Observing and Modeling Cellulosic Substrate Depolymerization by Commercial Enzyme Cocktails Using Confocal Fluorescence Microscopy

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Understanding and modeling the depolymerization mechanisms of cellulosic and lignocellulosic substrates by cellulase cocktails is a critical step towards further optimizing the production of monosaccharides from biomass. The Spezyme CP cellulase cocktail combined with the Novo 188 b-glucosidase blend was used to depolymerize bacterial microcrystalline cellulose (BMCC), filter paper cellulose and untreated and H2O-CO2 pretreated lignocellulosic substrates (hardwood and switchgrass), which were immobilized on a glass surface. The enzyme mixture was supplemented with a small fraction of fluorescently labeled T. Reseii CBH I, which served as a reporter to track cellulase diffusion into and binding onto the internal physical structure of the cellulosic substrate. All reported experiments were conducted at 50°C, the optimal temperature for maximum hydrolytic activity of the enzyme cocktail. BMCC was observed throughout degradation by labeling it with a fluorescent dye. Native autofluorescence was used in a similar way to track filter paper and lignocellulosic substrates. This method allowed us to measure the binding of cellulases in situ and follow the temporal morphological changes of cellulose and lignocellulosic substrates during their depolymerization by a commercial cellulase mixture.

In the case of BMCC, a kinetic model was developed and fitted to fluorescence intensity data obtained through image processing. This model was successfully used to predict the bulk sugar concentrations that were liberated over time from BMCC in a separate experiment and measured by liquid chromatography. A more complete mass transfer and kinetic model is currently under development for the more complex substrates (filter paper and lignocellulosic biomass).

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