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.
The overall aim of this project was to develop a strategy to broadly improve the stability of enzymes in non-native solvent environments that are conducive to processing biomass. This work has specifically led to a novel approach to prevent the inactivation of cellulase enzymes by ionic liquids through altering enzyme surface charge. Such improvements in cellulase stability correlated with the inhibition of denaturing interactions between the ionic liquid and enzyme. While investigating the effect of charge on cellulase stability in ionic liquids, we also found that altering charge reduces non-productive adsorption of insoluble matrix components to cellulase. The reduction in non-productive adsorption is the result of disrupting hydrophobic and electrostatic interactions between cellulase and the biomass matrix. As a result of such improvements in stability and activity, this approach opens the door to efficiently convert cellulose to biofuels without the need for biomass pretreatment. By eliminating the need for pretreatment, the use of ionic liquids to solubilize biomass can significantly increase the economic and environmental viability of biomass-derived fuels and thus improve the widespread commercialization of biofuels as a sustainable fuel alternative. Moreover, these results provide evidence that naturally evolved cellulases with highly negatively charged surfaces may have enhanced utility in biomass conversion in ionic liquids.
