UBC Theses and Dissertations
UBC Theses and Dissertations
The potential of using carbohydrate-binding modules (CBMs) for the characterization and modification of cellulose surfaces Aïssa, Kevin
Cellulose is a structural material that, through its association with lignin and hemicellulose, is recalcitrant to degradation. Although the effectiveness of cellulose hydrolysis is usually assessed via glucose release, typically, cellulase accessibility to the cellulosic substrate is the key limitation that restricts effective enzymatic hydrolysis and has proven much harder to quantify. A novel method, which has the potential to better elucidate the mechanisms involved, involves the use of carbohydrate-binding modules (CBMs). In the work described here, CBM production was optimized, yielding g.L-1 quantities of the specific proteins, which were subsequently used to both characterize the surface morphology of lignocellulosic substrates and functionalize cellulose surfaces. A combination of type A and type B CBMs (CBM2a and CBM17) were primarily employed, as they showed binding preferences towards different morphologies within the cellulosic structure. Compared to more established methods the CBM method more accurately predicted enzyme accessibility, indicating that refining did not significantly improve enzyme accessibility at the microfibril level of the cellulosic substrate. In subsequent work, fluorescence-tagged carbohydrate binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Quantitative image analysis, supported by 13C NMR, SEM imaging, and fiber length distribution analysis, indicated that enzymatic degradation predominated in the more disorganized zones during the initial phase of the hydrolysis reaction. This resulted in rapid fiber fragmentation and an increase in cellulose surface crystallinity. Drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate cellulose morphology and its impact on enzyme accessibility. This in turn provided novel insights into the mechanisms involved in enzyme-mediated cellulose deconstruction. As well as using CBMs as an analytical tool, the affinity of CBMs for cellulosic surfaces was also used to introduce functionality. When CBM2a-alkyne bioconjugation was used to link polyethylene glycol (PEG) to CNC surfaces via Click reactions, the CBM-PEG modification of cellulosic surfaces increased CNC redispersion after drying and improved suspension stability.
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