Our long-term goal is to develop a methodology to improve enzyme activity in non-aqueous media in order to create safer and mo

Our long-term goal is to develop a methodology to improve enzyme activity in non-aqueous media in order to create safer and more effective pharmaceuticals to treat human diseases. The major objective of this proposal is to develop enzyme-dissolving ionic liquids, evaluate the enzyme activity, stability and enantioselectivity in these ionic liquids under microwave radiation, and determine the protein structure changes that occur under these conditions. Enzymes are effective biocatalysts in producing drugs and related molecules, but enzymatic reactions are often conducted in volatile organic solvents. The use of volatile organic solvents is very harmful to researchers and workers; more importantly, their residues may be carried over to the final product and create problems for the public health. On the other hand, using ionic liquids instead can eliminate these problems caused by organic solvents. Thus, this objective is consistent with the NIH mission to benefit human health and the quality of life, in this proposal by conducting research using innovative solvents and techniques that will support the efficient production of pharmaceuticals. The central hypothesis is that enzymes will maintain high activities in ionic liquids under low-energy microwave radiation through systematically varying the ionic-liquid systems and microwave conditions. Our rationale for this project is that its successful completion would provide a fundamental understanding of enzyme behaviors in ionic liquids under microwave radiation, and potentially allow implementation of such systems for many important biotransformations. In order to test the hypothesis and achieve our objective, two Specific Aims are proposed: (1) Identify strategies to solubilize and stabilize enzymes in non-aqueous ionic liquids; (2) Determine the mechanism of enzyme stabilization in ionic liquids under microwave radiation. The research design is based on the unique combination of ionic liquids and microwaves for the enzymatic applications: ionic liquids can dissolve and stabilize enzymes, while microwave radiation can activate enzymes. Through a careful selection of various ionic liquid systems, the enzyme stability and activity will be examined in these solvents through esterification or hydrolysis reactions. Furthermore, the mechanism of enzyme behaviors in ionic media will be investigated by determining the protein structure changes through a real-time technology, in situ infrared spectroscopy, under microwaves, as well as through fluorescence and far-UV circular dichroism spectra. Biology and chemistry major undergraduates will participate in the project.