Jeremy S. Luterbacher and Jose M. Moran-Mirabal contributed equally to this work.
Observing and modeling BMCC degradation by commercial cellulase cocktails with fluorescently labeled Trichoderma reseii Cel7A through confocal microscopy†
Article first published online: 18 JUL 2012
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 110, Issue 1, pages 108–117, January 2013
How to Cite
Luterbacher, J. S., Walker, L. P. and Moran-Mirabal, J. M. (2013), Observing and modeling BMCC degradation by commercial cellulase cocktails with fluorescently labeled Trichoderma reseii Cel7A through confocal microscopy. Biotechnol. Bioeng., 110: 108–117. doi: 10.1002/bit.24597
- Issue published online: 20 NOV 2012
- Article first published online: 18 JUL 2012
- Accepted manuscript online: 5 JUL 2012 07:33AM EST
- Manuscript Accepted: 20 JUN 2012
- Manuscript Revised: 14 JUN 2012
- Manuscript Received: 15 FEB 2012
- Northeast Sun Grant Initiative
- fluorescence microscopy;
- confocal microscopy;
- enzymatic hydrolysis;
- kinetic modeling;
Understanding the depolymerization mechanisms of cellulosic substrates by cellulase cocktails is a critical step towards optimizing the production of monosaccharides from biomass. The Spezyme CP cellulase cocktail combined with the Novo 188 β-glucosidase blend was used to depolymerize bacterial microcrystalline cellulose (BMCC), which was immobilized on a glass surface. The enzyme mixture was supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A, which served as a reporter to track cellulase binding onto the physical structure of the cellulosic substrate. Both micro-scale imaging and bulk experiments were conducted. All reported experiments were conducted at 50°C, the optimal temperature for maximum hydrolytic activity of the enzyme cocktail. BMCC structure was observed throughout degradation by labeling it with a fluorescent dye. This method allowed us to measure the binding of cellulases in situ and follow the temporal morphological changes of cellulose during its depolymerization by a commercial cellulase mixture. Three kinetic models were developed and fitted to fluorescence intensity data obtained through confocal microscopy: irreversible and reversible binding models, and an instantaneous binding model. The models were successfully used to predict the soluble sugar concentrations that were liberated from BMCC in bulk experiments. Comparing binding and kinetic parameters from models with different assumptions to previously reported constants in the literature led us to conclude that exposing new binding sites is an important rate-limiting step in the hydrolysis of crystalline cellulose. Biotechnol. Bioeng. 2013; 110: 108–117. © 2012 Wiley Periodicals, Inc.