Comparisons of catalytic efficiency during the reduction of lignocellulosic substrates between free enzyme versus enzyme bound to mobile enzyme sequestration platforms (mESP)

Abstract

Plants represent a vast, renewable source of biomass. Plant biomass serves as a major substrate for sustainable biofuel production. However, to overcome the food‐versus‐fuel dilemma, effective use of plant residues for cellulosic (versus corn‐ or sugarcane‐based) ethanol must be realized. Effective deconstruction of lignocellulosic matrix and efficiently accessing and reducing cellulose to fermentable sugars (i.e., glucose) would overcome current constraints that prevent cellulosic ethanol from becoming an economically viable alternative for liquid/transportation fuel. Cellulosic feedstocks consist of cellulose (20–50%), hemicellulose (15–35%), and lignin (5–30%). In previous work, we demonstrated that the use of thermotolerant and acidotolerant engineered mobile enzyme sequestration platforms (mESP) could enhance lignocellulosic deconstruction of two acid pretreated (and alkaline pretreated) feedstocks. Specifically, acid‐pretreated wheat straw and corn pericarp were subjected to a suite of cellulases, an enzyme cocktail, under two conditions: (a) pretreated substrate plus enzymes free in solution (experimental control); and, (b) pretreated substrate plus enzymes bound to a prototype mESP. The mESP‐bound enzymes showed enhanced ability to convert complex carbohydrates to glucose (i.e., sugar reduction) when compared to the free enzyme in solution control (at equimolar enzyme concentrations). Although the mESP was known to be thermotolerant and acidotolerant, enhancement of sugar reduction efficiency was also observed with alkaline pretreated substrate. Still, these preliminary experiments only demonstrated a 2‐ to 3‐fold increase in enzymatic efficiency over controls. Indeed, with some enzyme ‘cocktails’, sugar reduction efficiency actually decreased when employing mESPs. It was suggested that steric interference between enzymes due to the close proximity of enzyme attachment points on the platform and the use of enzymes with different target points (exo‐versus endo‐) on the substrate (i.e., cellulose) may limit the magnitude of enhancement that can be achieved. After re‐engineering the mESP system to separate attachment points and strategic development of free enzyme cocktails and enzyme composition on charged mESPs, this study demonstrates that next‐generation mESPs can improve catalytic efficiency more than 3‐fold. In addition, alternative substrates have been tested (e.g., rice straw) with similar results. These results and implications for mESP technology are discussed in detail.Support or Funding InformationU.S. National Science Foundation Molecular and Cellular Biosciences (MCB) grant (Award No. 1818346; PI‐Ceballos); U.S. National Science Foundation Research Coordination Network (RCN) grant (Award No. 1624171; PI‐Ceballos)