Characterizing the Degradation of Cellulose by Combinations of Cellulolytic Enzymes

Open Access
- Author:
- Starr, Thomas
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Charles T Anderson, Thesis Supervisor
Teh-Hui Kao, Thesis Honors Advisor - Keywords:
- Biomass Conversion
Cellulases
Cellobiohyrdolases
Cellulose
Enzyme Synergy
Endoglucanases - Abstract:
- Cellulose is the most abundant biomolecule on Earth and is composed of chains of glucose, but its depolymerization for use in the production of renewable bioenergy and biomaterials is inefficient. To better understand the mechanisms of cellulose degradation by cellobiohydrolases, a class of enzymes that cleave cellulose into cellobiose dimers, we characterized accessory enzymes that act on cellulose in combination with Trichoderma reesei Cel7A, a cellobiohydrolase that targets the reducing ends of glucan chains in cellulose. The accessory enzymes studied include endo-1,4-β-D-glucanase (Cel7B) from Trichoderma longibrachiatum and Cel6A, a cellobiohydrolase II. Cel7B randomly cleaves cellulose to produce additional binding sites for other cellulases, whereas Cel6A cleaves cellobiose units from the nonreducing ends of glucan chains in cellulose. By measuring reducing end production in reactions between cellulose from Acetobacter and these enzymes, we found that although the addition of Cel7B with Cel7A significantly enhances cellulose hydrolysis, the enzyme appears to act in an additive manner rather than a synergistic (multiplicative) one. Conversely, Cel6A appears to act synergistically with Cel7A at varying concentrations, resulting in more cellulose hydrolysis by the enzyme mixture than the sum of the two enzymes alone. Further experiments to test three-enzyme cocktails containing each of the previous enzymes indicate that combining all three cellulases increases synergy above that observed for each two-enzyme combination. These data will be used to develop biochemical models to help explain the single-molecule behaviors of these enzymes as they degrade cellulose alone or in concert, as observed using a newly developed SCATTIRSTORM microscope.