Entrapment of Proteins into Reverse Micelles

Proteins (enzymes) can be entrapped into surfactant reverse micelles in organic solvent using one of the following procedures. The first procedure has been proposed by ourselves (14) and is now used most extensively. It is known as the "injection" method. According to this method, a small amount of aqueous

Fig. 1. Some special cases of aqueous organic two phase systems: (A) the simplest system consisting of water and water-immiscible organic solvent; (B) water-in-oil emulsion; and (C) surfactant-stabilized microemulsion (reverse micelles).

protein solution (typically, several percent by volume) is added to a solution of surfactant in organic solvent (dry or slightly hydrated). The actual volume ratio of the aqueous to organic solutions is determined by experimental conditions (e.g., the degree of hydration of the surfactant, its concentration, etc.). The resulting mixture is vigorously shaken until an optically transparent solution is obtained. This procedure is very simple and effective, although, in some instances, the question of time required to attain equilibrium in this system has been raised (see, for example, ref. 22). Generally, long equilibration times (as well as undesirable side reactions), if a problem, can be avoided by separately preparing micellar solutions of reagents with the required degree of hydration and then mixing them in different proportions. As a result, there is no change in the hydration of the surfactant molecules; hence, the size of micelles remains constant. (The change in the size of micelles is believed to be the main reason for slow equilibration and the occurrence of side reactions.)

The second procedure, proposed by Menger and Yamada (23), consists of the initial introduction of the required amount of water into the surfactant solution in the solvent in order to attain the required degree of hydration (w0). This is followed by dissolution of dry (lyophilized) protein in the micellar solution under vigorous shaking (see Note 2). Usually, an excess of the protein is used and the undissolved material is removed by centrifugation. A new portion of the protein is then added to the supernatant and the abovementioned procedure is repeated several times with careful control of the surfactant and water content of the supernatant (see Note 3). One of the drawbacks of this method is a prolonged contact between the enzyme molecules and the organic solvent/sur-

Fig. 2. The dependence of the equilibrium constant of the reaction of oxidation of iso-butanol into iso-butyraldehyde (catalyzed by horse liver alcohol dehydrogenase) on the volume ratio of organic to aqueous phase (Vorg/Vwater). Curve 1: Two-phase system (not containing surfactant) octane/water (0.02 M phosphate buffer, pH 8.8); Curve 2: the system of reverse micelles of AOT-water (0.02 M phosphate buffer, pH 8.8)-octane at initial AOT concentration of 0.7 M, 20°C. (From ref. 1.)

Fig. 2. The dependence of the equilibrium constant of the reaction of oxidation of iso-butanol into iso-butyraldehyde (catalyzed by horse liver alcohol dehydrogenase) on the volume ratio of organic to aqueous phase (Vorg/Vwater). Curve 1: Two-phase system (not containing surfactant) octane/water (0.02 M phosphate buffer, pH 8.8); Curve 2: the system of reverse micelles of AOT-water (0.02 M phosphate buffer, pH 8.8)-octane at initial AOT concentration of 0.7 M, 20°C. (From ref. 1.)

factant, as this may lead to partial denaturation of the former. By using this procedure, it is difficult to control the changes in the content of impurities (and contaminants in the proteins preparation), such as other proteins or salts. However, the method allows one to obtain a much higher final concentration of enzymes in the system as compared to the one described previously.

The third procedure, first described by Hanahan (13) and developed by Luisi and coworkers (24,25) (see also reviews in refs. 2 and 26), is based on the phenomenon of spontaneous interfacial transfer of proteins in a two-phase system that consists of approximately equal volumes of an aqueous protein solution and an organic solvent containing surfactant (i.e., micellar system). However, it is relatively slow to equilibrate (see Note 4) and the interaction of proteins with the interface could lead to their inactivation. The degree of protein inclusion into the micellar phase can be regulated by, for example, pH and ionic strength. In our opinion, this method is more useful in the context of protein purification than for applications to bio-organic synthesis.

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