Immobilization of cellulose fibrils on solid substrates for cellulase‐binding studies through quantitative fluorescence microscopy

Cellulases, enzymes capable of depolymerizing cellulose polymers into fermentable sugars, are essential components in the production of bioethanol from lignocellulosic materials. Given the importance of these enzymes to the evolving biofuel industry considerable research effort is focused on understanding the interaction between cellulases and cellulose fibrils. This manuscript presents a method that addresses challenges that must be overcome in order to study such interactions through high-resolution fluorescence microscopy. First, it is shown that cellulose can be immobilized on solid substrates through a polymer lift-off technique. The immobilized cellulose aggregates present characteristic morphologies influenced by the patterned feature size used to immobilize it. Thus, through a variety of pattern sizes, cellulose can be immobilized in the form of cellulose particles, cellulose mats or individual cellulose fibrils. Second, it is shown that both cellulose and Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A can be fluorescently tagged and that the labeling does not inhibit the capability of these cellulases to depolymerize cellulose. The combination of the immobilization technique together with fluorescence labeling yields a system that can be used to study cellulose-cellulase interactions with spatial and temporal resolution not available through more conventional techniques which measure ensemble averages. It is shown that with such a system, the kinetics of cellulase binding onto cellulose fibrils and mats can be followed through sequences of fluorescence images. The intensity from the images can then be used to reconstruct binding curves for the cellulases studied. It was found that the complexity of cellulose morphology has a large impact on the binding curve characteristics, with binding curves for individual cellulose fibrils closely following a binding saturation model and binding curves for cellulose mats and particles deviating from it. The behavior observed is interpreted as the effect pore and interstice penetration play in cellulase binding to the accessible surface of cellulose aggregates. These results validate our method for immobilizing nanoscale cellulose fibrils and fibril aggregates on solid supports and lay the foundation for future studies on cellulase-cellulose interactions.