Solubilization limit of lysozyme into DODMAC reverse micelles

Dioctyldimethyl ammonium chloride (DODMAC) was used to form reverse micelles and to extract lysozyme from an aqueous solution into an organic phase. The solubilization behavior of lysozyme into a DODMAC reverse micellar phase was examined in terms of the temperature, the type of cations in the aqueous phase, and the surfactant concentration in the organic phase. Complete removal of lysozyme from the aqueous phase was obtained when the pH was set one unit higher than the pI of the protein. However, it was found that there is a solubilization limit of lysozyme in the organic phase. Not all the lysozyme extracted out of the initial aqueous phase was solubilized into the DODMAC reverse micellar phase, resulting in the formation of white precipitate at the aqueous-organic interface. Temperature has a negligible effect on the solubilization limit of lysozyme. The value of the solubilization limit is a strong function of the type of cations present in the aqueous phase, indicating an important role of lysozyme-cation interactions on the extraction process. An increase in the DODMAC concentration from 100-200 mM resulted in little change in the highest concentration of lysozyme obtained in the organic phase.     

Kinetic behavior of Desulfovibrio gigas aldehyde oxidoreductase encapsulated in reverse micelles

We report the kinetic behavior of the enzyme aldehyde oxidoreductase (AOR) from the sulfate reducing bacterium Desulfovibrio gigas (Dg) encapsulated in reverse micelles of sodium bis-(2-ethylhexyl) sulfosuccinate in isooctane using benzaldehyde, octaldehyde, and decylaldehyde as substrates. Dg AOR is a 200-kDa homodimeric protein that catalyzes the conversion of aldehydes to carboxylic acids. Ultrasedimentation analysis of Dg AOR-containing micelles showed the presence of 100-kDa molecular weight species, confirming that the Dg AOR subunits can be dissociated. UV-visible spectra of encapsulated Dg AOR are indistinguishable from the enzyme spectrum in solution, suggesting that both protein fold and metal cofactor are kept intact upon encapsulation. The catalytic constant (k(cat)) profile as a function of the micelle size W(0) (W(0)=[H(2)O]/[AOT]) using benzaldehyde as substrate showed two bell-shaped activity peaks at W(0)=20 and 26. Furthermore, enzymatic activity for octaldehyde and decylaldehyde was detected only in reverse micelles. Like for the benzaldehyde kinetics, two peaks with both similar k(cat) values and W(0) positions were obtained. EPR studies using spin-labeled reverse micelles indicated that octaldehyde and benzaldehyde are intercalated in the micelle membrane. This suggests that, though Dg AOR is found in the cytoplasm of bacterial cells, the enzyme may catalyze the reaction of substrates incorporated into a cell membrane.     

Improving cutinase stability in aqueous solution and in reverse micelles by media engineering

The stability of a recombinant cutinase from the fungus Fusarium solani was evaluated in aqueous media and in reverse micelles. Thermal unfolding in aqueous solution is a two-state process at the pH values tested and trehalose increased the temperature at the mid-point of the unfolding transitions. Irreversible inactivation is a first-order process at pH 9.2, but two inactivation phases were resolved at pH 4.5. Trehalose did not change the irreversible inactivation pathway but increased the kinetics of the irreversible inactivation step. Unfolding of cutinase induced by guanidine hydrochloride was more complex, showing a stable intermediate, molten globule in character, within the transition region. Trehalose did not change the three-state nature of the unfolding process. Encapsulation of cutinase in AOT reverse micelles induced unfolding at room temperature due to an enzyme location at the micellar interface. The presence of 1-hexanol as co-surfactant delayed or even prevented the unfolding of cutinase by promoting the establishment of a new equilibrium in the system. Cutinase is encapsulated in a 10-fold larger AOT/hexanol reverse micelle built up by the fusion of empty reverse micelles. When tested in a membrane reactor in the presence of 1-hexanol, an operational half-life of 674 days was achieved.     

Improved activity of enzymes in mixed cationic reverse micelles with imidazolium-based surfactants

This work reports the significant enhancement in performance of interfacially active enzymes, Chromobacterium viscosum (CV) lipase and horseradish peroxidase (HRP) in mixed reverse micelles of cetyltrimethylammonium bromide (CTAB) and imidazolium-based amphiphiles having varying tail lengths. Lipase activity in these mixed systems was always higher than that in the individual cationic reverse micelles of CTAB or any imidazolium surfactant, highest being observed in the mixed system of CTAB (50 mM) and 6 (1-tetradecyl-3-methyl imidazolium bromide, 40 mM)/water/isooctane/n-hexanol (0.24 M), second-order rate constant, k2=1301+/-5 cm3 g(-1)s(-1), approximately 200% higher compared to that in CTAB and approximately 65% more than the most popular AOT-microemulsion. Activity increased with concentration of imidazolium surfactant and also with its alkyl tail length. To have a more profound view on the structure-activity relationship, CTAB was replaced by cetyltriethylammonium bromide (CTEAB) and cetyltripropylammonium bromide (CTPAB) with subsequent increase in the headgroup size. The generalized influence of these mixed cationic systems on surface-active enzyme was also verified using HRP, where the activity improved approximately 100%. This enhancement in enzyme activity is presumably due to the activating effect of the imidazolium cation in the enzymatic reactions by improving the nucleophilicity of interfacial water in vicinity of enzyme through hydrogen bonding.     

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ₀ (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C₁₂E₄), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C₁₂E₄ addition. The stabilizing effect of C₁₂E₄ resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C₁₂E₄ may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.     

Cutinase-AOT interactions in reverse micelles: the effect of 1-hexanol

Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution.Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.     

Conformational analysis of bioactive peptides in reverse micelles as mimics of cell membrane environments

Summary Conformational preferences of secretin as a model peptide have been analyzed by CD and IR spectroscopy in reverse micelles of AOT/isooctane/water and compared to those in aqueous TFE, in SDS micelles and in DMPG vesicles. Among the systems examined, reverse micelles and phospholipid vesicles displayed almost identical conformational equilibria. Very high lipid-to-peptide ratios can be obtained in reverse micelles with full retention of optical transparency, even at millimolar peptide concentrations, thus indicating this system to be an interesting mimic of cell membrane environments for spectroscopic analysis of bioactive peptide conformations.Abbreviations TFE trifluoroethanol - SDS sodium dodecyl sulfate - DMPG dimyristoylphosphatidylglycerol - AOT bis(2-ethylhexyl)sulfosuccinate - CMC critical micellar concentration - VIP vasoactive intestinal peptide     

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio (W₀), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters (V_max, K_m and K_ad), and the values of K_m and K_ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.     

Stability and Enzymatic Studies of Glucose Dehydrogenase from the Archaeon Haloferax mediterranei in reverse micelles

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w₀=[H₂O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP⁺ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.     

Extraction of bovine serum albumin with reverse micelles from glucosylammonium and lactosylammonium surfactants

Reverse micelle extraction is still in the stage of laboratory. Major limitation associated with use of synthetic surfactants in reverse micelle extraction process is the unfolding or denaturation of proteins. Sugar surfactants are thought non-toxic and environmentally benign, and can exhibit interesting interfacial properties, but the application of sugar-based surfactants in protein extraction is still limited. In the present study, we extracted bovine serum albumin (BSA) by using reverse micelles from glucosylammonium (GA) and lactosylammonium (LA) surfactants (with dicarboxylate as counter ion). It was found that under optimum condition, (1) the maximum forward extraction efficiency was ca. 86% with GA, while only around 50% with LA, and (2) almost all BSA solubilized in reverse micelles prepared from GA could be recovered into aqueous phase, while the recovery of BSA from the reverse micelles of LA was lower. In addition, the optimum extraction parameters were closely related to surfactant structure. Therefore, the electrostatic interaction, H-bonding and sugar head size should be important for BSA transfer.     

Purification of nattokinase by reverse micelles extraction from fermentation broth: effect of temperature and phase volume ratio

Nattokinase is a novel fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. In this study, reverse micelles extraction was applied to purify and concentrate nattokinase from fermentation broth. The effects of temperature and phase volume ratio used for the forward and backward extraction on the extraction process were examined. The optimal temperature for forward and backward extraction were 25°C and 35°C respectively. Nattokinase became more thermosensitive during reverse micelles extraction. And it could be enriched in the stripping phase eight times during backward extraction. It was found that nattokinase could be purified by AOT reverse micelles with up to 80% activity recovery and with a purification factor of 3.9.     

Unsaturation at the surfactant head: influence on the activity of lipase and horseradish peroxidase in reverse micelles

Influence of unsaturation present at the surfactant head on the activity of interfacially located enzyme, lipase, and horseradish peroxidase (HRP) is investigated in cationic reverse micelles of a series of surfactants having unsaturated (allyl and pyridinium moieties) as well as analogous saturated (n-propyl and piperidinium moieties) polar head. Lipase activity increases with n-propyl (saturated) substitution as the increase in the headgroup area (A(min)) presumably provides greater space for the enzyme to attain flexible conformation and increases the local concentrations of enzyme and substrate at the interface. In contrast, activity of lipase decreases with increasing number of allyl (unsaturated) substitution though A(min) gradually increased. Similar trend in deactivation was observed when unsaturation is present in cyclic ring (pyridine) at the surfactant head in comparison to the saturated analogue, piperidine. Circular dichroism (CD) spectra of lipase in reverse micelles indicate that ellipticity in the far-UV region increases with increasing unsaturation. Thus, lipase probably loses its alpha-helix content and thereby its activity. Inhibition of biocatalyst with increasing unsaturation at the polar head of surfactant is also observed in case of HRP, an oxidoreductase enzyme.     

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     

Interaction of 3,7-diamino-2,8-dimethyl-5-phenyl phenazinium chloride with model biological membranes and reverse micelles of lipid: a spectroscopic study

The interaction of 3,7-diamino-2,8-dimethyl-5-phenyl phenazinium chloride (Safranine T) with the aqueous as well as reverse micellar solution of a phospholipid 1,2-diacyl-sn-glycero-3-phosphocholine (Azolecithin), a major structural phospholipid in brain, comprising approx 15% of total lipid, primarily localized in grey matter have been studied by absorption and fluorescence spectroscopic studies. The results show the evidence of complex formation of the dye in the ground and in the excited state. The interaction of the dye with the lipid in reverse micellar state is more compared to that in liposomes. An attempt has been made to determine the polarity of the microenvironment of the dye in liposomes or reverse micelles from the spectral studies of the dye in different solvents of known polarity. The polarity functions of the phosphatidylcholine (PC) liposomes are slightly lower compared to that of PC reverse micelles.     

Recovery of protein from reverse micelles through gas hydrate formation

The recovery of cytochrome c and ribonuclease A from di-2-ethylhexyl sodium sulfosuccinate (AOT) reverse micelles have been examined by the gas hydrate formation. The recovery of cytochrome c depended upon the kind of gas and the water content (w0=[H₂O]/[AOT]) of reverse micellar solution containing cytochrome c prepared. Recoveries of cytochrome c and ribonuclease A were more than 80%, when 1,1,1,2-tetrafluoroethane (TFE) was used as a hydrating gas. The activity of cytochrome c recovered from reverse micelles was maintained perfectly.     

Chirality of reverse micelles

Four chiral analogues of the surfactant Aerosol-OT (AOT) have been synthesized and characterized. All of them form reverse micelles in apolar solvents in the w₀ range 0–30 (w₀ = [water]/[tenside]). Reverse micellar solutions have been investigated by UV absorption and circular dichroism spectroscopies with the aim of clarifying whether the formation of the macromolecular micellar structure induces the appearance of new chromophoric bands or perturbs the existing ones. Methanolic solutions of the surfactants, in which no micellar aggregates are formed, were taken as references. One of the products 1(S),1′(S)-dimethylbisheptylsulphosuccinate sodium salt (MH-AOT) was capable of forming reverse micelles of relatively high water content (w₀ up to 40) and this process was accompanied by a specific increase in the intensity of the circular dichroism band associated with the ester absorbance of the molecule. As no concomitant changes were seen in the UV absorbance spectrum, it was concluded that this observation reflected conformational events occurring within the surfactant rather than chromophoric perturbation. These results are qualitatively similar to those found recently for lecithin reverse micelles which, however, form gels at sufficiently high water contents. The chiroptical properties of these supramolecular aggregates are compared with those of covalent macromolecular systems such as polypeptides.     

The effect of demulsifiers on lysozyme extraction from hen egg white using reverse micelles

The liquid–liquid extraction of protein from buffered aqueous phases using reverse micelles (RM) has been extensively researched from a fundamental point of view. However, very little effort has been expended at scaling up this process for the extraction of real fermentation broth. When real broths are used with reverse micellar phases there are major problems with emulsion formation. In this study the effect of a variety of demulsifiers on lysozyme extraction was evaluated in terms of their influence on the separating properties of the emulsion, water content (W _o ), and, extraction yield and kinetics from both buffer and hen egg white. In addition, the use of a low shear contactor (a Graesser or `raining bucket') was assessed in terms of its suitability as a RM contactor. It was found that most of the demulsifiers reduced the settling time of the emulsion, and enhanced the yield and kinetics of lysozyme extraction from hen egg white. It was hypothesised that this was due to the demulsifier displacing the lysozyme from the interface and preventing the protein unfolding and precipitating. This effect was found to depend on both the generic type of demulsifier, and its concentration.     

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.     

Chiroptical properties of lecithin reverse micelles and organogels

The ultraviolet absorbance and circular dichroism (CD) spectra of lecithin reverse micelles and gels were investigated in order to establish whether the formation of these noncovalent macromolecular aggregates, which was induced by the addition of water to solutions of lecithin in organic solvents, was accompanied by specific spectroscopic changes. Systems containing the synthetic short-chain lecithins, 1,2-hexanoyl-, 1,2-diheptanoyl-, 1,2-dioctanoyl-, and 1,2-dinonaoyl-sn-glycero-3-phosphatidylcholines were used as models for the long-chain lecithins, soybean phosphatidylcholine and palmitoyl-oleoyl-phosphatidylcholine. All the molecules studied had asymmetric centres, formed reverse micelles under appropriate conditions, and, while both the long-chain lecithins also formed gels, none of the short-chain molecules did. As well as having CD spectra that were simpler to interpret, spectroscopic observations on solutions of the short-chain lecithins could be carried out over a large water content range. The ester chromophore of these compounds was shown to be highly sensitive to variation in both the solvent environment and the temperature, and components of both direct solvent effects and conformational change upon the addition of water were detected in the spectra. The spectra of the longer chain lecithins were complicated by the presence of double bonds although, here again, it was found that significant changes occurred as the water content increased, as monitored by the ester chromophore. However, no specific effect that could be ascribed to gelation alone was detected. The overall picture that emerged was that the ester chromophore of anhydrous micelles gave rise to a specific negative band in the CD spectrum (λ_max ≈ 210 nm) whereas a positive CD signal (λ_max ≈ 233 nm) was associated with the same chromophore in filled (i.e., hydrated) micelles. The two signals correspond to two different conformational states of the lecithin molecule, the hydrated state being not only more conformationally restricted but also providing a less polar environment for the ester groups, while the addition of water to the system shifts the conformational equilibrium. These observations have been interpreted as showing that only a limited range of lecithin conformation is compatible with the formation of the micellar structure and that it is this constraint, together with those introduced by the overall geometry of the aggregated state, that gives rise to the changes observed in the CD spectrum.     

Decontamination of surfaces by lysozyme encapsulated in reverse micelles

Cells and enzymes can be used to decontaminate soil, water supplies, personal equipment, weapons and hospital equipment that have been exposed to bacteria, toxins or viruses. One of the problems associated with the use of microorganisms and enzymes for decontamination purposes is that the presence of water is not acceptable for some applications such as electronic equipment. One way of circumventing this problem is to allow the enzyme to distribute between a water phase and an organic phase-containing surfactant and then use the encapsulated enzyme in reverse micelles directly into the device to be clean. Reverse micelles were used to deliver the enzyme (lysozyme) to the cell-surface interface. They serve as a way to increase the local concentration of lysozyme and decrease the amount of water delivered. Specifically, we explored the lysis by free lysozyme and lysozyme encapsulated in reverse micelles of Klebsiella pneumoniae and Staphylococcus epidermidis attached to steel, glass, and hydroxyapatite. These two bacteria have been selected because they are known to be pathogenic and because of their differences in cell wall structure. Lysozyme was added to the surfaces in either reverse micelles or as a free solution and was tested under conditions of stirring and no stirring. Stirring was implemented to study the interplay between mass transfer limitations and surface roughness. We have shown that free lysozyme or lysozyme encapsulated in reverse micelles is capable of decontaminating surfaces of different texture. Lysis of the cells is slower when the encapsulated enzyme is used but lysis is more complete.