Immobilization of Enzymes for Use in Supercritical Fluids

Pedro Lozano, Teresa de Diego, and José L. Iborra

Summary

Supercritical fluids (SCFs) are environmentally benign solvents that enable efficient ability to dissolve and/or transport many hydrophobic compounds. Because the solvent properties of SCFs can be adjusted by changing either the pressure or the temperature, they are widely used in a industrial extractive clean processes. For enzymatic biotransformation, several criteria must be considered to select an SCF as the reaction medium, such as the critical parameters or the safety and cost advantages. The importance of strategies for stabilizing enzymes toward SCFs, as well as the appropriate design of reactors is also discussed. Four different high-pressure reactors (i.e., stirred tank, packed bed, cross-flow membrane, and membrane with recirculation) used for enzymatic synthetic processes utilizing SCFs are described in detail. Protocols for operation with these reactors to carry out butyl butyrate synthesis or the kinetic resolution or rac-1-phenylethanol are described in detail, including procedures to obtain membranes with immobilized enzymes and a list of notes of special interest for researchers.

Key Words: Biotransformations; enzyme reactors; green chemistry; lipases; nonconventional media; supercritical fluids.

1. Introduction

Supercritical fluids (SCFs), in their role as nonconventional media for enzymatic reactions, are an interesting alternative for developing clean processes because of their interesting physical properties. SCFs are materials which operate at pressures and temperatures higher than their critical points (Pc and Tc) (see Fig. 1), having densities comparable to those of liquids, while their diffusivities and viscosities are similar to those of gases (1,2). These characteristics, which suit them as ideal solvents for use in extraction processes, also make them attractive as media for biocatalytic transformations, especially when reactions are limited by the rate of diffusion, rather than by any intrinsic kinetics (3,4). However the key feature of

Supercritical Fluid

Fig. 1. Phase diagram of pure substances.

Temperature (°C)

Fig. 1. Phase diagram of pure substances.

SCFs as solvents is their tunability with changes in pressure and temperature, for which reason they can be used to recover products from reagents and to modify the selectivity of reactions (5-7). The density of SCFs is highly sensitive to both temperature and pressure and so all their density-dependent solvent properties (e.g., dielectric constant, relative permittivity, Hildebrand solubility parameter) may be substantially modified by small changes in pressure or temperature. This provides a potential for controlling reactions by precipitation of a product, or for purification by the selective precipitation of products. Different pure substances can be used as supercritical fluids, some of them are used already in biocatalysis (Table 1). However, several criteria must be considered before selecting a given SCF as enzyme-reaction medium, including the critical parameters and safety and cost advantages. Supercritical carbon dioxide (scCO2) is the most popular SCF because of its relatively low critical parameters, its low toxicity, and nonflammability; furthermore, it is chemically inert in most conditions, has excellent solvent properties for nonpolar solutes, and is considered as a green solvent (7). Carbon dioxide is clearly a "greenhouse gas," however, it is produced at an industrial scale as a by-product (e.g., ammonia plants). It is therefore cheap. Furthermore, at atmospheric pressure, CO2 is gaseous, which means that simple depressurization is necessary to separate solutes from scCO2, after which it can be pressurized for reuse as a solvent. The solvent power of scCO2 can be modified by increasing the bulk density or by adding a modifier (e.g., MeOH or toluene). Cosolvents can therefore be used to increase or reduce polarity, or to enhance affinity for aromatic substances, although, of course, the more cosolvent that is added, the further scCO2 moves away from being a truly green solvent (1). The most important limitation to

Table 1

Critical Parameters of Some SCFs (2)

Fluid

Ethylene*

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