A kinetic theory is proposed for enzymatic reactions proceeding in reversed micellar systems in organic solvents, and involving SUbStrdteS capable of partitioning among all pseudophases of the micellar system, i.e. aqueous cores of reversed micelles, micellar membranes and organic solvent. The theory permits determination of true (i.e. with reference to the aqueous phase, where solubilized enzyme is localized) catalytic parameters of the enzyme, provided partition coefficients of the substrate between different phases are known. The validity of the kinetic theory was verified by the example of oxidation of aliphatic alcohols catalyzed by horse liver alcohol dehydrogenase in the system of reversed sodium bis(2-ethylhexyl)sulfosuccinate (AOT, aerosol OT) micelles in octane. In order to determine partition coefficients of alcohols between phases of the micellar system, flow microcalorimetry technique was used. It was shown that in the first approximation, the partition coefficient of the substrate in a simple biphasic system consisting of water and corresponding organic solvent can be used as an estimate for the partition coefficient of the substrate between aqueous and organic solvent phases of the micellar system. True values of the Michaelis constant of alcohols in the micellar system, determined using suggested approach, are equal to those obtained in aqueous solution and differ from apparent values referred to the total volume of the system. The results clearly show that the previously reported shift in the substrate specificity of HLADH, observed on changing from aqueous solution to the system of reversed aerosol OT micelles in octane, is apparent and can be explained on the basis of partitioning effects of alcoholic substrates between phases of the micellar system. Micellar enzymology, which deals with enzymes entrapped in surfactant reversed micelles in orgarlic solvents, has received increasing attention during the last decade (for recent reviews see [l -41). The kinetic description of enzymatic reactions in reversed micellar systems often represents a rather complicated problem because of the microheterogeneity of the micellar medium: micellar solution is composed of tiny surfactantstabilized aqueous microdroplets (with diameter up to 200 nm) suspended in organic solvent. Solubilized enzyme molecules are localized exclusively inside these droplets (micelles), whereas the substrate can be distributed throughout the whole volume of the system. Evidently, the latter fact should be taken into account in kinetic analysis. To allow for substrate partitioning, the pseudophase approach can be used, according to which micellar systems can be regarded as being composed of two phases, namely the phase of organic solvent and a pseudophase of micelles. The pseudophase approach was successfully used for description of the kinetics of nonenzymatic [5] as well as enzymatic [6 -91 reactions in micellar systems. However, the notion that a micellar solution can be viewed as a pseudobiphasic system is to a certain extent on ...

Physicochemical characteristics of previously suggested surface-modified polymeric nanogranules (SMPN) and catalytic and stability properties of a-chymotrypsin entrapped into such nanogranules in a nonpolar solvent were investigated in more details. SMPN were obtained by polymerization of an acrylamide/N,N'-methylene-bisacrylamide mixture in a mixed reversed micellar system composed of Aerosol OT [sodium di(2-ethylhexyl)sulfosuccinate] and the polymeric surfactant Pluronic F-108 modified with polymerizable groups, followed by the chromatographic removal of the auxiliary surfactant, Aerosol OT. An optimal solvent system was found providing the required orientation of the polymeric surfactant in starting mixed micelles, i. e. with polar fragments immersed into the micellar interior and apolar fragments protruding into organic solvent. The hydrodynamic diameter of SMPN in benzene solution was estimated by means of quasi-elastic light scattering to be 84 f 1 nm. Catalytic and stability properties of a-chymotrypsin entrapped into SMPN strongly depended on conditions of preparation of SMPN. The optimal concentration of acrylamide monomers in the micellar interior and hydration degree of starting reversed micelles were found to be 20% by mass and w, = 15, respectively. a-Chymotrypsin-containing SMPN were used as a catalyst in the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and ethanol, performed in a membrane reactor Biocatalysis in non-aqueous media has received growing attention during the last decade due to its obvious potentials, such as conversion of hydrophobic compounds and favorable shifts of reaction equilibrium [l, 21. One of the main problems encountered when one tries to conduct enzymic reactions in organic solvents is how to protect the biocatalyst from denaturation by such an unnatural environment. A number of approaches have been developed to overcome this obstacle [3]. Many of them are based on the simple idea that denaturation can be prevented by spatial separation of the organic solvent on a macrolevel or microlevel inside the limits of a non-aqueous reaction system. This approach is realized, for example, in systems of reversed surfactant micelles in nonpolar organic solvents [4, Abbreviations. SMPN, surface-modified polymeric nanogranules ; AOT, sodium bis(2-ethylhexyl sulfosuccinate); AcTyrEt, N-acetyl-L-tyrosine ethyl ester; AcTyr, N-acetyl-L-tyrosine; TLC, thin-layer chromatography; Pluronic F-108, poly(ethy1ene oxide)-poly(propy1ene oxide)-poly(ethy1ene oxide) triblock copolymer.Enzyme. a-Chymotrypsin (EC 3.4.21.1).in this way the unfavorable direct contact with the surrounding organic solvent is avoided. Enzyme-containing reversed micellar systems seem to be quite promising in many respects [4,5]. However, their practical application is severely restricted by considerable difficulties in enzyme and especially reactionproduct recovery caused by the presence of high concentrations of low-molecular-mass surfactants which are notoriously resistant to any separation proce...

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The catalytic activity and stability of laccase in the detergentless ternary system hexane - 2-propanol - water were studied. In the microemulsion region of the phase diagram the enzyme exhibited the highest catalytic activity and stability, which were comparable to those in aqueous solution. The character of the microenvironment inside microemulsion droplets was studied using nitrate anion as spectral probe and its polarity was found to correspond to that of 75% v/v aqueous 2-propanol. In principle, laccase can be recovered many times from the microemulsion without loss of catalytic activity.