Solubilizing water involved in protein extraction using reversed micelles

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

Liquid-liquid extraction of commercial glucose oxidase by reversed micelles

Aim of this work was to establish the optimum operating conditions for the extraction and recovery by cationic reversed micelles of commercial glucose oxidase (GOX) from Aspergillus niger, in view of possible application to raw cell homogenates. The influence of pH, temperature, electric conductivity and solvent/co-solvents ratio on the extraction was investigated by a fractional factorial design of 2(3-1) type, conjugated with a mixture experimental design, using the residual enzyme activity to evaluate the results. The best conditions for GOX extraction were ensured using isooctane as solvent and hexanol and butanol as co-solvents at 76/6/18 volume ratio, pH 6.0, 0.2 M cetyl trimethylammonium bromide (CTAB) as cationic surfactant, and electrical conductivity (kappa) of 4.8 mS cm-1. The highest yield of GOX activity recovery (about 90%) was in fair accordance with the value predicted by the model.     

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.     

Fluorescence of hemoproteins in reversed micelles of surfactants in octanes

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

Catalysis by hemoproteins and their structural organization in reversed micelles of surfactants in octane

The cytochromes $\text{P-450}$ LM-2 and $\text{b}{}_5$ from rabbit liver microsomes have been entrapped into reversed micelles of surfactants in octane. The optimum conditions providing for the maximum stability of the hemoproteins have been found: pH and concentration of the buffer solution, the glycerol addition, the surfactant concentration, the [H₂O]/[surfactant] ratio and, finally, the reversed micelles composition including aerosol OT and its mixture with Triton X-45, Tween 20 and cetyltrimethylammonium bromide (CTAB). The transformation kinetics of the hemoproteins solubilized by detergents has been studied by monitoring the absorbance of hemoproteins in the Soret band region. Significant changes in tryptophan fluorescence of cytochrome $\text{b}{}_5$ and in CD spectra of myoglobin in reversed micelles and their dependence on the [H₂O]/[aerosol OT] ratio have been shown. The three hemoproteins in reversed micelles have been found to exhibit high catalytic activity with respect to their reaction with cumene hydroperoxide. The kinetic and spectral data reveal the structural transformations of the proteins entrapped into the micelles due to the interactions of the lumenal surface of the micelles and the protein molecule surface.     

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.     

Liquid-liquid extraction of xylitol dehydrogenase from Candida guilliermondii homogenate by reversed micelles

The intracellular enzyme xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugarcane bagasse hydrolysate, was separated by reversed micelles of BDBAC [N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride] cationic surfactant. An experimental design was employed to evaluate the influence of the following factors on the enzyme separation: temperature, co-solvent concentration and surfactant concentration. The results showed that just the temperature did not show significant effect on XD recovery. A model was used to represent the activity recovery and fit the experimental data. Under optimized conditions, the recovery of total activity was about 121%, and the purity increased 2.3-fold.     

Purification of glucose oxidase from Aspergillus niger by liquid-liquid cationic reversed micelles extraction

The aim of this work was to select the operating conditions for the extraction and recovery of glucose oxidase (GOX) by reversed micelles from mixtures of commercial enzyme and Aspergillus niger homogenates. For this purpose, the influence of the main operating parameters (pH, surfactant concentration, and presence of cell debris or not) on GOX extraction was investigated at 25 degrees C. Without cell debris, the highest yield of GOX activity recovery (90.8%) was obtained performing (a) the forward extraction in isooctane as solvent and hexanol and butanol as cosolvents at 76/6/18 ratio, pH 7.0, 0.2 M cetyl trimethylammonium bromide as cationic surfactant, and electric conductivity of 5.0 mS cm(-1) and (b) the backward extraction at pH 5.5. Forward and backward extractions furnished comparable results when using raw homogenate, which demonstrates a negligible impact of the presence of cell debris on the process. The highest extraction yield (94%) was obtained under the same forward and backward conditions adopted without cell debris. The promising results of this work suggest that the proposed methodology could be profitably exploited at an industrial level.     

Effects of ionic surfactants used in reversed micelles on cutinase activity and stability

The effects of aqueous surfactant solutions on the kinetics and stability of cutinase from Fusarium solani pisi were studied. The surfactant sodium bis[2-ethylhexyl]ester sulfosuccinic acid (AOT) acts as a pseudo-competitive inhibitor within a limited concentration range relative to the hydrolysis of short-chain p-nitrophenyl esters. For higher concentrations a hyperbolic mixed inhibition takes place. A pseudo-activation of hydrolysis in presence of AOT and hexadecyltrimethyl-ammonium bromide (CTAB) was observed. CTAB has similar effects on kinetics of cutinase. Cutinase revealed to be stable in CTAB solutions, with activity retention as high as 80%. AOT has a deleterious effect on the enzyme in the time course, resulting in acute loss of activity possibly related with unfolding of the protein structure. A relation between deactivation rate constants and AOT/cutinase concentration ratios is suggested. The presence of the linear alcohol, 1-hexanol, was included in these solutions, in the attempt to interpret the deactivation of cutinase when encapsulated in reversed micelle systems in the absence of this co-surfactant.     

Liquid-liquid extraction of lectin from Cratylia mollis seeds using reversed micelles

Extraction of lectin from seeds ofCratylia mollis, camaratu bean, with reversed micelles of 100 mM sodium di(2-ethylhexyl) sulfosuccinate/isooctane was performed firstly with affinity-purified lectin. The best conditions were extraction of the seed extract at pH 5 and back-extraction at pH 10, giving yields of 38% and 100%, respectively.     

High pressure EPR studies of protein mobility in reversed micelles

We have investigated the effect of pressure on structural properties of subtilisin solubilized in reversed micelles of Tween-85/isopropanol in hexane. Electron paramagnetic resonance (EPR) spectra of spin-labeled enzyme indicate a reduction in spin-label mobility when the enzyme is transferred from aqueous solution to the microemulsion. One explanation for the spectral broadening is a change in the protein's active-site conformation and/or dynamics. However, over a W(0) range of 80 to 180, EPR spectroscopy could detect no change in the enzyme's environment, conformation, or molecular dynamics. The EPR spectra also contained a contribution from free spin label located in an environment with a polarity roughly between that of propanol and bulk water. No changes in the polarity surrounding the free spin label nor in the enzyme's structural properties were evident at pressures up to 10,000 psi. Previous work has demonstrated that pressure can be used to manipulate the size of some reversed micelles, and the EPR data indicated that for this system such pressure tuning of micellar properties will not adversely affect the structure of solubilized enzyme. (c) 1994 John Wiley & Sons, Inc.     

Fluorescence dynamics of green fluorescent protein in AOT reversed micelles

We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the organic solvents were isooctane and (the more viscous) dodecane, respectively. The water content of the water pools could be controlled through the parameter w0, which is the water-to-surfactant molar ratio. With steady-state fluorescence, it was observed that subtle fluorescence changes could be noted in reversed micelles of different water contents. EGFP can be used as a pH-indicator of the water droplets in reversed micelles. Time-resolved fluorescence methods also revealed subtle changes in fluorescence decay times when the results in bulk water were compared with those in reversed micelles. The average fluorescence lifetimes of EGFP scaled with the relative fluorescence intensities. Time-resolved fluorescence anisotropy of EGFP in aqueous solution and reversed micelles yielded single rotational correlation times. Geometrical considerations could assign the observed correlation times to dehydrated protein at low w0 and internal EGFP rotation within the droplet at the highest w0.     

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

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

Liquid-liquid extraction of a recombinant cutinase from fermentation media with AOT reversed micelles

This work reports the extraction and back-extraction of an intracellular recombinant cutinase from complex biological media using AOT reversed micelles in isooctane. Cutinase was recovered from different complex media namely, fermentation broths and supernatants after cell disruption by osmotic shock and sonication. The application of the AOT reversed micellar system to the extraction of cutinase allowed activity yields and purification factors ranging from about 5% to 50% and 1.2 to 10.2, respectively, depending on the biological medium.Maria das Graças Carneiro da Cunha, from ITEP-Instituto Tecnológico do Estado de Pernambuco, acknowledges a Ph.D fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Centro de Pesquisa Aggeu Magalhães, Recife — PE — Brasil. E. P. Melo thanks Junta Nacional de Investigação Científica, Lisboa, Portugal, for providing a Ph.D. fellowship. The scientific support given by Prof. Sílvia M. B. Costa for the spectroscopic data and further discussions are particularly acknowledged.This work was partly financed by the BRIDGE and BIOTECHNOLOGY Programmes (Contracts BIOT-CT91-0274(DTEE) and BIOT 2 CT-943016).     

蚜虫全蛋白提取方法的比较研究

为建立适于SDS-PAGE分析的蚜虫蛋白质样品制备平台,以便为蚜虫蛋白质的双向电泳分析奠定基础,本研究比较了TCA/丙酮沉淀、PEG提取、饱和酚抽提和直接裂解4种蛋白质提取方法.结果表明:不同样品制备方法的蛋白提取率有显著的差异,其中直接裂解法的提取率最高,为17.43 mg/g;其次是饱和酚抽提法,提取率为12.30 mg/g;而PEG制备法提取率最低,只有7.96 mg/g.利用SDS-PAGE电泳对不同的蛋白质样品进行了分析,发现在凝胶图谱上显现的条带也有明显的差异,其中饱和酚抽提法显现的条带数最多,为36条,且从14.4 kDa～116.0 kDa范围有广泛分布,条带清晰;PEG提取法条带数为30条,一些蛋白条带丢失或不明显;TCA/丙酮沉淀法的蛋白条带集中分布在25.0 kDa～67.0 kDa区域;直接裂解法条带数仅为24条,且小分子量的条带可辩率很低.通过以上结果可以得出,饱和酚抽提法最适用于蚜虫全蛋白样品的制备.     

A new strategy for the study of oligomeric enzymes: gamma-glutamyltransferase in reversed micelles of surfactants in organic solvents

A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.     

Stabilisation of tyrosinase by reversed micelles for bioelectrocatalysis in dry organic media

The enzymatic and bioelectrocatalytic activity of tyrosinase from mushrooms was studied in a system of reversed micelles formed by Aerosol OT (AOT) in hexane. The optimal catechol oxidising activity of tyrosinase incorporated in reversed micelles was found at a hydration degree of w(0)=25. The catalytic activity was comparable with tyrosinase activity in aqueous media. When immobilized at an Au electrode, either directly or in reversed micelles, tyrosinase exhibited a similar efficiency of the bioelectrocatalytic reduction of O(2) mediated by catechol; however, a rapid decrease in the activity correlated with the destruction of reversed micelles and/or the removal of tyrosinase from the electrode surface. The system containing tyrosinase in reversed micelles with caoutchouk, spread on the surface of the Au electrode and successively covered with a Nafion membrane layer, was found to result in stable tyrosinase-modified electrodes, which were resistant to inactivation in dry acetonitrile. The proposed technique offers possibilities for further development of highly active and stable surfactant/enzyme-modified electrodes for measurements carried out in organic solvents.     

The principles of enzymes stabilization: VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents

• 1.1.|The possibility of stabilizing water-soluble enzymes againsts the inactivating action of organic solvents by means of surfactants has been studied. Several enzymes (α-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity.
• 2.2.|A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of the charges in the substrate molecules and on interphase (++, +−, −−).
• 3.3.|The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.