Similarly, high-speed atomic force microscopy utilizes a high aspect ratio probe that is brought into close proximity and scanned across the sample to visualize the surface topography and its evolution over time. Both SM techniques have been recently applied to the study of cellulase-cellulose interactions and used to probe enzyme-binding orientation, affinity and reversibility, non-catalytic and catalytic surface motion, and the effect of molecular crowding on enzyme mobility. This review aims to showcase SM techniques and how they have been applied to study cellulose structure and cellulose depolymerization by cellulases. While the study of cellulase-cellulose interactions and cellulose depolymerization through SM microscopy is still a young field, these methods have already contributed to our understanding of the nanoscale processes involved in biomass conversion. Further application of SM techniques could elucidate molecular mechanisms involved in enzyme synergism, as well as the molecular changes that take place as cellulose fibrils are converted into soluble sugars.