Biocatalytic synthesis of acrylates in supercritical fluids: tuning enzyme activity by changing pressure.

Abstract

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 ...

Reactions at supercritical conditions: Applications and fundamentals

“…This extensive research into these systems has identified and elucidated the effects of many of the important variables, such as pressure (Miller et al, 1990(Miller et al, , 1991Erickson et al, 1990;Ikushima et al, 1993). Reaction rates in SC CO, and in organic solvents (typically hexane) have been compared (Martins et al, 1991;Bolz et al, 1991;Marty et al, 1990Marty et al, , 1992aKamat et al, 1992Kamat et al, , 1993Chi et al, 1988;Pasta et al, 1989). Kinetics parameters and reaction mechanisms have been reported (Miller et al, 1991;Bolz et al, 1991;Marty et al, 1992b;Dumont et al, 1992).…”

“…SC CO,, either pure or with a cosolvent, has been employed as the SCF solvent in nearly all investigations. The few exceptions are Hammond et al (1985) using fluoroform, and work at the University of Pittsburgh (Kamat et al, 1992(Kamat et al, , 1993) that employed SC ethane, ethylene, SF,, and CHF,. Several different enzymes have been shown to maintain their activity after exposure to SC CO,, pure and with added water and ethanol (Taniguchi et al, 1987).…”

“…This discovery motivated a great deal of research, beginning in the early 1980s, aimed at better understanding the role of the solvent in enzyme catalysis and at developing new biocatalytic processes for the synthesis of chemicals. This attempt to identify solvent effects on enzyme catalyzed reactions and manipulate them to achieve a desired goal has been termed "solvent engineering" (Kamat et al, 1992(Kamat et al, , 1993. It is within this context that supercritical fluids have been examined as potential media for enzyme catalysis.…”

“…Work at the University of Pittsburgh (Kamat et al, 1992(Kamat et al, , 1993 has explored the use of compounds other than CO, as SCF solvents for enzyme catalysis. The Pittsburgh group found that SC SF, gave the highest rates for the lipase-catalyzed transesterification of methylmethacrylate and 2-ethoxyhexanol ( Figure 20).…”

“…SC CO, was not a good solvent for this reaction, presumably because it altered the pH of the microaqueous enzyme environment or because it formed covalent complexes with free amine groups on the enzyme. Kamat et al (1993) used these different SCFs to explore the effects of pressure-induced variations in solvent properties on the reaction rate. They found that when increasing pressure had an effect, it was to decrease the rate.…”