Andrei V. Levashov

The principles of enzymes stabilization: VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents☆

1. The possibility of stabilizing water-soluble enzymes against the inactivation action of organic solvents by means of surfactants has been studied. Several enzymes (alpha-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity. 2. A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of charges in the substrate molecule and on interphase (++, +-, --). 3. The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.

The Kinetic Theory and the Mechanisms of Micellar Effects on Chemical Reactions

A few years ago we suggested, as an explanation of micellar effects on chemical reactions, a comprehensive kinetic theory which takes into consideration a partition of the reagents between the bulk and micellar “phases”, the simultaneous course of the reaction in the two phases and the shift of the apparent ionization constant of one of the reagents under the action of the surface micelle charge. In terms of this theory, from “surfactant concentration versus overall rate” profiles one can obtain partition coefficients of reagents between the bulk and micellar phases and a true rate constant of the reaction going in a micellar medium. That the kinetic equations are true is confirmed by the fact that the partition coefficients obtained in this way are in conformity with the values obtained by other methods (gel filtration, solubilization, spectrophotometric titration, etc.).

Enzymes Entrapped Into Reversed Micelles Of Surfactants In Organic Solvents: Key Trends In Applied Enzymology (Biotechnology)

This paper discusses applications of enzymes solubilized by surfactants in organic solvents to fine organic syntheses, in clinical and chemical analysis, and in therapy, as well as some future trends in biotechnology.

A new strategy for the study of oligomeric enzymes: γ-glutamyltransferase in reversed micelles of surfactants in organic solvents

A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.

Solubilization and refolding of inclusion body proteins in reverse micelles.

Today, many valuable proteins can be obtained in sufficient amounts using recombinant DNA techniques. However, frequently the expression of recombinant proteins results in the accumulation of the product in dense amorphous deposits inside the cells, called inclusion bodies. The challenge then is to transform these inactive and misfolded protein aggregates into soluble bioactive forms. Although a number of general guidelines have been proposed, the search for proper reconstitution conditions can be very laborious and time consuming. Here, we suggest a new versatile approach for solubilization and refolding of inclusion body proteins using a water-sodium bis-2-ethylhexyl sulfosuccinate-isooctane reverse micellar system. Instead of amorphous aggregates, a transparent solution is obtained, where refolded protein is entrapped inside the micelles. The entrapped enzyme has native-like secondary structure and catalytic activity. This approach has been implemented with Fusarium galactose oxidase and Stigmatella aurantiaca putative galactose oxidase.

Fixation of a highly reactive form of α-chymotrypsin by micellar matrix

Using reversed micelles of surfactants solvated by water‐organic cosolvent mixtures as a matrix for enzyme entrapping, it is possible to fix the highly reactive α‐chymotrypsin form. The reactivity of α‐chymotrypsin towards nonspecific substrates increases to the extent comparable with that observed in reactions involving specific substrates.

Binding-catalysis relationship in α-chymotrypsin action as revealed from reversible inhibition study of phenylalkylboronic acids

Studies of the reversible inhibition of a-chymotrypsin (CT) by n-alkylboronic acids have supplied important Information on the topography of the enzymes active centre [1, 2]. The hydrocarbon moiety of these bifunctional inhibitors interacts with the hydrophobic region of the CT active centre, whereas the borate grouping forms a complex with its catalytic functional groups. The following facts point to participation of the catalytically active histidine-57 residue in the complex formation: (1) both HaB03 [3] as well as the alkylboronic acids [1 ,2] , and also phenylboronic [4] and phenylethylboronic [5] acids are reversible, pH-dependent inhibitors of CT, the pK a of the protein group controlling the degree of inhibition being 6.5-7; (2) HaBO a competes with Cu 2+ ions for binding by the CT active centre [6] ; also it has been unequivocally demonstrated that Cu 2÷ ions interact with the activesite imidazole [7, 8] ; (3) HaBO3 reversibly inhibits alkylation of the catalytically active imidazole by 1,4-dibromo-2-phenylacetoin [9, 10]. In the light of present knowledge of borate complex chemistry (see e.g. [11 ]) and of the structure and mode of action of the CT active centre, these facts have led to the proposal that the OH group of the serine-195

CATALYSIS BY LACCASE (FROM Coriolus uersicolor) IN MICROHETEROGENEOUS MEDIA OF THE WATER/ORGANIC SOLVENT/SURFACTANT TYPE

Catalysis by laccase from Coriolus uersicolor solubilized in the ternary systems of surfactant/water/organic solvent type, namely, Aerosol OT/water/octane, Brij 56/water/cyclohexane and egg lecithin/water/octane + pentanol + methanol mixture, has been studied. The laccase activity is found to depend, in principle, not only on the water/surfactant molar ratio, but on the surfactant concentration (with its hydration degree being constant) as well. The following inferences should be emphasized. Firstly, in all the systems under study, the catalytic activity (kcat) of laccase entrapped into surfactant reversed micelles increases more than 50 times (when the surfactant concentration is extrapolated to zero) compared with the kcat value in aqueous solution. Secondly, the catalytic activity (kcat) of laccase entrapped in hydrated Aerosol OT aggregates, having lamellar, reversed cylindrical (hexagonal) and reversed micellar structure, depends greatly on the aggregate type. In other words, the phase transitions, i...

On the modes of interaction between competitive inhibitors and the α-chymotrypsin active centre

The “binding specificity” of cwchymotrypsin (CT) for different amino acid side-chains has been postulated to arise simply from a hydrophobic interaction between enzyme and substrate [l] . Any present day binding model of the enzyme-substrate or enzyme-inhibitor complex formation should take into account correlations between affinity of substrate or inhibitor for the enzyme and parameters relevant to the hydrophobic characteristics of these compounds [l-3] . The most correct approach to the study of hydrophobic interaction is that of comparing the free energy of process (1):

Applied Aspects of Micellar Enzymology

Applications of enzyme catalysis in different practical fields can be widened on the basis of the novel scientific trend, micellar en~ymology.’-~ Micellar enzymology uses, as reaction medium, the colloidal solution composed of surfactant, water, and organic solvent. The remarkable feature of such microheterogeneous systems is their ability to dissolve both polar and nonpolar compounds. Polar compounds, including water, are incorporated into inner cavities of reversed micelles (FIG. 1). The dimensions of the aqueous cavity and its physicochemical properties depend on the degree of surfactant hydration; the latter value can be easily varied. The location of the solubilized enzyme molecule in the interior of the reversed micelle depends on the nature of the enzyme: water-soluble enzymes are buried in the aqueous cavity; surface-active enzymes tend to contact with the layer formed by polar surfactant groups; and finally, hydrophobic (membrane) enzymes can interact with the organic solvent composing the bulk phase. Nonpolar substances are either dissolved in the bulk (organic) phase or can be embedded in the outer shell of the micelle (FIG. 1) . Micellar systems exhibit highly dynamic behavior, thus providing conditions for the rapid exchange of reagents. Some particular examples will be discussed below to demonstrate the advantages of reversed micellar enzyme-containing systems. In fine organic synthesis such systems provide the possibility of the biocatalytic conversion of water-insoluble compounds, such as steroids, prostaglandins, alkaloids, lipids, etc.”” The most illustrative example of the process is, in our view, the stereospecific reduction of steroids by molecular hydrogen or electrochemically with the use of a multienzyme system with cofactor regeneration.**’ Second, in the conventional medium for enzymatic reactions, i.e. in water, the equilibrium of many practically important reactions is strongly shifted in favor of initial reactants. This refers first of all to such processes where the initial reactants are ionized and for this reason are strongly hydrated, or else one of the products is water, as in the polymerization of carbohydrates and amino acids (peptide synthesis), in dehydration reactions, etc. This unfavorable situation may be improved by using a reaction medium with a low water content. The reversed micellar systems represent a possible version of such a medium.’ For example, the oxidation of isobutanol to the respective aldehyde, catalyzed by alcohol dehydrogenase in reversed micelles of sodium diisooctyl sulfosuccinate (Aerosol OT, AOT) in octane is characterized by the equilibrium constant differing from that in aqueous solution by the factor of 106.12 The same idea has been recently used to carry out peptide synthesis in the reversed micellar m e d i ~ m . ’ ~