This renewal project is concerned with two areas of importance for increasing the impact of biological processes on production of specialty chemicals: use of non-aqueous solvents for enzyme catalyzed reactions and development of novel separations processes based on aqueous two-phase systems. The first aspect will focus on enzyme reactions in supercritical solvents: In the previous grant period, the stability of enzymes, both free and immobilized in dense carbon dioxide, has been demonstrated, and kinetic data have been obtained for the oxidation of cholesterol and the interesterification of triacylglycerides. This research expands the possible range of substrate concentrations which can be used with supercritical carbon dioxide by exploring its use with entrainers; it also provides a more detailed study of the kinetic and mass transfer effects of importance for both enzymes studied. In addition, the conformation of enzymes in this non-aqueous medium is examined by the use of EPR spectroscopy. A nitroxide spin-labelled cholesterol probe is employed to monitor the spatial coordinates at the active site of cholesterol oxidase. The second aspect will focus on aqueous two-phase systems for product recovery: This research is directed toward establishing the fundamental molecular thermodynamics of aqueous two-phase systems. An important objective is to determine what polymer-polymer systems may be best suited for bioprocessing. Toward that end, the PIs continue their measurements using light scattering and differential vapor pressure to obtain thermodynamic parameters for aqueous mixtures containing dextran, polyethyleneglycol, proteins and combinations of polymer-polymer and polymer protein pairs. These data, combined with information on electrical-potential effects due to salt partitioning and a molecular-thermodynamic model based upon the osmotic virial equation, allow the PI's to predict polymer-polymer-water phase diagrams and protein partition coefficients for process design and optimization.
