EAGER: Rational Modification of Enzyme Charge for Enhanced Biocatalyst Stability in Ionic Liquids

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Widespread interest in the use of ionic liquids (ILs) as solvents for biocatalytic reactions, which stems from their exceptional solvent properties, has largely been met with underwhelming results. Such results can be directly attributed to the current lack of understanding of how ILs interact with enzymes at the molecular level. The PI, Prof. Joel Kaar of the University of Colorado, hypothesizes that altering the charge state of residues on the enzyme surface will provide a route to mediate electrostatic interactions between ILs and enzymes. Moreover, altering the surface charge of an enzyme should also prevent hydrophobic interactions that can influence IL-enzyme binding as well as enzyme aggregation in ILs. The National Science Foundation Catalysis & Biocatalysis Program will offer an EAGER Award to support this highly exploratory experimentation which will specifically enable preliminary demonstration of the role of electrostatic interactions in the association of enzymes and ILs at the molecular level. The role of electrostatic interactions in the association of enzymes and ILs will be quantified as a function of enzyme surface charge via fluorescence quenching assays. The potential use of charge modification to increase the tolerance of cellulases to ILs will also be demonstrated. This has implications in biomass conversion technology.This research will begin to uncover the mechanisms by which enzymes are inactivated in ILs by characterizing the biophysical basis for the impact of charge modification on IL-induced inactivation. The results of this work will ultimately support the use of enzyme charge modification as a means to enhance the utility of ILs for biocatalysis in general.This research will enable opportunities to train students in molecular biology, biochemical, and biophysical techniques while providing practical laboratory experience. Training in new protein engineering techniques developed in this work will be extended to students in the University of Colorado iGEM (international Genetically Engineered Machines) program, which the PI has helped grow. These techniques may be implemented in student iGEM projects for the purpose of developing novel biological systems with utility in areas of medicine, energy, and sustainability.