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

Abstract

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.

Observing Thermobifida fusca cellulase binding to pretreated wood particles using time-lapse confocal laser scanning microscopy

“…Indeed, extracted k 1 values for experiments done at higher enzyme concentration solutions are larger, since a smaller fraction of enzyme was lost to non-specific adsorption. Furthermore, the binding rates at the highest concentrations we measured, are similar to results obtained in previous studies with BMCC, whose bindings sites are easily accessible (Moran-Mirabal et al 2008). Nevertheless, given the large variations seen in these data, only order-of-magnitude estimation can be Fig.…”

“…Thus, any effort to resolve cellulase binding, mobility and synergistic interactions must provide nano-scale resolution of the behavior of these interesting molecular machines. Recently, fluorescence microscopy was used to observe temporal cellulase binding to micro-patterned BMCC substrate (Moran-Mirabal et al 2008). In the present study of cellulase binding to pretreated wood particles, time-lapse CLSM was used to explore the adsorption of three T. fusca cellulases to lignocellulose substrates.…”

Surface Engineering and Patterning Using Parylene for Biological Applications

“…When combined with high-resolution fluorescence microscopy techniques, biomolecules can be manipulated with nanoscale precision using these patterned surfaces, potentially elucidating the inner workings of cells and shedding new light on biophysical processes. In a study by Moran-Mirabal and coworkers, the binding and activity of cellulases have been quantified by fluorescence microscopy of Alexa Fluor 647 labeled cellulases moving along Alexa Fluor 488 labeled cellulose fibrils [65]. These fibrils were immobilised onto glass coverslips using parylene “peel-off” stencils.…”

Addressing the Recalcitrance of Cellulose Degradation through Cellulase Discovery, Nano-scale Elucidation of Molecular Mechanisms, and Kinetic Modeling

“…Throughout this project we have endeavored to develop methods for the comprehensive study of the binding and diffusion behavior of cellulases on cellulose fibrils, bundles, and mats. Method development focused on immobilization of cellulose onto solid support [3], managing photo-bleaching of fluorphores [4], and developing tracking software for measuring and analyzing the surface diffusion on insoluble cellulose [5]. Our approach has been to use these methods to explore hypotheses regarding cellulases surface diffusion and binding along the cellulose fibrils.…”