UBC Theses and Dissertations
The role of accessory enzymes in enhancing the effective hydrolysis of the cellulosic component of pretreated biomass Hu, Jinguang
To achieve effective cellulose hydrolysis requires the synergistic cooperation of various cellulases and accessory enzymes/proteins. Most of previous synergism studies have used “model” cellulosic substrates, such as cotton or Avicel, and focused on the initial stages of hydrolysis. Previous studies have also demonstrated that the extent of synergism was influenced significantly by the composition and concentration of “cellulase” mixture and the nature of cellulosic substrate. To gain a better understanding of “cellulase synergism”, the actions of individual and combinations of cellulases, β-glucosidase and “accessory” enzymes (such as xylanases, xyloglucanases and AA9) were assessed on various pretreated lignocellulosic substrates at different enzyme loadings. The synergistic cooperation between cellulases and xylanases was found to enhance the extent of hydrolysis of steam pretreated corn stover and dramatically reduced the required cellulase dosage (about 7 times) needed to achieve reasonable cellulose hydrolysis (>70%). Xylanases appeared to act cooperatively with cellulases by solubilising the xylan and consequently increasing fibre swelling and cellulose accessibility. However, the observed synergism between the cellulase monocomponents and hemicellulases was highly substrate dependent. Those hemicellulases with broader substrate specificities, such as family 10 xylanase and family 5 xyloglucanase, promoted the greatest improvement in the hydrolytic performance of cellulases on a broader range of substrates. The “boosting effect” of AA9 on cellulase hydrolytic performance was highest on substrates showing a higher degree of accessible crystalline, rather than amorphous cellulose. The synergistic cooperation was probably, at least in part, due to AA9s oxidative cleavage, resulting in negatively charged sites on the cellulose. This likely increased the more rapid turn-over of processive enzyme CBHI. A greater degree of synergism among cellulase components was demonstrated at lower enzyme concentrations and on pretreated substrates containing relatively accessible/disordered cellulose. Higher xylanase loadings were required to derive an “optimized mixture” when high solid loadings and substrates with a higher xylan content were used, while the addition of low amounts of AA9 (2mg/g cellulose) was beneficial in all cases. Determining the optimum enzyme composition for a particular substrate was shown to be a key strategy for reducing the protein loading required to achieve effective hydrolysis of pretreated biomass substrates.
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