Micellar Organic Phase Research Articles

Reverse micellar extraction of fungal glucoamylase produced in solid-state fermentation culture.

Partial purification of glucoamylase from solid-state fermentation culture was, firstly, investigated by reverse micellar extraction (RME). To avoid back extraction problems, the glucoamylase was kept in the original aqueous phase, while the other undesired proteins/ enzymes were moved to the reverse micellar organic phase. The individual and interaction effects of main factors (i.e., pH and NaCl concentration in the aqueous phase, and concentration of sodium bis-2-ethyl-hexyl-sulfosuccinate (AOT) in the organic phase) were studied using response surface methodology. The optimum conditions for the maximum recovery of the enzyme were pH 2.75, 100 mM NaCl, and 200 mM AOT. Furthermore, the optimum organic to aqueous volume ratio (Vorg/Vaq) and appropriate number of sequential extraction stages were 2 and 3, respectively. Finally, 60% of the undesired enzymes including proteases and xylanases were removed from the aqueous phase, while 140% of glucoamylase activity was recovered in the aqueous phase and the purification factor of glucoamylase was found to be 3.0-fold.

Preparation of protein nanoparticles using AOT reverse micelles

Biopolymeric nanoparticles made of protein via cross-linking reaction was prepared using sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelle system as a nanoreactor. The prepared ribonuclease A nanoparticles were stably dispersed in aqueous solutions and the size of the nanoparticles was controlled in the range of 6–100 nm with water content in the system. Solvent species formulating the reverse micellar organic phase also can control the nanoparticle size. These effects on the nanoparticle size would be attributed to the dynamic behavior of reverse micelles. In contrast, cytochrome c nanoparticles were stably dispersed in ethanol while completely precipitated in both aqueous solutions and non-polar organic solvents. These findings indicate that a specific interaction of protein with AOT, which depends on the solubilization site of each protein in reverse micelles, is a significant factor to affect the surface properties of protein nanoparticles.

Preparation of water-soluble palladium nanocrystals by reverse micelle method: Digestive ripening behavior of mercaptocarboxylic acids as stabilizing agent

Worm-like palladium nanoparticles are formed in the reverse micellar organic phase by means of chemical reduction in the water pools of reverse micelles. Water-soluble, spherical Pd nanocrystals are extracted from the worm-like Pd nanoparticles through the digestive ripening behavior of mercaptocarboxylic acids as stabilizing agents. It was found that during the reaction the production of spheres occurs in parallel with the formation of worm-like structure by association of the spheres on increasing the reaction time and the crystallization process occurs even during the extraction.

Extraction of water miscible organic dyes by reverse micelles of alkyl glucosides

The extraction of methyl orange or methylene blue from an aqueous phase to an organic phase of reverse micelles of alkyl glucosides was investigated. Dodecyl glucoside, a biodegradable and biocompatible surfactant, was employed as a kind of alkyl glucosides, since a stable Winsor II system consisting of the water-in-oil type microemulsion and aqueous phases was formed when an organic solution containing dodecyl glucoside was contacted with an aqueous solution. The water content in the reverse micellar organic phase increased with an increase in the concentration of dodecyl glucoside. The extraction ratio of dyes also increased with increasing the concentration of dodecyl glucoside. Furthermore, the extraction ratio of dyes was dramatically dependent upon the pH of an aqueous phase and temperature.

Partition of polyhydroxy compounds of biological and pharmacological significance between AOT reverse microemulsions and aqueous salt solutions

AbstractThe partition behavior of shikimic acid, gallic acid, gallotannic acid, rutin and quercetin between reverse microemulsions of the surfactant bis(2‐ethylhexyl) sulfosuccinate (AOT) and aqueous salt solutions of LiCl, NaCl and KCl at different salt concentrations and pH was studied. Quantification of the extracted analytes was performed by HPLC on an octadecyl silica column using a methanol–buffer mobile phase at pH 3.0. Clean‐up before injection was not needed because the micellar organic phase was compatible with the HPLC mobile phase. A solvent gradient was used to elute all compounds and the surfactant in a reasonable time. The extraction from the aqueous media is fast and reproducible. Different solubilization behavior was observed by changing the salt concentration and the type of cation in the aqueous phase. By proper selection of the experimental conditions, it is observed that shikimic acid and gallic acid remain mostly in the aqueous phase, whereas significant amounts of gallotannic acid and rutin are transferred to the micellar phase in only one contact and quercetin is completely solubilized in this phase. Thus, the different solubilization behavior that these compounds exhibit in the AOT micellar media can be very useful for extraction and preconcentration purposes. Copyright © 2006 John Wiley & Sons, Ltd.

How can temperature affect reverse micellar extraction using sucrose fatty acid ester?

The reverse micellar extraction of lysozyme using sucrose fatty acid ester was found to be greatly affected by the temperature in the extraction process. For example, lysozyme was perfectly extracted from the feed aqueous phase to the reverse micellar organic phase at 25°C, while it was not extracted at 5°C at all. After entrapping lysozyme into the reverse micelles, lysozyme was recovered from the reverse micellar organic phase to the recovery aqueous phase only by decreasing the temperature in the backward extraction. Moreover, lysozyme solubilized in the reverse micellar organic solution could be recovered without the recovery aqueous solution at 3°C, and its activity was retained at 95%.

Application of sucrose fatty acid ester to reverse micellar extraction of lysozyme

Reverse micellar extraction of lysozyme has been carried out using an organic solution containing a mixture of monoester and polyester of sucrose fatty acid ester. The forward extraction of lysozyme from the feed aqueous phase to the reverse micellar organic phase of the mixture of monoester and polyester of sucrose fatty acid ester at pH 7.2 was strongly dependent upon the weight fraction of monoester, while any amount of lysozyme was not extracted only by using monoester or polyester. The forward extraction ratio dramatically increased with an increase in the concentration of fatty acid ester, and was high around neutral pH and at low ionic strength. The backward extraction of lysozyme from the reverse micellar organic phase to the recovery aqueous phase exhibited high efficiency at acidic pH value or at high ionic strength. The addition of sucrose into the recovery aqueous phase promoted the backward extraction ratio, and caused the activity of lysozyme recovered from the reverse micellar phase to be retained perfectly.

Forward and backward extractions of cytochrome c using reverse micellar system of sucrose fatty acid ester

The effects of solvent, pH, and ionic strength on the reverse micellar extraction of cytochrome c have been examined, when sucrose fatty acid esters were employed as surfactants of reverse micelles. The transparent and stable reverse micellar organic phase was formed, when the mixture of isooctane and n-butanol (7:3 v/v) was used as the bulk organic phase. The high forward extraction ratio was obtained under mild alkaline and low ionic strength conditions, while the high backward extraction ratio was obtained for acidic pH values or at high ionic strength. The activity of cytochrome c recovered from the reverse micellar phase was sufficiently retained.

Liquid‐liquid extraction of α‐lactalbumin using reverse micellar organic solvent

The liquid-liquid extraction of alpha-lactalbumin based on reverse micellar organic solvents was investigated. Forward extraction of the protein in the reverse micellar organic phase from aqueous feed solutions was strongly dependent on the initial pH of the feed solution and the complete forward extraction of 0.03 mM alpha-lactalbumin was successfully achieved at pH 6.0. The forward extraction percentage steeply decreased with increasing KCl concentration, while in the NaCl system the forward extraction was independent on the salt concentration below 1 M. From the circular dichroic measurement, higher order structure of the recovered alpha-lactalbumin through the extraction process was well preserved.

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.

Efficacy of guanidium salts in protein recovery from reverse micellar organic media

The efficacy of guanidium salts in the recovery of extracted lysozyme from aerosol-OT (AOT) reverse micellar organic phase was investigated. Adding guanidium salt at a low concentration as pretreatment reagent in the feed solution led to successful protein recovery, and the enzymatic activity of the recovered lysozyme was well maintained. Among the electrolytes tested, caotropic guanidine thiocyanate (GuHSCN) was the most effective in recovering lysozyme as well as in preserving its activity. The presence of guanidium salt in the micellar organic phase markedly lowered the water content, apparently by reducing or eliminating accompanying water arising from lysozyme solubilization. CD data showed that the α-helix content of the lysozyme in the micellar phase in the presence of dilute guanidium salt was smaller than that in a guanidium-free micellar phase. These results indicated that the guanidium salt expelled lysozyme molecules from the micro-interface of the reverse micelles into the hydrophilic micro-water pool.

Esterification by Rhizopus delemar lipase in organic solvent using sugar ester reverse micelles

The esterification of oleic acid with octyl alcohol catalyzed by Rhizopus delemar lipase was investigated in reverse micellar system of sugar ester DK-F-110. High initial reaction rate was obtained by preparing the micellar organic phase with extremely low water content. The maximum rate was obtained at a W 0 (=[ H 2 O]/[ amphiphile]) of 2.5. The highest reaction rate occurred at pH 6. With decreasing DK-F-110 concentration, the initial reaction rate was slightly decreased. The optimal temperature for lipase activity was around 313 K (40°C). Apparent activation energy of the esterification in the system was 43.7 kJ/mol. The lipase showed 40% of its esterification activity after 28 h incubation in the DK-F-110 micellar organic phase. The reaction kinetics in this system were recognized as ping-pong bi–bi mechanism. From a Michaelis–Menten analysis, apparent kinetic parameters were determined. The turnover number of the DK-F-110 system was larger than that of the sodium bis(2-ethylhexyl)sulfosuccinate (AOT) system.

Formation and Characterization of Reversed Micelles Composed of Phospholipids and Fatty Acids

The formation of reversed micellar systems composed of phosphatidylcholine (PC) and fatty acid was newly demonstrated by a significant increase in water content in the organic ethyl oleate phase when the micelles were prepared by the contact method. The solubilized water concentration in the reversed micellar organic phase reached 3 wt%. The new systems are expected to be used as highly biocompatible reversed micellar systems. The structure of the reversed micelles composed of PC and oleic acid was characterized by determining the water concentration and by small-angle X-ray scattering analysis. The reversed micelles composed of PC and oleic acid formed in ethyl oleate were spherical. The radius of gyration was between 30 and 50 Å. The size of the reversed micelles decreased with an increase in the oleic acid concentration and was independent of the PC concentration. Experimental results indicated that the structure of the reversed micellar system was determined by the oleic acid concentration. An increase in the PC concentration caused an increase in the number of reversed micelles of the same size. These reversed micellar systems are expected to be used as solubilization media in pharmaceutical and food industries because they are not toxic.

逆ミセル有機溶媒における電気的パーコレーション現象とタンパク質の可溶化状態

Electric percolation phenomena of reverse micellar organic phase of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) were investigated. Solubilization of electrolytes into the micellar water pool depressed the percolation between reverse micelles and increased the percolation temperature. In particular, caotropic GuHSCN and strong electrolyte KCl increased the percolation temperature more effectively. Solubilization of low-molecular-weight proteins into the micellar phase induced the percolation processes and lowered the percolation temperature, which was dependent on the protein concentration solubilized. The magnitude order of effectiveness of the protein on the percolation was cytochrome c > lysozyme > ribonuclease A. The efficiency of each protein in promoting percolation corresponded to its local solubilizing state in the micelles. In the cases of solubilizing both the electrolyte and the protein, the percolation processes were mutually affected by each additive. In the reverse micellar systems formed by different organic solvents (C6-10 alkane), the percolation temperature was linearly decreased with molecular volume of the solvent. The shift of the percolation temperature due to protein solubilization (ΔTp) depended upon the solvent molecular volume and the protein species. In the case of lysozyme, ΔTp increased with the molecular volume of solvent, while ΔTp of cytochrome c showed the minimum value at the molecular volume of C8-9 alkane.

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 KHCO 3/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.

Protein extraction using sugar ester reverse micelles

In protein extraction, using the nonionic surfactant sugar ester DK-F-110, the critical micelle concentration (CMC) of a DK-F-110/isopropyl alcohol(IPA)/hexane system was found to be at a DK-F-110 concentration of 0.5 g dm -3 , indicating that the sugar ester reverse micelles could be formed in hexane. At concentrations higher than the CMC, cytochrome c was extracted into the DK-F-110 reverse micelles as judged from UV spectra of the DK-F-110/IPA/hexane solution after it was contacted with the aqueous protein solution. In extraction of cytochrome c using this system, forward extraction was found to be pH-dependent, with high extraction percentage being obtained at pH 8. The forward extraction percentage was reduced by an increase in the buffer concentration, and at a buffer concentration of 0.5 mol dm -3 was ca 25% as high as that of sodium bis(2-ethylhexyl) sulfosuccinate(AOT) systems. An optimal DK-F-110 concentration was found to give the maximum forward extraction percentage. Addition of alcohol, especially IPA, to the micellar organic phase enabled the highly backward extraction of cytochrome c to be achieved without the formation of insoluble aggregates. The esterification reaction rate by Rhizopus delemar lipase in the DK-F-110 reverse micellar system had a higher maximum value than that of AOT and lecithin systems.

Extraction of flexibly structured protein in AOT reverse micelles: the flexible structure of protein is the dominant factor for its incorporation into reverse micelles

The extraction of flexibly-structured protein in Aerosol-OT (AOT)/isooctane reverse micelles was investigated. A flexibly-structured lysozyme was prepared by reduction and carboxymethylation of the disulfide bonds in the lysozyme molecule. For a comparison, lysozymes whose surface hydrophobicity was modified by monoacylation of the amino groups were also used. The extraction rate of the flexibly-structured lysozyme into the micellar phase was greater than that of the native and monoacylated lysozymes, although the free energy change of the lysozyme prepared by breaking the disulfide bonds was smaller than that of the lysozymes whose surfaces were monoacylated. Viscosity measurement of the micellar organic phase containing the modified lysozymes indicated that extraction of the flexibly-structured lysozyme changed the micelle–micelle interaction, while measurement of the interfacial tension between the AOT/isooctane and protein aqueous systems showed the flexibly-structured lysozyme to be the most amphiphilic in character. These results indicated that the flexible structure of a protein was more dominant than its surface hydrophobicity for its incorporation into reverse micelles, and that it leads to greater micelle–micelle interaction.

Protein extraction using non-ionic reverse micelles of Span 60

In protein extraction using non-ionic Span 60 reverse micelles, the critical micelle concentration (CMC) of a Span 60/IPA/hexane system was at Span 60 concentration of 3.5 g l −1. At concentrations higher than the CMC, cytochrome c was extracted into the Span 60 reverse micelles as judged from UV spectra of the Span 60/IPA/hexane solution after it contacted with the protein aqueous solution. In extractions of cytochrome c and lysozyme using this system, forward extraction was found to be pH-dependent, with high extraction percentages being obtained at neutral pHs. The forward extraction percentage was drastically reduced by only a small increase in the buffer concentration (even up to 0.1 M). In cytochrome c extraction using various means of phase contacting, high extraction percentages were obtained with rotary and reciprocal shaking, but in well-mixed extraction by magnetic stirring, extraction percentages were much lower. Addition of alcohol, especially isopropyl alcohol, to the micellar organic phase enabled the backward extraction of cytochrome c to be achieved without the formation of insoluble aggregates.

Transport between an aqueous phase and a CTAB/hexanol-octane reversed micellar phase

Using bovine serum albumin (BSA) as a model protein, protein transport between an aqueous phase and a reversed micellar organic phase was examined. From near-infrared spectra and UV absorption spectra, it was confirmed that CTAB (cetyl trimethyl ammonium bromide)/hexanol octane reversed micellar solution is a suitable medium for BSA solubilization. It seems that the forward transfer efficiency depends not only on the parameters of the two phases (for example: the pH value, the concentration and species of ions in the aqueous phase, the content of surfactant and co-solvent in reversed micellar solution), but also the titration behaviour and the concentration of the protein in aqueous phase. The experimental results lead to the conclusion that the major driving force in the protein extraction is electrostatic interaction. A modified phenomenological thermodynamic model for reversed micellar extraction was presented, and shown to correlate the data on extraction efficiency of BSA with both pH value and surfactant concentration. The effects of various factors, such as pH, and the type and concentration of salt in stripping solution, on BSA back transfer were also studied. It was found that the backward transfer efficiency of BSA is strongly influenced by the type of ions in stripping solution. When the operation runs under optimized conditions and with suitable phase ratio, the recovery and concentration of protein were successfully obtained. In addition, the problems how long the reversed micellar solutions can be recycled and how much of the surfactant will be lost to the aqueous phase have been examined.

Parameters affecting the efficiency of affinity-based reversed micellar extraction and separation (ARMES) in glycoprotein purification.

Affinity-based reversed micellar extraction and separation (ARMES) is an effective method for purifying both low and high molecular weight glycoproteins via liquid-liquid extraction. A range of extraction conditions were examined to gain insight into the mechanism of ARMES. Concanavalin A (Con A) was used as the model affinity ligand to bind soybean peroxidase (SBP) and beta-galactosidase as model glycoproteins. Factorial design was used to investigate the effect of various system variables on the extraction of SBP via ARMES. A quadratic model described the systems well, resulting in a standard deviation of 7% between calculated and experimental extraction efficiencies. Sensitivity analysis suggested that the key criteria in ARMES were the NaCl concentration and pH of the aqueous feed phase. Extraction of both glycoproteins decreased above pH 7 but fell to zero only at pH values significantly above the pI of the model glycoproteins and the Con A affinity ligand. It is proposed that the complex of the affinity lectin with the glycoprotein results in a sufficiently hydrophobic species that can be extracted into a reversed micellar organic phase even at pH's far above the pI's of the individual proteins that comprise the complex. This finding has practical considerations for the use of ARMES in the resolution and purification of protein glycoforms.