Protein extration from an aqueous phase into a reversed micellar phase: Effect of water content and reversed micellar composition

In the system composed of the cationic surfactant TOMAC (10 mM), the nonionic (co)surfactant Rewopal HV5 (2 mM), and octanol (0.1% v/v) in isooctane, reversed micelles are formed upon contact with an aqueous phase containing 50 mM ethylene diamine. alpha-Amylase can be transferred from the aqueous phase into reversed micelles in the pH range 9.5 to 10.5 and re-extracted into a second aqueous phase of different composition. The size of the reversed micelles (as reflected in the water content of the organic phase) can be varied by changes in percentage of octanol, type of counterion in the aqueous phase, or in the number of ethoxylate head groups of the nonionic surfactant. An increase in size results in transfer at lower pH values. Experiments in which the charge density in the reversed micellar interface was changed by incorporation of charged derivatives of the nonionic surfactant, without influencing the water content, revealed that an increased charge density facilitated transfer, resulting in a broader transfer profile. Replacement of TOMAC by other quaternary ammonium surfactants differing in number and length of tails revealed that, of the 14 surfactants tested, only 2 gave appreciable amounts of transfer. The amount of transfer is related to the dynamics of phase separation of the surfactants: those giving a poor phase separation inactivate the enzyme. This inactivation is caused by electrostatic interactions between the charged surfactant head groups and charged groups on the enzyme. Electrostatic interactions are the first step of transfer, and can result in either incorporation in a reversed micelle, or, if reversed micelle formation is slow, in enzyme inactivation. (c) 1995 John Wiley & Sons, Inc.     

Catalytic activity of steroid conjugates of various types of peroxidases in aqueous and micellar media

The horseradish peroxidase (HRP) conjugates with the sheep antirabbit antibodies and cortisol (COR) or progesterone (PROG) containing 9 to 40 steroid molecules per HRP molecule were synthesized. In aqueous media all the conjugates have lower catalytic activity in the o-phenylenediamine oxidation than the native enzyme. In reversed Aerosol OT micelles in heptane the HRP-COR and HRP-PROG conjugates containing 12 and 9 steroid molecules, respectively, have catalytic constants 2.6 and 2.7 times higher than the unmodified enzyme. The influence of the HRP hydrophobisation and its inactivation on the course of modification on catalytic properties of the enzyme is discussed.     

Catalytic activity of conjugates of peroxidase with progesterone in aqueous and micellar media in the presence of antibodies against progesterone

At 22 and 41 degrees C the horseradish peroxidase (HRP) conjugates with progesterone, HRP-PROG-1 and HRP-PROG-2, were obtained by HRP reaction with N-hydroxysuccinimide ester of 3-O-carboxymethyloxime of progesterone. The interaction of these conjugates and unmodified enzyme with antibodies against progesterone was studied in phosphate-citrate buffer pH 3.6 and in the reversed micelles of Aerosol OT and Triton X-45 in heptane. Catalytic function of conjugates in buffered solutions and in reversed micelles increases as result of the conjugates' interaction with antibodies. The free progesterone in reversed micelles competes with HRP-PROG-1 for antibody binding, which may be the basis for homogeneous enzyme-immunoassay of progesterone, depending on the micelle composition, the antibody concentration and temperature.     

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.     

Reverse micelles‐mediated transport of lipase in liquid emulsion membrane for downstream processing

This work deals with the downstream processing of lipase (EC 3.1.1.3, from Aspergillus niger) using liquid emulsion membrane (LEM) containing reverse micelles for the first time. The membrane phase consisted of surfactants [cetyltrimethylammonium bromide (CTAB) and Span 80] and cosolvents (isooctane and paraffin light oil). The various process parameters for the extraction of lipase from aqueous feed were optimized to maximize activity recovery and purification fold. The mechanism of lipase transport through LEM consisted of three steps namely solubilization of lipase in reverse micelles, transportation of reverse micelles loaded with lipase through the liquid membrane, and release of the lipase into internal aqueous phase. The results showed that the optimum conditions for activity recovery (78.6%) and purification (3.14‐fold) were feed phase ionic strength 0.10 M NaCl and pH 9.0, surfactants concentration (Span 80 0.18 M and CTAB 0.1 M), volume ratio of organic phase to internal aqueous phase 0.9, ratio of membrane emulsion to feed volume 1.0, internal aqueous phase concentration 1.0 M KCl and pH 7.0, stirring speed 450 rpm, and contact time 15 min. This work indicated the feasibility of LEM for the downstream processing of lipase. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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.     

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.     

Mixed micelles and other structures in the solubilization of bilayer lipid membranes by surfactants

The solubilization of lipid bilayers by surfactants is accompanied by morphological changes of the bilayer and the emergence of mixed micelles. From a phase equilibrium perspective, the lipid/surfactant/water system is in a two-phase area during the solubilization: a phase containing mixed micelles is in equilibrium with bilayer structures of the lamellar phase. In some cases three phases are present, the single micelle phase replaced by a concentrated and a dilute solution phase. In the case of non-ionic surfactants, the lipid bilayers reach saturation when mixed micelles, often flexible rod-like or thread-like, start to form in the aqueous solution, at a constant chemical potential of the surfactant. The composition of the bilayers also remains fixed during the dissolution. The phase behavior encountered with many charged surfactants is different. The lamellar phase becomes destabilized at a certain content of surfactant in the membrane, and then disintegrates, forming mixed micelles, or a hexagonal phase, or an intermediate phase. Defective bilayer intermediates, such as perforated vesicles, have been found in several systems, mainly with charged surfactants. The perforated membranes, in some systems, go over into thread-like micelles via lace-like structures, often without a clear two-phase region. Intermediates in the form of disks, either micelles or bilayer fragments, have been observed in several cases. Most noteworthy are the planar and circular disks found in systems containing a large fraction of cholesterol in the bilayer. Bile salts are a special class of surfactants that seem to break down the bilayer at low additions. Originally, disk-like mixed micelles were conjectured, with polar membrane lipids building the disk, and the bile salts covering the hydrophobic rim. Later work has shown that flexible cylinders are the dominant intermediates also in these systems, even if the disk-like structures have been re-established as transients in the transformation from mixed micelles to vesicles.     

Development of a new reversed micelle liquid emulsion membrane for protein extraction

A new type of liquid emulsion membrane containing reversed micelles for protein extraction is introduced. A three-step extraction mechanism is proposed including solubilization, transportation, and release of the protein. The surfactants Span80 and sodium di(2-ethylhexyl)sulfosuccinate (AOT) are used to stabilize the membrane phase and to build up the reversed micelles, respectively. alpha-Chymotrypsin was used as the model protein. The condition in the internal phase inhibits the solubilization process of the already extracted protein back into reversed micelles. Concerning the solubilization, we studied the influence of the AOT concentration in the membrane phase and the ionic strength in the external phase. The extraction rate increases with higher AOT concentration and decreases with higher ionic strength. Using NaCl in the external phase led to better extraction results than using KCl. Maximum extraction results of 98% into the membrane phase and 65% into the internal phase were obtained. This condition retained 60% of the enzyme's activity. The concentration of KCl in the internal phase does not affect the solubilization rate but the release into the internal phase. By this way the ionic strength in the internal phase is used as the driving force for the protein release. The solubilization process is much faster than the diffusion and the releasing process, as found by variation of the extraction time. The influence of the operating conditions on the membrane swelling is also discussed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 267-273, 1997.     

Protein refolding by reversed micelles utilizing solid-liquid extraction technique

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates.     

Mixed reverse micelles facilitated downstream processing of lipase involving water‐oil‐water liquid emulsion membrane

Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water‐oil‐water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0‐fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1–17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream processing of enzymes/proteins. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1084–1092, 2014     

Purification of lysozyme by affinity-based reversed micellar two-phase extraction

A dye-affinity reversed micellar system was used for lysozyme purification from a crude solution of chicken egg white. The dye-affinity reversed micelles consisted of Cibacron Blue F-3GA (CB; 0.1 mM) modified lecithin (50 g/l) in n-hexane. Starting with a crude egg white solution containing lysozyme of 0.0381 mg/mg protein, lysozyme purity was increased by 16 to 20 times, reached 0.62 to 0.76 mg/mg protein. The affinity micellar system was recycled and used three times. Addition of polyoxyethylene (20) sorbitan trioleate (Tween 85) as a cosurfactant could increase the capacity of the affinity-based reversed micelles. A lysozyme recovery yield of over 70% was obtained at a forward aqueous phase pH of 9.16 using the reversed micelles additionally containing 20 g/l of Tween 85.     

反胶束萃取胰蛋白酶的研究

本文以含有反胶束的有机溶剂作为萃取剂,进行了将胰蛋白酶从水相传入有机相,再从有机相传入另一水相的研究。结果表明：影响萃取率的主要因素为水相ｐＨ值、离子强度和种类,以及反胶束溶液中表面活性剂浓度等;在适宜的条件下,酶的单级萃取和反萃取率都很高,显示了良好的工业应用前景。     

Protein transfer from an aqueous phase into reversed micelles. The effect of protein size and charge distribution

Proteins are spontaneously transferred from an aqueous solution into reversed micelles, provided the aqueous phase has the proper composition. Besides the composition of the aqueous phase, the composition of the organic phase and the properties of the proteins also play a role. We studied uptake profiles of 19 proteins as a function of pH of the aqueous solution. The organic phase consisted of trioctylmethylammonium chloride and nonylphenol pentaethoxylate (Rewopal HV5) as surfactant, octanol as cosurfactant and isooctane as continuous phase. In all cases, except for rubredoxin, proteins were transferred at pH values above their isoelectric point. The pH where maximal solubilization takes place can be described by the relationship: pHoptimum = isoelectric point +0.11 x 10(-3) Mr -0.97. So, the larger the protein, the more charge is needed to provide the energy required for the adaptation of the micellar size to the protein size. For protein transfer into sodium di-(2-ethylhexyl)sulphosuccinate (AOT) reversed micelles a similar relationship was found. The percentage of protein transferred could be related to the symmetry of charge distribution over the protein. This symmetry was expressed as the % of random electric moments on a protein that is larger than the effective electric moment of the protein (% S) [Barlow, D. J. and Thornton, J. M. (1986) Biopolymers 25, 1717]. The larger the value of % S, the more homogeneously the charges are distributed and the lower the percentage transfer.     

Catalytic and structural properties of surfactant-horseradish peroxidase complex in organic media

A surfactant-horseradish peroxidase (HRP) complex that is catalytically active in organic media has been successfully prepared by a method utilizing water-in-oil (W/O) emulsions. To optimize conditions for preparation of the HRP complex, the effects of some key parameters in the aqueous phase of W/O emulsions were investigated. The surfactant-HRP complex prepared with a nonionic surfactant exhibited a high catalytic activity compared to those with a cationic or anionic surfactant in anhydrous benzene. At the preparation step, the pH of the aqueous solution had a prominent effect on the enzymatic activity of the HRP complex in organic media. Several kinds of salts present in the HRP complex could be employed to enhance the catalytic performance in organic media. However, anionic ions present in the preparation process appeared to lower the catalytic activity owing to the complexation with heme iron. UV-visible absorption spectra of the HRP complex in benzene, which were prepared from a KCN solution (pH 7.0) or an alkaline solution (pH 12), were comparable with those of native HRP in aqueous solution under the same conditions. Resonance Raman spectroscopic studies also revealed that no significant change in the coordination state of the heme iron occurred even after coating the enzyme with surfactant molecules, lyophilization, and solubilization in nonaqueous media.     

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.     

Design of surfactants suitable for protein extraction by reversed micelles

New surfactants have been synthesized for potential use in reversed micellar protein extraction operations. Preferential solubility of the surfactant in an aliphatic solvent such as hexane, heptane, or isooctane and the formation of reversed micelles accompanied with solubilization of significant quantities of water can be achieved by using strongly hydrophobic, twin alkyl chains as the hydrophobic moiety. Different surfactants having identical water-solubilizing capacities can have significantly different behavior in protein extractions, where extraction efficiency appears to be governed by the nature of the interfacial complex that forms between surfactants and proteins. Bulky surfactant chains provide a steric hindrance to the adsorption of the surfactant to the protein surface, thus inhibiting solvation of the protein/surfactant complex, and hence protein extraction. Under these conditions, a precipitate forms either in the bulk aqueous phase or at the interface. Surfactants that can form a close-packed complex with the protein are excellent protein-solubilizing agents. Dioleyl phosphoric acid (DOLPA) appears to be the best surfactant currently available for protein extraction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 26-32, 1997.     

Stabilization of diluted aqueous solutions of horseradish peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55 degrees C and infinite dilution, HRP was inactivated with a rate constant of 2.86 x 10(-3) s-1. CaCl2, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the "glycerol-based" one containing 10% ethanol and 20% glycerol, or the "protein-based" one containing 0.1% Kathon CG and 0.2 g/l of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl2 were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.     

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.     

Purification of intracellular enzymes from whole bacterial cells using reversed micelles

A new, rapid pre-chromatography isolation procedure for intracellular enzymes from whole bacterial cells has been developed using reversed micelles. The method involves two relatively simple steps. In the first step, bacterial cells are disintegrated by the surfactants in the reversed micellar medium, and in the second step the liberated enzymes are extracted from the reversed micellar phase into an aqueous phase. The feasibility of using reversed micelles as a bioseparation tool has been demonstrated by following the activities and recoveries of three different dehydrogenases from Azotobacter vinelandii.