Nuno Fontes M Conceiao Almeida and Susana Barreiros 1 Introduction

A substance is said to be supercritical (SC) above a singular point on the phase diagram, the so-called critical point. The use of SC fluids as solvents for enzymatic transformations is a relatively new area of research (1,2) that is expected to expand in the future. Close to the critical point, small changes in temperature or pressure can effect large changes in the density/solvation ability of SC-fluid. This property of SC-fluids is currently used in a wide range of extraction applications. It can also be fruitfully exploited for the integration of biotransformation and downstream processing steps in a single bioreactor. In addition, lower viscosity and higher diffusivity of SC-fluids as compared to most organic solvents enable better mass transfer, which is often a limiting factor in reaction systems where the enzyme and reactants are not contained in the same phase.

Water plays multiple roles in nonaqueous biocatalysis by enhancing the molecular mobility, and hence the catalytic activity of enzymes (3), by stabilizing charged transition states (4) and by shifting the equilibrium between hydrolysis and synthesis. Thus, the properties of an enzyme are largely dependent on the amount of water directly associated with it; consequently, the degree of biocatalyst hydration must be carefully controlled. A convenient way to do this is by fixing the water activity (aw) of the system (5). This may be accomplished by, for example, pre-equilibrating separately enzyme, solvent, and reactants with saturated salt solutions (3), although this approach is not practical when the solvent is a gas at normal temperature and pressure. However, if the system is equilibrated with water, the value of the aw will be the same in all the phases present. Therefore, the aw of the whole system can be set and measured in the most convenient phase (5). When using supercritical fluids, the direct addition to the reaction mixture of pairs of salt hydrates that confer a certain aw is particularly useful, and guidelines for selecting adequate salt hydrates are available (6). Sodium phosphate salts have been used often in organic media, and we find that they are also effective in SC-CO2. Using these salts, it is possible to establish the dependence of water concentration in a given solvent as a function of the aw. The extrapolation of the data to aw = 1 (water saturation) gives values that are in good agreement with those available in the literature (7,8).

The protocol described here is a general method for biotransformations in supercritical fluids (e.g., SC-CO2, SC-ethane) using biocatalyst suspensions in the form of freeze-dried powders, an immobilized enzyme, or enzyme microcrystals, and it is an update of an earlier version (9).

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