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Solving a Sticky Problem: Understanding Enzyme Binding to Lignocellulosic Biomass during Biofuel Production

PI: Whitehead, Tim Proposal Number: 1236120 Institution: Michigan State University Title: Solving a Sticky Problem: Understanding Enzyme Binding to Lignocellulosic Biomass during Biofuel Production Biological-mediated conversion of cellulosic biomass to useful fuels and chemicals is a promising avenue towards energy sustainability. A critical impediment for this avenue is the cost of the cellulase enzymes needed to deconstruct biomass to fermentable sugars. At the current cellulase-to-substrate loading the cost per gallon of fuel produced from lignocellulose would still be at least an order of magnitude higher than the enzymes required to degrade starch, even if the enzymes could be produced as cheaply as soy protein. One strategy to reduce enzyme requirements is to recycle the cellulases in a continuous process, but enzyme inactivation during biomass saccharification severely limits this approach. PI?s recent findings show that enzyme inactivation occurs through non-productive interactions with residual lignin (i.e. the cellulases adsorb to hydrophobic lignin surfaces hastening their denaturation) for most pretreatment chemistries and biomass sources. The PI hypothesizes that minimizing the potential of mean force between the cellulases and lignin polymer will reduce surface adsorption between the two, hence minimizing enzyme inactivation. Thus, the primary objective of this project is to test the hypothesis that selective variation of cellulase surface chemistry can decrease lignin-mediated inactivation due to reduced protein surface adsorption to lignin. The hypothesis will be tested by using computational protein design to identify mutations that result in different enzyme surface potentials predicted to minimize enzyme-lignin adsorption, and then genetically program these mutations in two representative cellulases from the workhorse microbe Trichoderma reesei. A large number (ca. 1000) of cellulase variants will be recombinantly expressed and screened for solubility and activity on cellulosic substrates. Surviving variants will be tested for retention of structure, solubility, and stability. Adsorption isotherms will be recorded for the variants for natural and synthetic lignin of varying surface chemistry and microstructure. The PI will test this hypothesis for cellulase catalytic domains, carbohydrate binding modules, and the full-length cellulases. The project will provide fundamental quantitative understanding of protein-surface adsorption effects with significant implications for commercial-scale bioenergy production using lignocellulosic biomass. More generally, this work will supply datasets for identifying critical parameters governing enzyme-surface interactions crucial for disparate applications such as separations of single wall carbon nanotubes, laundry detergents, biomaterials for medical uses and immobilized biocatalysts. This project will engage students from K-12, undergraduate, and graduate levels from various interdisciplinary programs and under-represented groups to broaden their understanding of the role of industrial biotechnology in mitigating bioenergy related problems in an environmentally sustainable manner. The project will utilize the established outreach educational programs at the Great Lakes Bioenergy Research Center to create bioenergy-relevant educational materials for science teachers within the greater mid-Michigan school community.

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