Accessory enzymes play a key role in reducing the protein loading required to achieve effective hydrolysis of pretreated biomass

Although considerable progress has been made in reducing the cost of cellulase enzymes, relatively high protein loadings are still required to achieve effective cellulose hydrolysis. This restricts the economic viability of “biomass-to-sugar” processes as 5-10 times higher protein/enzyme loadings are required to break down cellulose as to hydrolyze starch. A major challenge is the limited accessibility of cellulases to the cellulosic substrate. We and others have shown that effective pretreatment and the addition of accessory enzymes can greatly increase cellulose accessibility and significantly enhance the hydrolytic potential of “cellulase” cocktails.

The effectiveness of enzyme synergism is highly substrate dependent as hemicellulases with broader substrate specificities, such as family 10 xylanases and family 5 xyloglucanases typically are more effective at enhancing the hydrolytic performance of cellulases over a broader range of substrates. The observed “boosting effect” of lytic polysaccharide monooxygenase AA9 on the hydrolytic potential of cellulases was highest on substrates showing a higher degree of accessible crystalline cellulose, rather than amorphous cellulose. Similarly, a greater extent of cellulase synergism was demonstrated at lower enzyme concentrations and on pretreated substrates containing relatively accessible/disordered cellulose while higher xylanase loadings were required to derive an “optimized mixture” when high solid loadings and substrates with higher xylan content were used. The addition of low amounts of AA9 was beneficial in all cases.

The overall protein/enzyme loading required to achieve effective cellulose hydrolysis can be significantly reduced by modifying the enzyme mixture in response to the biomass substrate and the type of pretreatment used.