Engineering enhanced systems for the deconstruction of cellulose is essential to the cost-effective utilization of biomass for production of liquid fuels and other value added chemicals. Unfortunately, the insolubility of the crystalline cellulose substrate and resulting limitations incurred by heterogeneous catalysis leads to high reaction times that are not suitable for large scale industrial processes. Task specific ionic liquids (IL) are a promising ?green? super solvent that enable the rapid dissolution of crystalline cellulosic biomass while also retaining cellulase enzyme activity thereby providing a promising route to a single step, homogeneous phase degradation of cellulosic biomass. However, much remains in regards to understanding the underlying IL-enzyme interactions affecting enzyme structure, activity, and stability. In this combined computational and experimental project we propose a two pronged approach to evaluate the interactions between three ILs on two cellulase enzymes. The cellulase enzymes being studied have shown varying degrees of activity in IL-H2O mixtures, allowing for the identification of specific enzyme features necessary for functionality while the different IL-H2O mixtures enable the identification of specific IL features that favorably affect enzyme functionality. The identification of specific IL-enzyme interactions leading to altered enzyme functionality and activity will be accomplished through the following objectives: (1) MD simulations on IL-H2O-enzyme systems. These simulations will reveal specific IL-enzyme interactions and their impact on dynamic fluctuations and the overall structure of the enzyme, (2) Conduct Thermodynamic Integration calculations on IL-H2O-enzyme and IL-H2O-cellulose oligomer systems. These calculations probe the ability of the IL to enter the active site of cellulose enzymes and illuminate the role of competing solvation energies, (3) Conduct CpHMD simulations on IL-H2O-enzyme systems. These simulations will reveal the impact of IL mixtures on the pKa of critical residues in the active site, and (4) Conduct kinetic analysis of IL-H2O-enzyme degradation of cellulose. These experiments will reveal the impact of IL-H2O mixtures on the observed enzyme activity.
The computational and experimental studies will provide an increased molecular-level understanding of the IL-enzyme interactions and the resulting effect on structure, dynamical fluctuations, pKa values, and solvation will elucidate how the solvent environment impacts the functionality of enzymes and other macromolecule systems relevant to industrial processes. In addition, evaluation of the enzyme and IL properties that facilitate structural stability and activity will enable the use of rational design to create tailored enzymes and ILs for a wide range of industries. Closely integrated with the research is the education of the next generation of scientists and engineers across multiple education levels. Engaging underrepresented groups in STEM fields is accomplished through the Research Experience for Pre-Collegiate Students (REPS) outreach program with Martin Luther King Jr. Early ollege, a Denver high school. The research effort will be broadly disseminated through research articles, regional and national meetings, in addition to the incorporation into computational workshops at the regional AAAS Pacific Division meetings and Boise State University, a primarily undergraduate institution.
