Higher order structure of Mucor miehei lipase and micelle size in cetyltrimethylammonium bromide reverse micellar system

The higher order structure of Mucor miehei lipase and micelle size in a cationic cetyltrimethylammonium bromide (CTAB) reverse micellar system was investigated. Circular dichroic (CD) measurement revealed that the lipase far-UV CD spectra changed markedly, going from buffer solution to the reverse micellar solution, and were very similar for any organic solvent used. The ellipticity of the solubilized lipase in the far-UV region markedly decreased with increasing water content (W(0): molar ratio of water to CTAB), indicating that the secondary structure of lipase changed with the water content. The linear correlation between the W(0) and the micelle size was obtained by measuring dynamic light scattering. From the linear correlation between the micelle size and W(0), the higher order structure of the solubilized lipase appears to be affected directly by the micellar interface. The species and concentration of alcohol as a cosurfactant had an inferior effect on lipase structure. Especially, at ratios of 1-pentanol to CTAB of less than 8, the secondary and tertiary structures of lipase were preserved in the reverse micelles. The CTAB concentration had little effect on the lipase structure in the micelles. The catalytic activity of the lipase solubilized in the CTAB reverse micelles increased with increasing the W(0).     

Enzymatic production of L-tryptophan in a reverse micelle reactor

The enzymatic production of tryptophan from indole and serine was investigated in a micellar solution of the surfactant Brij 56 in cyclohexane. An anion exchanger was employed to facilitate the transfer of tryptophan and serine between the water pool of the reverse micelle and the bulk organic phase. The influence of potassium ion, water content, pH, and co-surfactant on enzyme activity is reported. Kinetic studies indicate that the enzyme is not inhibited by indole in the micellar system and that the enzyme is more stable in reverse micelles than in bulk water. The design of a continuous reverse micelle reactor, which accommodates both product recovery and enzyme reactivation, is discussed.     

Use of reverse micelles in membrane protein structural biology

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conceptualisation of articular cartilage as a giant reverse micelle: a hypothetical mechanism for joint biocushioning and lubrication

Phospholipid (PL) molecules form the main structure of the membrane that prevents the direct contact of opposing articular cartilage layers. In this paper we conceptualise articular cartilage as a giant reverse micelle (GRM) in which the highly hydrated three-dimensional network of phospholipids is electrically charged and able to resist compressive forces during joint movement, and hence loading. Using this hypothetical base, we describe a hydrophilic-hydrophilic (HL-HL) biopair model of joint lubrication by contacting cartilages, whose mechanism is reliant on lamellar cushioning. To demonstrate the viability of our concept, the electrokinetic properties of the membranous layer on the articular surface were determined by measuring via microelectrophoresis, the adsorption of ions H, OH, Na and Cl on phospholipid membrane of liposomes, leading to the calculation of the effective surface charge density. The surface charge density was found to be -0.08+/-0.002cm(-2) (mean+/-S.D.) for phospholipid membranes, in 0.155M NaCl solution and physiological pH. This value was approximately five times less than that measured in 0.01M NaCl. The addition of synovial fluid (SF) to the 0.155M NaCl solution reduced the surface charge density by 30% which was attributed to the binding of synovial fluid macromolecules to the phospholipid membrane. Our experiments show that particles charge and interact strongly with the polar core of RM. We demonstrate that particles can have strong electrostatic interactions when ions and macromolecules are solubilized by reverse micelle (RM). Since ions are solubilized by reverse micelle, the surface entropy influences the change in the charge density of the phospholipid membrane on cartilage surfaces. Reverse micelles stabilize ions maintaining equilibrium, their surface charges contribute to the stability of particles, while providing additional screening for electrostatic processes.     

The use of reverse micelles for the simultaneous extraction of oil and proteins from vegetable meal

A method for the simultaneous extraction of oil and proteins from vegetable meals is presented. The method uses hydrocarbon reverse micelles, so that the oil is extracted directly into the hydrocarbon phase and the proteins are solubilized in the water pools of the reverse micelles. The surfactant used is bis (2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane at variable w(0) values (w(0) measures the amount of water in the system, where w(0) = [H(2)O]/[AOT]). A comparison with the usual extraction methods is offered. It is shown that with the micelle system the extraction of oil is as large as with the usual methods, and it is independent of w(0). However the amount and type of proteins extracted depends strongly on w(0). At w(0) values below 6, no protein and only low molecular weight compounds (i.e. chlorogenic acid) are extracted, at larger water content (i.e. by increasing the dimension of the micelle water pool), also proteins are solubilized in a significant amount and with a molecular weight which increases by increasing W(0). The protein solubilized in the microemulsion system can be recovered into an aqueous phase with a back-transfer step.     

Mechanisms of protein solubilization in reverse micelles

Solubilization properties of alpha-chymotrypsin and alcohol dehydrogenase (LADH) in reverse micelles are reported for three different solubilization techniques. The solubilization properties for these two proteins depend on the method used for protein addition. The addition of a dry protein powder to a reverse-micelle-containing organic phase does not appreciably solubilize the protein until the diameter of the reverse micelle is similar to that of the protein. However, when an aqueous protein solution is injected an organic phase, protein solubilization is not strongly dependent on micelle size. For chymotrypsin, multiple protein occupancy occurs at large micelle size, with as many as 11 chymotrypsin molecules solubilized in one reverse micelle. The solubilization of chymotrypsin using a phase-transter technique with a positively charged surfactant follows the expected traned based on protein-surfactant electrostatic interactions. When a negatively charged sufactants is used for phase transfer, at low pH the solubilization data do not fit this electrostatic interaction mechanism. In this case, proteinsurfactant aggregation may be occurring at the aqueousorganic interface.     

Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles

A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes.     

Ethylene glycol and the thermostability of trypsin in a reverse micelle system

The influence of ethylene glycol (EG) on the kinetics of hydrolysis of N-alpha-benzoyl-L-arginine ethyl ether catalyzed by trypsin encapsulated in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT)-based reverse micelles was studied at different temperatures. Ethylene glycol was shown to shift the range of the trypsin activity in the reverse micelles towards higher temperatures. Infrared spectroscopy showed a stabilizing effect of EG on the secondary structure of the protein in the system of reverse micelles. Electron spin resonance spectroscopy showed that the solubilized protein affected the interactions of EG with the polar head groups of AOT and altered the rigidity of the micellar matrix. The results indicate that EG increases the thermostability of the solubilized enzyme in microemulsion media by two mechanisms.     

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.     

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.     

Conceptualisation of articular cartilage as a giant reverse micelle: A hypothetical mechanism for joint biocushioning and lubrication

Phospholipid (PL) molecules form the main structure of the membrane that prevents the direct contact of opposing articular cartilage layers. In this paper we conceptualise articular cartilage as a giant reverse micelle (GRM) in which the highly hydrated three-dimensional network of phospholipids is electrically charged and able to resist compressive forces during joint movement, and hence loading. Using this hypothetical base, we describe a hydrophilic–hydrophilic (HL–HL) biopair model of joint lubrication by contacting cartilages, whose mechanism is reliant on lamellar cushioning. To demonstrate the viability of our concept, the electrokinetic properties of the membranous layer on the articular surface were determined by measuring via microelectrophoresis, the adsorption of ions H, OH, Na and Cl on phospholipid membrane of liposomes, leading to the calculation of the effective surface charge density. The surface charge density was found to be −0.08 ± 0.002 cm⁻² (mean ± S.D.) for phospholipid membranes, in 0.155 M NaCl solution and physiological pH. This value was approximately five times less than that measured in 0.01 M NaCl. The addition of synovial fluid (SF) to the 0.155 M NaCl solution reduced the surface charge density by 30% which was attributed to the binding of synovial fluid macromolecules to the phospholipid membrane. Our experiments show that particles charge and interact strongly with the polar core of RM. We demonstrate that particles can have strong electrostatic interactions when ions and macromolecules are solubilized by reverse micelle (RM). Since ions are solubilized by reverse micelle, the surface entropy influences the change in the charge density of the phospholipid membrane on cartilage surfaces. Reverse micelles stabilize ions maintaining equilibrium, their surface charges contribute to the stability of particles, while providing additional screening for electrostatic processes.     

Biocatalytic oxidation of bisphenol A in a reverse micelle system using horseradish peroxidase

Horseradish peroxidase (HRP) was used to catalyze the oxidation of bisphenol A (BPA) in a reverse micelle system consisting of water, sodium bis(2-ethylhexyl)sulfosuccinate (AOT) as the surfactant, and n-octane as the organic solvent phase. In order to achieve maximal BPA transformation, a water-to-surfactant molar ratio greater than 15 was required, above which no further increase in conversion was observed. BPA transformation was catalyzed in the reverse micelle system over a pH range of 6-9 with an optimum at pH 7 and was enhanced with increasing temperatures up to 40 degrees C. The stoichiometric ratio of moles of bisphenol A transformed per mole of peroxide consumed was 0.46 when the initial BPA concentration was 0.01 mM, which is significantly less than the theoretical value of 2 based on the known catalytic cycle of the enzyme. However, the stoichiometric ratio increased and approach the theoretical value with higher BPA concentrations. Over the course of the catalytic reaction, the enzyme became inactivated. Hydrogen peroxide strongly inhibited the enzyme and, thus, when the oxidant was present in quantities in excess of the stoichiometric amount, BPA transformation was significantly reduced.     

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.     

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.     

A theoretical study on the expression of enzymic activity in reverse micelles.

The present work deals with a theoretical model of catalysis by enzymes entrapped in reverse micelles. Three aspects of the enzyme-reverse-micelle system have been considered: structure, dynamics and enzyme distribution and catalysis in reverse micelles. A proposed structural model of reverse micelles [El Seoud (1984) in Reverse Micelles (Luisi, P. L. & Straub, B. E., eds.), p. 81, Plenum Press, New York] consists of three domains: surfactant apolar tails, bound water and free water. Dynamics are based on a dynamic equilibrium of association-dissociation that lead one to consider the dispersed polar phase as a pseudo-continuous phase [Luisi, Giomini, Pileni & Robinson (1988) Biochim. Biophys. Acta 947, 207-246]. Enzyme is distributed among the reverse-micelle domains and it expresses a catalytic constant for each one of them. The overall activity is calculated taking into account the volume in which enzyme is solubilized, and expressed as a function of the whole volume (V). The characteristic parameters of reverse micelles, omega 0 (= [H2O]/[surfactant]) and theta (= % water, v/v), were investigated as modulators of enzymic activity. Three basic patterns of modulation by omega 0 were found depending on which domain the enzyme expressed the highest catalytic constant. Combinations of those basic patterns lead to other modulation types that can be found experimentally, such as superactivation. Other combinations predict behaviour patterns not described to date, such as superinhibition. Dependence of catalytic activity on theta was only stated at omega 0 values around a critical value, which coincides with the appearance of free water.     

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. Copyright 1998 John Wiley & Sons, Inc.     

Variations in the activity of microsomal palmitoyl-CoA hydrolase in mixed micelle solutions of palmitoyl-CoA and non-ionic detergents of the triton X series

The kinetics of palmitoyl-CoA hydrolase were influenced by both the availability of the substrate and formation of micelles. At palmitoyl-CoA concentrations below the critical micelle concentration, addition of non-ionic detergent increased the activity until the critical micelle concentration of the mixed micelles was reached. At palmitoyl-CoA concentrations above the critical micelle concentration, inhibitor of the activity was observed, but addition of detergents of the Triton X series reversed the inhibition. Maximum palmitoyl-CoA hydrolase activity was found when the ratios (w/v) of palmitoyl-CoA: Triton X-100 and palmitoyl-CoA: Triton X-405 were approximately 0.35 and 0.05, respectively. At these above the mixed critical micelle concentration. The results indicate that monomer palmitoyl-CoA is the substrate and that monomer forms of the non-ionic detergents of the Triton X series activate the enzyme. Isolated microsomal lipids activated the microsomal palmitoyl-CoA hydrolase, suggesting that a hydrophobic environment is advantageous for interaction between enzyme and substrate in vivo. The maximum activity in the presence of mixed micelles is discussed in relation to a model where mixed micelles are regarded as artificial membranes to which the enzyme may adhere in an equilibrium with the monomer substrate and detergent in the monomer form. It is suggested that intracellular membranes may resemble mixed micelles in equilibrium with detergent-active substrates such as palmitoyl-CoA.     

Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures

Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-β-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K_m for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.     

Comparison of the size and physical properties of gamma-glutamyltranspeptidase purified from rat kidney following solubilization with papain or with Triton X-100.

gamma-Glutamyltranspeptidase is associated with the brush border membrane of kidney proximal straight tubule cells. It can be solubilized qualitatively by treatment with papain or Triton X-100. Neither procedure affects its catalytic activity but the two resulting forms of the enzyme differ considerably in their physical properties. The papain-solubilized transpeptidase is soluble in aqueous buffers and was purified 430-fold. It has an s20,w of 4.9 S, a Stokes radius of 36 A, and a calculated molecular weight of 69,000. It appears homogeneous by sedimentation equilibrium centrifugation (Mr=66,700). In contrast, the Triton-solubilized transpeptidase is soluble only in the presence of detergents and was purifed 300-fold. This form of the enzyme has a Stokes radius of 70 A but an s20,w of only 4.15 S. Aggregation of the enzyme just below the critical micelle concentration of Triton X-100 and its ability to bind 1.16 mg of Triton X-100-protein complex was calculated to be 169,000, but the glycoprotein portion of the complex is 52% of the total mass (87,000). The mass of Triton X-100 (82,000) is consistent with its reported micelle molecular weight. Treatment of the Triton-purified transpeptidase with papain or bromelain results in a form of the enzyme identical in all respects with the papain-purified enzyme. Both the Triton- and papain-purified transpeptidase exhibit two protein bands on sodium lauryl sulfate-polyacrylamide gel electrophoresis. The smaller subunits of the two forms appear identical (Mr=27,000), while the larger subunits of the Triton- and papain-purified enzyme have apparent molecular weights of 54,000 and 51,000, respectively. These data suggest that a peptide (3,000 to 19,000) in the larger subunit of gamma-glutamyltranspeptidase is responsible for its binding to Triton micelles and probably for holding the enzyme in the brush border membrane.