Time-resolved fluorescence and circular dichroism of porphyrin cytochrome c and Zn-porphyrin cytochrome c incorporated in reversed micelles

Interactions between fluorescent horse heart cytochrome c derivatives (e. g. porphyrin cytochrome c and Zn-porphyrin cytochrome c) with surfactant interfaces in reversed micellar solutions have been studied, using different spectroscopic techniques. Anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] and cationic (cetyltrime-thylammonium bromide, CTAB) surfactant solutions have been used in order to investigate the effects of charge interactions between proteins and interfaces. Circular dichroism reveals that much of the protein secondary structure is lost in AOT-reversed micelles, especially when the molar water/surfactant ratio, wo, is high (wo = 40), whereas in CTAB-reversed micelles secondary structure seems to be preserved. Time-resolved fluorescence measurements of the porphyrin in the cytochrome c molecule yields information about the changes in structure and the dynamics of the protein upon interaction with surfactant assemblies both in aqueous and in hydrocarbon solutions. With AOT as surfactant a strong interaction between protein and interface can be observed. The effects found in aqueous AOT solution are of the same kind as in hydrocarbon solution. In the CTAB systems the interactions between protein and surfactant are much less pronounced. The measured effects on the fluorescence properties of the proteins are different in aqueous and hydrocarbon solutions. In general, the observations can be explained by an electrostatic attraction between the overall positively charged protein molecules and the anionic AOT interface. Electrostatic attraction can also occur between the cytochrome c derivatives and CTAB because there is a negatively charged zone on the surface of the proteins. From the fluorescence anisotropy decays it can be concluded that in the CTAB-reversed micellar system these interactions are not important, whereas in an aqueous CTAB solution the proteins interact with surfactant molecules.     

Spectroscopic properties of horse liver alcohol dehydrogenase in reversed micellar solutions

Catalytic and spectroscopic properties of alcohol dehydrogenase from horse liver, incorporated in reversed micellar media, have been studied. Two different reversed micellar systems have been used, one containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT], the other containing a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. With 1-hexanol as substrate the turnover number of the enzyme in AOT-reversed micelles is strongly dependent on the water content of the system. At low wo ([H2O]/[surfactant]) (wo less than 20) no enzymatic activity can be detected whereas at high wo (wo = 40) the turnover is only slightly lower than in aqueous solution. In CTAB-reversed micelles the dependence of the turnover number on wo is much less. The enzymatic activity is in this case significantly lower than in aqueous solution and increases only slightly with an increasing water content of the reversed micelles. Possible interactions of the protein with the surfactant interfaces in the reversed micellar media were studied via circular dichroism and fluorescence measurements. From the circular dichroism of the protein backbone it is observed that the protein secondary structure is not significantly affected upon incorporation in the reversed micelles since the far-ultraviolet spectrum is not altered. Results from time-resolved fluorescence anisotropy experiments indicate that, especially in AOT-reversed micelles, interactions between the protein and the surfactant interface are largely electrostatic in nature, as evident from the dependence on the pH of the buffer used. In CTAB-reversed micellar solutions such interactions appear to be much less pronounced than in AOT.     

Fluorescence of hemoproteins in reversed micelles of surfactants in octanes

The fluorescence of myoglobin, cytochromes b5 and c in the reversed aerosol OT (AOT) micelles in octane has been investigated. The fluorescence intensity of all the three hemoproteins is higher than that in aqueous solutions. The maxima and intensities of fluorescence in the AOT micelles depend on the [H2O]/[AOT] ratio and reflect the protein structure. Aliphatic alcohols and secondary amines (piperidine and morpholine) quench the cytochrome c fluorescence in the AOT micelles, whereas dipolar aprotic solvents (dimethylsulfoxide, dimethylformamide) significantly increase the intensity of cytochrome c fluorescence in the same micelles. The transformations of the proteins solubilized by the reversed micelles of a surfactant are discussed.     

The effect of reversed micelles of cetyltrimethylammonium bromide on equilibrium constants and kinetics of oxidation-reduction reactions with the participation of cytochrome c

To model the effect of membrane environment on the electron transfer reactions we studied the thermodynamics and kinetics of the reactions of cytochrome c and 2,6-dichlorophenolindophenol with ferri- and ferrocyanide in the reversed micelles cetyltrimethylammonium bromide in chloroform/octan mixture. Incorporation of the protein in micelles leads to increasing the equilibrium constant (K1) up to 300 times. This effect is mainly due to a decrease in the ferrocytochrome c oxidation rate constant in the reaction with ferricyanide. Micellar solubilization of the dye also leads to a marked increase in the equilibrium constant K2. The estimations of the values K1 and K2 in water-alcohol mixtures and in aqueous micellar solutions of surfactant together with kinetical and spectral data show that the increase of K1 and K2 in reversed micelles is caused generally by redox potential changes of low-molecular reagents. The latter change their environment after adsorption on the micellar surface.     

Mechanism of extraction of chymotrypsin into isooctane at very low concentrations of aerosol OT in the absence of reversed micelles

Chymotrypsin is easily extracted from an aqueous solution into isooctane containing the anionic surfactant aerosol OT (AOT). The concentration of AOT needed to efficiently extract 0.5 mg/mL CMT is as low as 1 mM and as low as 0.2 mM AOT was sufficient to extract the protein into isooctane. The extraction process was unaffected by 10% (v/v) ethyl acetate in the isooctane phase. Moreover, spectroscopic analysis by electron paramagnetic resonance indicated that CMT did not exist inside a discreet water pool of a reversed micelle. Calculations of the number of AOT molecules associated per extracted CMT molecule indicate that only ca. 30 surfactant molecules interact with the protein, a value too low for reversed micellar incorporation of the protein in isooctane. These studies suggested that reversed micelles do not need to be involved in the actual transfer of the protein from the aqueous to the organic phase and protein solubilization in the organic phase is possible in the absence of reversed micelles. Based on these findings, a new mechanism has been proposed herein for protein extraction via the phase transfer method involving ionic surfactants. The central theme of this mechanism is the formation of an electrostatic complex between CMT and AOT at the aqueous/organic interface between AOT and CMT, thereby leading to the formation of a hydrophobic species that partitions into the organic phase. Consistent with this mechanism, the efficiency of extraction is dependent on the interfacial mass transfer, the concentrations of CMT and AOT in the aqueous and organic phases, respectively; the ionic strength of the aqueous phase; and the presence of various cosolvents. (c) 1994 John Wiley & Sons, Inc.     

Kinetic Studies on the Interaction of Ferricytochrome c with Anionic Surfactants

The kinetics of absorbance and fluorescence changes of cytochrome c as induced by an aqueous solution of the anionic surfactant sodium dodecyl sulfate (SDS) or sodium bis(2-ethylhexyl)sulfosuccinate (AOT) are studied. The results are compared with far-UV circular dichroism (CD) spectra. Both surfactants cause similar alterations in the secondary structure of cytochrome c, while their influence on the heme environment of cytochrome c is different. In the presence of AOT below and above critical micellar concentration a conversion of the low-spin native cytochrome c to a denatured low-spin protein not having methionine ligand takes place. In the presence of SDS micelles conversion of the native protein to a denatured mixed-spin form occurs. The changes in the heme group induced by both surfactants occur independently of the alterations in tertiary structure.     

Protein refolding in reversed micelles: Interactions of the protein with micelle components

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, gamma-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since gamma-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.     

Enzyme kinetics in reversed micelles. 3. Behaviour of 20 beta-hydroxysteroid dehydrogenase

The kinetic parameters of 20 beta-hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine 'apparent' Michaelis constants and V values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium-di(ethylhexyl)sulphosuccinate-reversed micelles and the observed low activity in Triton-reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles.     

Hydrogen-bonding modulation of excited-state properties of flavins in a model of aqueous confined environment

The singlet and triplet excited states properties of lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in reversed micelles (RM) of sodium docusate (AOT) in n-hexane solutions were evaluated as a function of the water to surfactant molar ratio, w(0) = [H(2)O]/[AOT], by both steady-state and time-resolved absorption and fluorescence spectroscopy. The results indicated that hydrogen-bonding interactions between the isoalloxazine ring of the flavins with the water molecules of the micellar interior play a crucial role on the modulation of the excited state properties of the flavins. Fluorescence dynamic experiments in the RM, allowed the calculation of similar values for both the internal rotational time of the flavins (θ(i)) and the hydrogen-bonding relaxation time (τ(HB)), e.g.≈ 7 and 1.5 ns at w(0) = 1 and 20, respectively. In turn, the triplet state lifetimes of the flavins were also enlarged in RM solutions at low w(0), without modifications of their quantum yields. A hydrogen bonding relaxation model is proposed to explain the singlet excited state properties of the flavins, while the changes of the triplet state decays of the flavins were related with the global composition and strength of the hydrogen bonding network inside of the RM.     

Protein refolding in reversed micelles

A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant-free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.     

Extraction of lysozyme and ribonuclease-a using reverse micelles: Limits to protein solubilization

Experiments are reported here on the equilibrium partitioning of lysozyme and ribonuclease-a between aqueous and reversed micellar phases comprised of an anionic surfactant, sodium di-2-ethylhexyl sulfosuccinate (AOT), in isooctane. A distinct maximum, [P](rm,max) was found for the quantity of a given protein that can be solubilized in the reverse micelle phase by the phase-transfer method. This upper limit depended upon the size of the protein, the surfactant concentration, and the aqueous phase ionic strength, and was determined by complex formation between protein and surfactant molecules to form an insoluble interfacial precipitate at high values of [P](rm). In this work, it was found to be possible to dissociate the protein-surfactant complex and recover the precipitated protein. The kinetics of protein-surfactant complex formation depended upon the nature and concentration of the solubilized protein and on the surfactant concentration. Calculations of micellar occupancy and the relative surface areas of protein molecules and surfactant head-groups suggested that it was the exposure of the solubilized protein to the bulk organic solvent which promoted protein-surfactant complex formation as [P](rm) --> [P](rm,max). In the light of the experimental results and calculations described above, a mechanistic model is proposed to account for the observed phenomena. This is based upon the competing effects of increasing the solubilized protein concentration and the corresponding increase in the rate of protein-surfactant complex formation. The dynamic nature of the reverse micelles is inherent in the model, explaining the formation of the interfacial precipitate with time and its dependence on the internal phase volume of the micellar phase. Experiments on the co-partitioning of water and measurement ofthe AOT concentration in both phases verified the loss of protein, water, and surfactant from the organic phase at high values of [P](rm). (c) 1995 John Wiley & Sons Inc.     

Enzyme kinetics in reversed micelles. 2. Behaviour of enoate reductase

Enoate reductase (EC 1.3.1.31) can stereospecifically reduce a variety of alpha,beta-unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2-methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution. In aqueous solution the enzyme obeys a ping pong mechanism [Bühler et al. (1982) Hoppe-Seyler's Z. Physiol. Chem 363, 609-625]. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2-methylbutenoic acid are 20 microM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2-methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction. In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5-5 mM, implying that a concentration of substrate equal to the Km value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2-methylbutenoic acid concentrations.     

Solubilizing water involved in protein extraction using reversed micelles

The extraction of protein using reversed micelles was investigated in relation to the amount of solubilizing water in the reversed micellar organic phase. The minimal concentration of amphiphilic molecule di-2-ethylhexyl sodium sulfosuccinate (C(20)H(37)O(7)Na) (AOT) required for 100% cytochrome c extraction was recognized. This critical AOT concentration increased with protein concentration in the aqueous phase. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein was found to be a constant of 3500 under C(KCI) = 1.0 x 10(2) mol . m(-3) in this system. This ratio means the hydrophillic surroundings required for extracting one protein molecule into the micellar organic phase under the suitable pH and salt concentration for the forward extraction. In this regard, AOT molecules seemed to take the part of water solubilizing agent in the reversed micellar extraction. This role of AOT is important to extract protein under the suitable pH and salt concentration. The amount of solubilizing water in the protein-containing system was larger than in the protein-free system. This difference shows that the water molecules accompany the extracted protein into the reversed micellar organic phase at constant ratio 2200 under C(KCI) = 1.0 x 10(2) mol . m(-3), i.e., accompanying water molecules per one extracted protein. The minimal AOT concentration increased with ionic strength. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein also increased with ionic strength, so that in higher ionic strength, more solubilizing water was required. Then more AOT was required to provide the hydrophillic surroundings for protein. The pH affected the minimal AOT concentration required for 100% protein extraction.     

Optimization of Peroxidase-Catalyzed Oxidation of Luminol in Reversed Micelles of Surfactants in Octane

Luminol oxidation in the Aerosol OT (AOT) reversed micelles in octane catalyzed by horseradish peroxidase (HRP), or its conjugate with Cortisol (HRP-COR), was optimized. The chemiluminescence intensity during luminol oxidation was strongly dependent on the method of preparation of the reaction mixture and the addition of Triton X-45, cyclohexanol and the chemiluminescence “enhancer”, p-iodophenol, into the micellar system. Five procedures for the preparation of the reaction mixture were compared. The maximum chemiluminescence was observed in the micellar system containing all the reaction components excluding a biocatalyst, addition of which into the system started the reaction. Triton X-45, cyclohexanol or p-iodophenol added to the micellar system enhanced significantly the chemiluminescence intensity. The “enhancing” action of p-iodophenol in AOT reversed micelles was 10-fold less than in an aqueous medium.     

Forward and backward extraction rates of amino acid in reversed micellar extraction

The mass transfer characterization in reversed micellar extraction of amino acid phenylalanine (Phe) is presented. The mass transfer rates in forward extraction of Phe from aqueous KCl solutions (pH 1.4 – 2.3) to AOT/isooctane reversed micellar solutions and in backward extraction from the reversed micellar organic phase to KHCO₃/KOH buffer solutions (pH 9.0 – 11.0) were investigated using a stirred cell with a flat liquid–liquid interface. Both the forward and the backward extraction rates are controlled by the interfacial rate processes, i.e., the solubilization and the release processes. The solubilizing rate constants for the forward extraction of Phe increase with decreasing pH and initial Phe concentration and with increasing initial AOT concentration. On the other hand, the releasing rate constants for the backward extraction decrease with increasing initial AOT concentration and with decreasing ionic strength, but are little influenced by pH. The backward extraction rates are fairly slow compared to the forward extraction rates, and are accelerated by the addition of 2-methyl-2-propanol, similar to the extraction of protein lysozyme.     

Fluorescence quenching of beta-carboline alkaloids in micellar media. A study to select the adequate surfactant to use in analytical techniques.

The study of fluorescence quenching of the fluorophores allows the localization of the alkaloids (harmane and harmine) in the micelles (SDS, CTAB, Brij-35) to be established. In aqueous micellar solutions (SDS and Brij-35) at pH 13.0, emission corresponding to the neutral or zwitterionic forms can be observed. In the presence of CTAB (pH = 13.0) it was possible to observe the emission of anionic form. These species are not present in buffered aqueous solutions at these pH values. Bromide ion was added to the different surfactant solutions and the quenching effect was studied according to the Stern-Volmer equation. In the presence of SDS the quenching effect is considerably reduced compared to the aqueous solutions without surfactants, while for Brij-35 micelles were similar to those observed in homogeneous aqueous solution. For CTAB micelles a notable fluorescence quenching was observed for the different pH values studied. The fluorescence quenching studies show that the neutral species are associated inside the micelles, instead of the ionic species (cationic, zwitterionic or anionic) remaining on the surface of the micelles. The anionic surface of SDS micelles prevents the quenching effect by anionic quenchers for both neutral and charged species.     

Quenching of the triplet state of Safranine-O by aliphatic amines in AOT reverse micelles studied by transient absorption spectroscopy

The photophysics of Safranine-O (3,6-diamino-2,7-dimethyl-5 phenyl phenazinium chloride) (SfH(+)Cl(-)) was investigated in reverse micelles (RMs) of AOT (sodium bis(2-ethylhexyl)sulfosuccinate) with special emphasis on the triplet state processes. The triplet is formed in its monoprotonated form, independently of the pH of the water used to prepare the RMs. While the intersystem crossing quantum yields in RMs are similar to those in organic solvents, the triplet lifetime is much longer. Since the pH in the water pool of AOT RMs is close to 5 and the triplet state of the dye is subjected to proton quenching, the long lifetime indicates that the dye resides in a region where it cannot be reached by protons during its lifetime. All the measurements indicate that the dye is localized in the interface, sensing a medium of micropolarity similar to EtOH : water (3:1) mixtures. The quenching by aliphatic amines was also investigated. While the quenching by the hydrophobic tributylamine is similar to that in methanol, the hydro-soluble triethanolamine is one order of magnitude more effective in RMs than in homogeneous solution. In the latter case the quenching process is interpreted by a very fast intramicellar quenching, the overall kinetics being controlled by the exchange of amine molecules between RMs. Semireduced dye is formed in the quenching process in RMs in the di-protonated state with a comparable quantum yield to the monoprotonated state formed in homogeneous solvents. The results point to the advantage of the reverse micellar system for the generation of active radicals for the initiation of vinyl polymerization, since a much lower concentration of amine can be employed with similar quantum yields.     

Catalysis by enzymes entrapped into reversed micelles of surfactants in organic solvents. Peroxidase in the aerosol OT-water-octane system

Spectral and catalytic parameters of peroxidase solubilized in the aerosol OT-water-octane system have been studied. The spectrum of peroxidase solubilized in octane with AOT reversed micelles, a degree of surfactant hydration being above 12, is actually identical to that of the enzyme aqueous solution. On the other hand, significant spectral changes have been detected when transferring the enzyme from water to the reversed micelle medium at low degrees of surfactant hydration, precisely [H2O]/[AOT] less than 12. The reversed micelle-entrapped peroxidase catalyses the oxidation of pyrogallol with hydrogen peroxide much more actively (at [H2O]/[surfactant] approximately 13) than that in aqueous solution. The entrapment of peroxidase into surfactant reversed micelles increases precisely the catalytic constant of the reaction, i.e. the virtual reactivity of the enzyme increases ten and hundred times depending on degrees of surfactant hydration and concentration. The systems of reversed micelles may be considered as models of biomembranes. Our findings hence show that enzymes in vivo can be much more catalytically active then it appears possible to reveal in conventional experiments in vitro in aqueous solutions.     

Important parameters affecting efficiency of protein refolding by reversed micelles

Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W(o) value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W(o)) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.     

Activation of lignin peroxidase in organic media by reversed micelles

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media.