AOT Reverse Micellar System Research Articles

Effect of Key Parameters on Jacalin Extraction from Aqueous Phase into Anionic Reverse Micelles

This study demonstrates the extraction of jacalin from crude extract of jackfruit seeds using anionic surfactant based reverse micellar system, sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. Effects of various parameters, such as, NaCl concentration (0.05 – 1 M), pH of aqueous phase (pH 4 – 10), AOT concentration (5 – 150 mM), contact time (5 – 30 min) and phase volume ratio (0.5-5) on the transfer efficiency of jacalin was evaluated by changing one factor at a time (OFAT) while keeping the other parameters constant. A maximum of 83% of protein transfer was achieved after equal volume of organic and aqueous phase was stirred for 20 min using 20 mM AOT at pH 5 aqueous phase containing 0.1 M NaCl. The findings demonstrated AOT reverse micellar system as a promising and effective method to extract and purify jacalin from crude protein mixture.

Primary recovery of miraculin from miracle fruit, Synsepalum dulcificum by AOT reverse micellar system

Miracle fruit, Synsepalum dulcificum, contains a glycoprotein known as miraculin. After consuming this glycoprotein, sour foods taste sweet and the effect lasts for up to 4 h. With increasing demand for natural and “low-calorie” sweeteners, the use of miraculin as an additive is increasing enormously in the food, medicine and cosmetic industries. In this study, we used reverse micelles formed from a sodium di (2-ethylhexyl) sulfosuccinate (AOT)/isooctane system to purify miraculin from S. dulcificum. We studied factors affecting purification performance, such as surfactant (AOT) concentration and the pH of the crude during forward extraction. During backward extraction, we examined the effects of NaCl concentration, the pH of the aqueous phase and addition of isopropanol. We found that 0.1 mol/L AOT/isooctane solution mixed with crude extract at pH 8 during the forward extraction stage and 0.5 mol/L NaCl solution at pH 11 during the backward stripping stage were optimal purification conditions, from which 22% miraculin was recovered with a purity of 94.8%.

Improvement in extraction and catalytic activity of Mucor javanicus lipase by modification of AOT reverse micelle

Reverse micelles are formed in apolar solvents by spontaneous aggregation of surfactants. Surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) is most often used for the reverse micellar extraction of enzymes. However, the inactivation of enzyme due to strong interaction with AOT molecules is a severe problem. To overcome this problem, the AOT/water/isooctane reverse micellar system was modified by adding short chain polyethylene glycol 400 (PEG 400). The modified AOT reverse micellar system was used to extract Mucor javanicus lipase from the aqueous phase to the reverse micellar phase. The extraction efficiency (E) increased with the increase in PEG 400 addition and the maximum E in PEG 400 modified system was twofold higher than that in the PEG 400-free system. Upon addition of PEG 400, the water activity (a(w)) of aqueous phase decreased, whereas a(w) of reverse micellar phase increased. The circular dichroism spectroscopy analysis revealed that PEG 400 changes the secondary and tertiary structure of lipase. The maximum specific activity of lipase extracted in PEG 400-modified reverse micellar system was threefold higher than that in the PEG-free system.

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w0 (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.

Monomerization and photodynamic activity of Zn(II) tetraalkyltetrapyridinoporphyrazinium derivatives in AOT reverse micelles

Monomerization and photodynamic activity of cationic Zn(II) tetraalkyltetrapyridinoporphyrazinium derivatives bearing N-alkyl chains of different length (ZnPc 1 R: –CH 3, ZnPc 2 R: –(CH 2) 11CH 3, ZnPc 3, R: –(CH 2) 15CH 3) were evaluated in n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water micellar system. Absorption and fluorescence spectroscopic studies of these sensitizers were analyzed in n-heptane/AOT (0.1 M)/water system by varying the amount of water dispersed in the reverse micelles ( W 0 = [H 2O]/[AOT]). Under these conditions, the absorbance of the monomeric Q-band (∼686 nm) of ZnPcs 1– 3 increases rapidly until W 0 ∼ 30 and then the increase becomes steady. These results were also confirmed by fluorescence studies. Analysis of the Q-band at different AOT concentrations, keeping W 0 = 30 as constant, was carried out to determine the binding constant ( K b) between these sensitizers and AOT reverse micelles. The values of K of 210, 150 and 34 were found for ZnPcs 1, 2 and 3, respectively. Photodynamic activity of these sensitizers was evaluated in AOT system using 9,10-dimethylanthracene (DMA). The photooxidation rate of DMA sensitized by these phthalocyanines follow the order: ZnPc 1 > ZnPc 2 ∼ ZnPc 3. As shown by spectroscopic and photodynamic studies, the AOT reverse micellar system very effectively inhibits the aggregation of phthalocyanines, promoting the formation of photoactive monomeric species.

Characteristics of Nano-capsules Prepared Using Reverse Micellar System

The preparation of nano-capsules using an AOT reverse micellar system was performed by the in situ polymerization of styrene and divinylbenzene on the surface of the AOT reverse micelles. The effects of the preparation conditions on the capsule morphologies, encapsulation ratio of water and average diameter of the nano-capsules were investigated. The morphology of nano-capsules were very spherical, and average diameter of the nano-capsules, which was approximately 600 nm, was not affected by the preparation conditions such as surfactant concentration, sodium chloride concentration in the injection water, monomer ratio and pH of the injection water. The encapsulation ratio of water was affected by the surfactant concentration, the amount of the injection water and the sodium chloride concentration. On the other hand, the encapsulation ratio was not affected by the pH of the injection water. Based on this result, it is possible to prepare nano-capsules regardless of the injection water pH. Finally, it is proven that a high encapsulation ratio was obtained when a low surfactant concentration and low quantity of injection water.

Enhanced activity and stability of Chromobacterium viscosum lipase in AOT reverse micellar systems by pretreatment with acetone

The activation and stabilization of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) by a simple pretreatment with acetone was studied in AOT/water/isooctane reverse micellar systems, using the hydrolysis of olive oil as a model reaction. The maximum activity of acetone treated lipase was found to be 160% higher than that of native lipase. These observations were rationalized by the assumption based on the lipase conformational changes accompanied by the enhancement of lipase surface with hydrophobic amino acids. The dependence of catalytic activity on the various parameters relevant to the pretreatment procedure such as acetone content, the pH of water–acetone solution, agitation time of the lipase in water–acetone solution and freeze-drying time were investigated. The change in lipase conformation due to the treatment with acetone was confirmed by typtophyl fluorescence spectroscopy as well as lipase microencapsulation methods in reverse micelles. The acetone treated lipase entrapped in AOT reverse micelles was very stable, retaining over 83% of its initial activity after 40 days, whereas the half-life of native lipase was 38 days.

Analysis of protein back-extraction processes in alcohol- and carboxylic acid-mediated AOT reverse micellar systems based on structural changes of proteins and reverse micelles

The effects of the addition of alcohol and carboxylic acid on the back-extraction of several small globular proteins such as β-lactoglobulin (β-LG), bovine carbonic anhydrase (CAB), and lipase have been studied using reverse micellar systems (RVMS). The protein back-extraction induced by the addition of alcohol and carboxylic acid was markedly influenced by the structural changes of reverse micelles ( β t) and protein ( m). The alcohols and carboxylic acids suppressing the formation of reverse micelle clusters (positive value of β t) remarkably enhanced the back-extraction of proteins. On the other hand, the alcohols and carboxylic acids enhancing the formation of reverse micelle clusters (negative value of β t) suppressed the back-extraction of proteins. The markedly effective alcohol and carboxylic acid molecules on the denaturation of proteins (high values of m) suppressed the back-extraction of proteins. The m values were estimated by measuring the dependence of the free energy change of denaturation on the concentration of alcohol or carboxylic acid, and were correlated well with the solvent-accessible surface area (ASA) of alcohol and carboxylic acid molecules. We have constructed a simple equation on the basis of these factors for explaining the effect of alcohols and carboxylic acids in back-extraction processes of proteins of RVMS. These results suggested that the back-extraction processes were controlled by the formation of reverse micelle clusters as well as the denaturation of proteins.

Back-Extraction Processes of C.C.Lipase with Mediated AOT Reverse Micellar System

The relationship between the behaviors of c.c.lipase back-extraction and their percolation phenomena by using AOT reverse micellar systems (RVMS) has been studied by the addition of a small amount of additives to organic phase such as thiols and nonionic-surfactants focusing on micelle-micelle interactions. The values of <TEX>${\beta}_t$</TEX> defined by the variation of percolation processes and back-extraction behaviors of c.c.lipase have a good linear correlation. The hydrophobicity of additive molecules suppressing the cluster formation of reverse micelles (high values of <TEX>${\beta}_t$</TEX>) improved the back-extraction behavior of c.c.lipase. The back-extraction fraction and its rate of c.c.clipase are increased with decreasing of the value of hydrophilic lipophilic balance (HLB) and increasing of the hydrophobicity per additive molecules added to reverse micellar systems (RVSM) in the same additives concentration.

Forward and Backward Extractions of Hemoglobin in an AOT Reverse Micellar System

Forward and Backward Extractions of Hemoglobin in an AOT Reverse Micellar System

Solvent condition in triolein hydrolysis by Rhizopus delemar lipase using an AOT reverse micellar system

The hydrolysis of triolein catalyzed by Rhizopus delemar lipase was examined using AOT reverse micellar systems formulated with several straight-chain alkanes (C 6–C 10), isooctane, and cyclohexane as non-polar organic solvents. The highest initial reaction rate was obtained in the isooctane system. In the straight-chain systems, the initial reaction rate was correlated with the hydrophobicity of the solvent, log P, but not with the water content ( W 0: molar ratio of water to AOT) in the micellar organic phase. In the isooctane system, the initial reaction rate was strongly dependent on W 0 and was maximal when W 0 is equal to approximately 13. Since there is known to be a linear correlation between reverse micelle size and W 0, the degree of curvature of the micellar interface also appears to influence enzyme reactivity, especially with a bulky solvent like isooctane. The percolation temperature decreased with increasing W 0 more rapidly in the isooctane system than in the other systems, indicating enhancement of the attractive interaction between reverse micelles. A Michaelis–Menten analysis showed the isooctane system to have the highest specificity ratio ( k cat/ K m). Isooctane at W 0=13 was thus considered to be the best solvent condition for triolein hydrolysis in a 50 mM AOT reverse micellar system.

Solvent Effects on the Growth and Steric Stabilization of Copper Metallic Nanoparticles in AOT Reverse Micelle Systems

Organic and inorganic reactions within the aqueous cores of water-in-oil AOT reverse micelle systems are viable methods for the production of nanomaterials of controllable composition and geometry while maintaining narrow size distributions. Considerable research has been done in order to better understand the governing features which comprise the AOT reverse micellar system, particularly stability and the intermicellar exchange of the contents within the aqueous core. The intermicellar exchange rate is affected by the bulk solvent type, the contents dissolved within the core, and the size of the reverse micelle or the water content, referred to as the W value, where W is the molar ratio of the water to AOT surfactant concentrations. Synthesis of nanomaterials within the AOT reverse micelle system is a strong function of the intermicellar exchange process and the factors mentioned previously. This study examines the effects of varying the bulk liquid solvent and the W value on the growth rate and ultimate particle size of copper nanoparticles produced via reduction of CuAOT2 within the micelle core. Particle growth is measured in-situ using time-resolved UV−Vis absorbance spectroscopy, and the particle size is determined by both UV−Vis measurements and TEM analysis. A total interaction energy model is implemented to represent the attractive van der Waals forces acting between the metallic particles and the repulsive osmotic and elastic forces which result from the surfactant tail−tail and solvent−tail interactions responsible for the steric stabilization of the metallic particles within the microemulsion. The model is able to predict the ultimate particle sizes obtained experimentally for copper and silver nanoparticles synthesized using a variety of bulk liquid solvents including isooctane, cyclohexane, and n-alkanes ranging from pentane to dodecane.

Increased activity of Chromobacterium viscosum lipase in AOT reverse micelles in the presence of short chain methoxypolyethylene glycol

AbstractThe activity of Chromobacterium viscosum lipase (glycerol‐ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane and AOT/Tween 85/isooctane reverse micelles was significantly increased by the addition of short chain methoxypolyethylene glycols (MPEGs), taking the hydrolysis of olive oil as a model reaction. The molecular weight of MPEG had a strong effect on the lipase activity, and MPEG of nominal molecular weight 550 was found to be the most effective. To optimize the factors affecting enzymatic hydrolysis of olive oil in reverse micellar systems containing MPEG 550, the effect of various parameters, such as Wo (molar ratio of water to surfactant), pH, ionic strength, surfactant concentration and temperature were investigated. A kinetic model considering the substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was also successfully used to understand the enzyme activity in the presence of MPEG 550. Both the Michaelis constant and the substrate adsorption equilibrium constant were obviously reduced as compared with those obtained in the simple AOT reverse micellar system.© 2001 Society of Chemical Industry

Effect of Electrostatic Interaction on Reverse Micellar Extraction of Large Molecular Weight Proteins.

Reverse micellar extraction of various proteins of large molecular weight and oligomeric proteins, as well as small ones, was carried out using conventional AOT reverse micellar extraction systems at various pH values. The conformational change of the proteins by electrostatic interaction with AOT was also measured using a CD spectrum. Extraction behavior and conformational change of the proteins were discussed from their electric charge properties and electrostatic interaction between protein and AOT. Most of the proteins were effectively extracted into the reverse micellar phase at around zero surface net charge. Formation of aggregates and denaturation of proteins were observed in the region of positive net charge higher than 10.

Enzymatic activity ofChromobacterium viscosum lipase in an AOT/Tween 85 mixed reverse micellar system

To enhance the Chromobacterium viscosum lipase (glycerol-ester hydrolase; EC 3.1.1.3) activity for the reaction of water-insoluble substrates, the AOT/isooctane reverse micellar interface was modified by co-adsorption of a non-ionic surfactant. Polyoxyethylene sorbitan trioleate (Tween 85) was used as the non-ionic surfactant and olive oil as a water-insoluble substrate. An appreciable increase of lipase activity was observed and at higher Wo values (where Wo = molar ratio of water to total surfactants of the micellar system) there was no sharp fall of the enzyme activity such as a typical bell-shaped profile. The kinetic model for the lipase-catalysed hydrolysis of olive oil in AOT/isooctane reverse micellar system was applied to the enzymatic reaction in this mixed reverse micellar system. It was found that the predictions of the model agree well with the experimental kinetic results and that the adsorption equilibrium constant of olive oil molecules between the micellar phase and the bulk phase of the organic solvent is smaller in AOT/Tween 85 mixed reverse micellar systems than in simple AOT reverse micellar systems. © 1999 Society of Chemical Industry

Extraction of proteins and polymers using reverse micelles and percolation process

Specific interfacial properties, which affect protein extraction in AOT and AOT-lecithin reverse micellar systems (RVMS), have been studied by using their percolation processes. Solubilization of proteins or synthetic polymers into RVMS by the injection method and its effect on the percolation processes could be easily evaluated by the measurement of electrical conductivity. The percolation process is found to be a sensitive and convenient measure of micro-interface of RVMS solubilizing various polymers or proteins, which clearly reflects the polymer(protein)-micellar and micellar-micellar interactions. The stability of RVMS or micellar-micellar interaction was dependent on the kinds, concentration and molecular weight of solubilized polymers. The value of p, defined as the variation of percolation threshold with the concentration of solubilized polymers, can be utilized to evaluate the stability of RVMS solubilizing polymers or micellar-micellar interactions. The values of Β are affected by the hydrophobicity, molecular weight and absolute value of the net charge of the polymers solubilized into the AOT reverse micelles, which were evaluated by using Aqueous Two-Phase Systems (ATPS).

Relation between Exchange Process and Structure of AOT Reverse Micellar System

The decreases of the exchange micellar rate constant, determined from stopped flow kinetic study, and of the sticky parameter, determined by small angle X-ray scattering, with the water volume fraction are strongly correlated. This phenomenon is explained according to the contact value of the micelle−micelle correlation function and the surfactant monolayer rigidity. It is shown that the attractive interactions govern the decrease of the exchange process with the increase in the number of droplets.

Lipase-catalysed interfacial reactions in reverse micellar systems. Role of water and microenvironment in determining enzyme activity or dormancy

Studies of the effect of water content on the equilibrium position of a lipase-catalysed hydrolysis/esterification reaction in an AOT reverse micellar system show that the extent of reaction is apparently independent of ωo, the mole ratio of water to surfactant, over the studied range (2–40). Near-IR spectroscopy is shown to be a useful technique for the in situ monitoring of hydrolysis and synthesis reactions in reverse micelle–water-in-oil (w/o) microemulsion systems, and the methods applied are applicable to other low water systems. The hydrolysis and esterification activity of lipases tends to zero at low ωo where the thermodynamic water activity 1. It is shown that a state of reversible enzyme dormancy can be induced by dehydrating the enzyme-containing w/o microemulsion. Molecular sieve beads were considered unsuitable for this purpose because in addition to water, AOT and lipase were also adsorbed from the microemulsion. Molecular sieve powder was more effective, rapidly dehydrating w/o microemulsions whilst leaving AOT and most of the lipase in solution. Drying through vapour-phase equilibration of the microemulsion with a saturated salt solution in a closed system allowed the water activity to be precisely controlled. Although relatively slow, vapour phase drying is ‘clean’ in that it is specific only to water. AOT reverse micellar media containing dormant Chromobacterium viscosum lipase can be stored for at least a month with the lipase activity immediately regenerated on rehydration, with good recovery of catalytic activity. This phenomenon may have application in the food industry for the instant biogeneration of aroma and flavour compounds.

Extraction of enzymes and their activities in AOT reverse micellar systems modified with nonionic surfactants.

The extraction of Chromobacterium viscosum lipase and α-chymotrypsin from aqueous solution, and hydrolysis catalyzed by these enzymes were carried out in AOT/Span 60 and in AOT/Tween 85 mixed reverse micellar systems. Span 60 was likely to be solubilized in the water pool of AOT micelles without any change in micellar shape, while Tween 85 brought about an elongation of the micellar shape. The extracted fraction of the α-chymotrypsin at pH 6 was decreased by the addition of Span 60 or Tween 85, owing to the dilution of negative charge density of micelles. However, the extracted fraction of the lipase was increased by the addition of Tween 85, which seemed to act as an affinity ligand. The activity of α-chymotrypsin was improved by the addition of Span 60 or Tween 85, which decreases the electrostatic and hydrophobic interaction with AOT molecules. The activity of the lipase was significantly improved by the addition of Tween 85, especially in the higher range of water content, and the bell-shaped dependence of the activity on the water content disappeared. The lipase activity was maximum when the molar ratio of AOT to Tween 85 was 3:2–4:1.

Increased activity of Chromobacterium viscosum lipase in aerosol OT reverse micelles in the presence of nonionic surfactants.

Modification of AOT reverse micellar systems with various alkyl glucosides and nonionic surfactants was attempted in order to improve the activity to Chromobacterium viscosum lipase. Tweens and Tritons, having the poly(oxyethylene) chain, have been shown to be solubilized at the oil-water interface of AOT micelles. Alkyl glucosides were also solubilized at the interface. These additives form mixed micelles with AOT and increase the concentration of the micelles. In contrast, Spans were found to be solubilized in the micro water pool of AOT micelles. Addition of nonionic surfactants decreased the hydrophobicity of the reverse micellar systems. Improvement of the lipase activity was achieved by the addition of nonionic surfactants, because of suppression of the hydrophobic and electrostatic interaction between AOT and lipase. The addition of nonionic surfactant having a poly(oxyethylene) chain has been shown to effectively increase the activity of the lipase in reverse micelles.