Characteristics of antigen-antibody interaction in the reversed micelles of surface-active agents in heptane

The alterations in the catalytic activity of the horseradish peroxidase after its interaction with antibodies against this enzyme have been studied in buffered solution and in reversed Aerosol OT (AOT) micelles in heptane. The antibodies were obtained by immunizing the rabbits with electrophoretically homogeneous enzyme and were purified by affinity chromatography. In the AOT micelles and mixed micelles containing AOT and Triton X-45, the enzyme interacted with antibodies very rapidly (in less than 5 min), i.e. the micelles did not hinder effective interaction between the enzyme and antibodies. The decrease in the peroxidase catalytic activity upon its interaction with antibodies in a micellar medium was determined by [H2O]/[AOT] ratio, pH and molarity of polar nucleus, as well as by the initial concentration of antibody. In buffered solutions, the decrease n the peroxidase activity of the enzyme--antibody complex was only weakly dependent on pH and molarity of a buffer solution.     

Purification of intracellular enzymes from whole bacterial cells using reversed micelles

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

Tyrosinase activity in reversed micelles

The hydroxylase and oxidase activities of mushroom tyrosinase were studied in both sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane and cetyltrimethylammonium bromide (CTAB)/hexane/chloroform reversed micelles. The enzyme presented its highest activity when the water to surfactant molar ratio (W ₀) was 20 for both systems. When entrapped in the AOT reversed micelles, the enzyme activity decreased with the increase in AOT concentration at a constant W ₀, and the enzyme not only presented a higher reaction rate related to its oxidase activity but also a shorter lag period related to its hydroxylase activity. The relation between water activity and W ₀ revealed that enzyme activity in reversed micelles was more related to the size of the micelles which was determined by W ₀ and less to the water activity. Tyrosinase in CTAB reversed micelles showed potential for the analysis of o-diphenols.     

Protein refolding in reversed micelles

A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant-free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.     

Vesicle reconstitution from lipid-detergent mixed micelles

The process of formation of lipid vesicles using the technique of detergent removal from mixed-micelles is examined. Recent studies on the solubilization and reconstitution of liposomes participated to our knowledge of the structure and properties of mixed lipid-detergent systems. The mechanisms involved in both the lipid self assembly and the micelle-vesicle transition are first reviewed. The simplistic three step minimum scheme is described and criticized in relation with isothermal as well as a function of the [det]/[lip] ratio, phase diagram explorations. The techniques of detergent elimination are reviewed and criticized for advantages and disadvantages. New methods inducing micelle-vesicle transition using enzymatic reaction and T-jump are also described and compared to more classical ones. Future developments of these techniques and improvements resulting of their combinations are also considered. Proper reconstitution of membrane constituents such as proteins and drugs into liposomes are examined in the light of our actual understanding of the micelle-vesicle transition.     

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.     

Cell-free translation in reversed micelles.

Cell-free translation in reversed micelles (RM) of surfactants in organic solvents is demonstrated using as an example the synthesis of human interleukin-2 by the wheat germ translation system solubilized in Brij 96 (oleyl-poly(10)oxyethylene ether) RM in cyclohexane. The translation system components and the product were recovered from the RM system by acetone precipitation. The recovery and translation reaction yields depended on the degree of surfactant hydration. The translation yields in Brij 96 RM were close to that observed in regular aqueous solution. The Brij 96 RM system is regarded as a promising media for the cell-free synthesis of hydrophobic proteins. Meanwhile, no translation reaction was observed in Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) RM in octane, which presumably is due to the ability of Aerosol OT to bind Mg2+ ions necessary for the functioning of the translation apparatus.     

Substrate-induced stability of the lipase from candida cylindracea in reversed micelles

Summary Activity of lipase (candida cylindracea) in reversed micelles was found to be sustained over extended periods of time in the presence of amphiphilic substrates. Esterification of palmitic or oleic acid and octanol was studied to characterize the lipase activity in AOT/isooctane reversed micelles. Complete conversion was possible even in the presence of stoichiometric excess of water. In the absence of acyl substrates, the enzyme lost all its activity within a few hours in reversed micelles. Thermal effects on the enzyme activity were studied, and the enzyme stability in reversed micelles was compared to that in a bulk organic solvent.     

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.     

Recovery of proteins from reversed micelles using a novel ion-exchange material

Summary The recovery of proteins solubilised inside reversed micelles is generally a low yielding process. We have studied three methods for recovery of lysozyme. The protein laden organic phase was contacted with solutions of varying pH. Greater than 95% recovery was achieved at pH values close to the isoelectric point (10.9). The use of 2.5 M KCl gave close to 100% recovery of lysozyme. The organic phase was passed onto a ion exchange matrix. This resulted in disruption of the reversed micelle releasing 60–80% of the protein into the eluent.     

Selective recovery of xylanase from Penicillium janthinellum using BDBAC reversed micelles

A xylanase was removed from crude extract of the fungus Penicillium janthinellum under optimized conditions: 0.10M phosphate buffer, pH 7.0, 0.2 M BDBAC (N-benzyl-N-dodeceyl-N-bis (2-hydroxyethyl) ammonium chloride), 7.5% hexanole, 30°C and an agitation time of 1 minute. At 1.42 mg per ml protein concentration, 73% of the xylanase activity was recovered and a 7-fold enrichment factor was obtained. The enzyme had a molecular weight (MW) of 20.1 kDa and the isoelectric point (PI) revealed the presence of two protein bands with a PI of 6.0 and 6.5. The optimum pH and optimum temperature were 4.2 and 50°C, respectively. The low pH differential between the aqueous medium and the protein PI seemed to influence the xylanase transportation into the reversed micelles.     

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.

     

Engineering of functional supramacromolecular complexes of proteins (enzymes) using reversed micelles as matrix microreactors.

The size of the inner water cavity of reversed micelles formed in a triple system 'water-surfactant-organic solvent' can be widely varied by changing the degree of surfactant hydration. This gives grounds to use reversed micelles as matrix microreactors for the design of supramolecular complexes of proteins. Using ultracentrifugation analysis, it has been demonstrated that the oligomeric composition of various enzymes (ketoglutarate dehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) solubilized in reversed micelles of Aerosol OT [sodium bis(2-ethylehexyl)sulfosuccinate] in octane changes upon variation of the degree of hydration. An oligomeric complex forms under conditions when the radius of the micelle inner cavity is big enough to incorporate this complex as a whole. At lower degrees of hydration the micelles 'uncouple' such complexes to their components. The catalytic properties of various oligomeric complexes have been studied. Possibilities of using reversed micelles for the separation of subunits of oligomeric enzymes under non-denaturating conditions have been demonstrated. In particular, the isolated subunits of alkaline phosphatase, lactic dehydrogenase and glyceraldehyde-3-phosphate have been found to be active in Aerosol OT reversed micelles. The dependences of the catalytic activity of oligomeric enzymes represent saw-like curves. The maxima of the catalytic activity observed at these curves relate to the functioning of various oligomeric forms of an enzyme. The radii of the micelle inner cavity under conditions when these maxima are observed correlate with the linear dimensions of the enzyme oligomeric forms. Correlation of the position of a maximum with the shape of an oligomeric complex is discussed.     

Ester hydrolyses in reversed micelles using lipase

Lipases are enzymes which require a favourable reaction system for efficient catalysis of their hydrophobic substrates and reversed micellar environment is one such medium which offers many advantages. Hydrolytic studies of esters of paranitrophenol and glycerol using imidazole and four fungal lipases are studied in AOT/isooctane reversed micelles. The effect of water and surfactant concentration on the hydrolysis of rice bran oil is investigated and the overall potential of the reversed micellar system for hydrolytic reactions is assessed.     

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.