Supercritical fluids are a unique class of nonaqueous media in which biocatalytic reactions can occur. The physical properties ofsupercritical fluids, which include gas-like diffusivities and liquid-like densities, can be predictably controlled with changing pressure. This paper describes how adjustment ofpressure, with the subsequent predictable changes of the dielectric constant and Hildebrand solubility parameter for fluoroform, ethane, sulfur hexafluoride, and propane, can be used to manipulate the activity oflipase in the transesterification of methylmethacrylate with 2-ethyl-1-hexanol. Of particular interest is that the dielectric constant of supercritical fluoroform can be tuned from approximately 1 to 8, merely by increasing pressure from 850 to 4000 psi (from 5.9 to 28 MPa). The possibility now exists to predictably alter both the selectivity and the activity of a biocatalyst merely by changing pressure.When the temperature and pressure of a material exceed, or approach, the critical points for that material, the physical properties ofthe "solvent" become sharply dependent on the pressure encountered by the material. Since density is controllable via adjusting either pressure or temperature, it is possible to tune the solvent physical properties by changing reaction conditions, rather than the solvent itself. In addition to these benefits, supercritical fluids (SCFs) are recyclable solvents that aid significantly in sample preparation (1). SCFs are also ideal dispersants in which to investigate the effect of solvent physical properties on an enzyme-catalyzed reaction (2-7).Enzymes are known to function in both aqueous and organic media (for instance, see ref. 8 or ref. 9). Studies ofbiocatalysts in nonaqueous environments have led to a deeper understanding of how enzymes function in unnatural surroundings as well as in water (10). A detailed understanding of environment/ structure/function relationships for proteins suspended in organic media may enable predictive control of enzyme function merely by "solvent engineering." Much research has been dedicated to furthering our understanding of how enzymatic activity is related to the physical properties of the solvent in which it is placed. The driving force for much ofthis research is the fact that when a lyophilized enzyme particle is suspended in an organic solvent, the activity, specificity, and stability of the catalyst depend on the solvent. If one could predict how a particular solvent would affect an enzyme, then, as described above, rational solvent engineering would be a simple way to tune biocatalyst function.Although determining the effect of changing solvent on activity and specificity can suggest interesting correlations between solvent and enzyme properties, the question of why similar solvents can have such different abilities to support biocatalytic reactions always remains. For example, lipasecatalyzed transesterification of acrylates in hexane, heptane, octane, nonane, and decane gives significantly differing rates that no simple ...