Mechanism of extraction of chymotrypsin into isooctane at very low concentrations of aerosol OT in the absence of reversed micelles

Chymotrypsin is easily extracted from an aqueous solution into isooctane containing the anionic surfactant aerosol OT (AOT). The concentration of AOT needed to efficiently extract 0.5 mg/mL CMT is as low as 1 mM and as low as 0.2 mM AOT was sufficient to extract the protein into isooctane. The extraction process was unaffected by 10% (v/v) ethyl acetate in the isooctane phase. Moreover, spectroscopic analysis by electron paramagnetic resonance indicated that CMT did not exist inside a discreet water pool of a reversed micelle. Calculations of the number of AOT molecules associated per extracted CMT molecule indicate that only ca. 30 surfactant molecules interact with the protein, a value too low for reversed micellar incorporation of the protein in isooctane. These studies suggested that reversed micelles do not need to be involved in the actual transfer of the protein from the aqueous to the organic phase and protein solubilization in the organic phase is possible in the absence of reversed micelles. Based on these findings, a new mechanism has been proposed herein for protein extraction via the phase transfer method involving ionic surfactants. The central theme of this mechanism is the formation of an electrostatic complex between CMT and AOT at the aqueous/organic interface between AOT and CMT, thereby leading to the formation of a hydrophobic species that partitions into the organic phase. Consistent with this mechanism, the efficiency of extraction is dependent on the interfacial mass transfer, the concentrations of CMT and AOT in the aqueous and organic phases, respectively; the ionic strength of the aqueous phase; and the presence of various cosolvents. (c) 1994 John Wiley & Sons, Inc.     

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.     

Photosynthetic activity of plant cells solubilized in water-in-oil microemulsions

With the aim of possibly extending plant microbiology and photosynthesis beyond the usual applicability in aqueous solution, we investigated the solubilization of plant cells inorganic media with the help of water-in-oil microemulsions. Cells isolated from leaves of Rumex obtusifolius were solubilized in a water/2-ethyl-hexyl-sodiumsulfosuccinate/isooctane system, containing 20% water (v:v) and 240 mM surfactant, and the oxygen evolution/consumption was measured polarographically. Although no oxygen evolution was detectable in the organic medium, the cells were able to carry out photosynthetic oxygen consumption at the expense of ascorbate. To a lesser extent, photosynthetic oxygen consumption was measured using N, N, N', N'-tetramethyl-p-phenylenediamine as electron donor. The rate of ascorbate photooxidation was linearly related to the concentration of cells.     

Activation of lignin peroxidase in organic media by reversed micelles

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media.     

Solid-phase peptide synthesis by ion-paired alpha-chymotrypsin in nonaqueous media

Solid-phase synthesis of dipeptides in low-water media was achieved using AOT ion-paired alpha-chymotrypsin solubilized in organic solvents. Multiple solvents and systematic variation of water activity, a(w), were used to examine the rate of coupling between N-alpha-benzyloxycarbonyl-L-phenylalanine methyl ester (Z-Phe-OMe) and leucine as a function of the reaction medium for both solid-phase and solution-phase reactions. In solution, the observed maximum reaction rate in a given solvent generally correlated with measures of hydrophobicity such as the log of the 1-octanol/water partitioning coefficient (log P) and the Hildebrand solubility parameter. The maximum rate for solution-phase synthesis (13 mmol/h g-enzyme) was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.30. For the synthesis of dipeptides from solid-phase leucine residues, the highest synthetic rates (0.14-1.3 mmol/h g-enzyme) were confined to solvent environments that fell inside abruptly defined regions of solvent parameter space (e.g., log P > 2.3 and normalized electron acceptance index <0.13). The maximum rate for solid-phase synthesis was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.14. In 90/10 and 70/30 (v/v) isooctane/tetrahydrofuran environments with a(w) set to 0.14, seven different N-protected dipeptides were synthesized on commercially available Tentagel support with yields of 74-98% in 24 h.     

Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media

A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.     

Catalytic activity of lignin peroxidase and partition of veratryl alcohol in AOT/isooctane/toluene/water reverse micelles

The activity of lignin peroxidase (LiP) and the partition of its optimum substrate veratryl alcohol (VA) in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/toluene/water reverse micelles were studied in this paper to understand the microheterogeneous effect of the medium on the catalytic properties of LiP hosted in the reverse micelle. Results showed that LiP from Phanerochaete chrysosporium could express its activity in the reverse micelles, but its activity depended, to a great extent, on the composition of the reverse micelles. Optimum activity occurred at a molar ratio of water to AOT (ω₀) of 11, a pH value of 3.6, and a volume ratio of isooctane to toluene of 7–9. Under optimum conditions, the half-life of LiP was circa 12 h. The dependence of LiP activity on the volume fraction of water in the medium (θ), at a constant ω₀ value of 11, indicated that VA was mainly solubilized in the pseudophase of the reverse micelle. Based on the pseudobiphasic model and the corresponding kinetic method, a linear line can be obtained in a plot of apparent Michaelis constant of VA vs θ, and the partition coefficient of VA between the pseudophase and the organic solvent phase was determined to be 35.8, which was higher than that (22.3) between bulk water and the corresponding mixed organic solvent. H₂O₂ inhibited LiP at concentrations higher than 80 μM; this concentration value seems to be different from that in aqueous solution (about 3 mM). The differences mentioned above should be ascribed to the microheterogeneity and the interface of the AOT reverse micelle.     

Microbial Oxidation of Tetradecanols and Related Substances in Organic Solvents

Microbial oxidations of n-tetradecane, tetradecanols and tetradecanoic acid were investigated by using intact cells of Corynebacterium equi, a hydrocarbon-assimilating bacterium, in an aqueous phase and organic solvents. The bacterial cells were hydrophobic and could be well dispersed in all organic solvents employed to give homogeneous reaction mixtures, and among them, isooctane was found to be the best for the reaction. n-Tetradecane and tetradecanoic acid were completely oxidized in the aqueous phase, but not in isooctane. In contrast, 1-tetradecanol was oxidized much more readily in isooctane than in the aqueous phase, and an oxidation product identified as myristyl myristate was accumulated in isooctane at the conversion rate of 80%. 2-Tetradecanol was also readily oxidized in isooctane, and 2-tetradecanone was obtained at the conversion rate of nearly 100%. Similar results were obtained when toluene and n-hexane were used as the solvent in place of isooctane, while no reaction was observed when chloroform was employed.     

Study of the factors affecting the extraction of soybean protein by reverse micelles

In this work, the forward and back extraction of soybean protein by reverse micelles was studied. The reverse micellar systems were formed by anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT), isooctane and KCl solution. The effects of AOT concentration, aqueous pH, KCl concentration and phase volume ratio on the extraction efficiency of soybean protein were tested. Suitability of reverse micelles of AOT and Triton-X-100/AOT mixture in organic solvent toluene for soybean protein extraction was also investigated. The experimental results lead to complete forward extraction at the AOT concentration 120 mmol l⁻¹, aqueous pH 5.5 and KCl concentration 0.8 mol l⁻¹. The backward extraction with aqueous phase (pH 5.5) resulted in 100% extraction of soybean protein from the organic phase.     

Three Systems Used for Biocatalysis in Organic Solvents a Comparative Study

The activity and operational stability of horse liver alcohol dehydrogenase (HLADH) and α-chymotrypsin were investigated in three systems commonly used for biocatalysis in organic solvents:

1. enzyme adsorbed on a solid support (celite) and added to the organic solvent (isooctane)

2. enzyme powder directly added to the organic solvent (isooctane).

3. enzyme dissolved in a microemulsion (AOT/isooctane).

The activity and the operational stability in all systems were strongly dependent on the water content. The initial reaction rate was high in both the microemulsion and the celite system, but was much lower when adding the enzymes directly to the organic solvent. HLADH was observed to be more stable when added directly to the organic solvent or dissolved in the microemulsion than when adsorbed on celite, whereas for α-chymotrypsin stability was higher when adsorbed on celite or added directly to the organic solvent. For a hydrolytic reaction, a microemulsion was preferred due to the high water content. When adding the enzymes directly to the organic solvent both HLADH and chymotrypsin were adsorbed strongly to the glass walls of the reaction vessel. None of the systems were superior in all respects for the two enzymes studied.     

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.     

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

The extraction of solid-phase alpha-chymotrypsin, bovine serum albumin (BSA), and lysozyme by water-in-oil microemulsion (w/o-ME) solution containing Aerosol-OT (AOT) was thoroughly examined as a means to maximize protein solubilization in organic solvent media. Protein extraction occurred simultaneously with the adsorption of water and AOT by the solid protein. Water and AOT were desorbed at nearly equal rates, suggesting that both materials were desorbed together as micreomulsions. The solubilization of protein increased linearly with the ratio of solid protein to extractant solution except at a high value of the ratio, where most protein-containing microemulsions were desorbed. Based on our results, a mechanistic model was developed to describe the solid-phase extraction procedure. First, microemulsions are desorbed from solution by the solid protein, resulting in the formation of a solid protein-AOT-water aggregate. Second, when a protein in the solid phase binds to a sufficient number of microemulsions, the resulting aggregate's increased hydrophobicity drives its solubilization into lipophilic solvent. Third, through the exchange of materials between the solubilized precipitate and the remaining microemulsions, protein-containing w/o-MEs are formed. The presence of adsorption is further indicated by an isotherm existing between the water, AOT, and protein content of the resulting solid phase for each protein. The driving force behind adsorption is either AOT-protein interactions or the protein's affinity for microemulsion-encapsulated water, depending on the properties of the protein and the size of the microemulsions, in agreement with the model of P. L. Luisi [Chimia, 44: 270-282 (1990)]. The second step of our model is mass transfer limited for the extraction of solid alpha-chymotrypsin and BSA. The extraction of solid lysozyme was limited by the occurrence of an irreversible precipitation process. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 583-593, 1997.     

Reverse micellar extraction and precipitation of lysozyme using sodium di(2-ethylhexyl) sulfosuccinate

Sodium di(2-ethylhexyl) sulfosuccinate, referred to as Aerosol-OT or AOT, was used to remove lysozyme from an aqueous phase via reverse micellar extraction and precipitation method. For both methods, when the surfactant was in excess, a complete removal of lysozyme from the aqueous phase was obtained at the values of pH below the pI of lysozyme. However, for the reverse micellar method, a solubilization limit of lysozyme in the organic phase was observed, and a white precipitate was formed at the aqueous-organic interface. This observation suggested using AOT directly as a precipitating ligand. The lysozyme precipitated with AOT was fully recovered, with its original enzymatic activity, using acetone as a recovery solvent. A mechanism is suggested to explain the solubilization of lysozyme in an AOT reverse micellar system. It is shown that a direct precipitation method can be used with advantage instead of using the reverse micellar extraction method to recover lysozyme from an aqueous phase.     

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.     

Characteristics of Lipase-Catalyzed Glycerolysis of Triglyceride in AOT-Isooctane Reversed Micelles

Chromobacterium viscosum lipase, solubilized in microemulsion droplets of glycerol containing small amounts of water and stabilized by a surfactant, could catalyze the glycerolysis of triolein. Kinetic analysis of the lipase-catalyzed reaction was possible in the reversed micellar system. Among surfactants and organic solvents tested, bis(2-ethylhexyl)sodiumsulfosuccinate (AOT) and isooctane were respectively most effective, for the glycerolysis of triolein in reversed micelles. Temperature effects, pH profile, K_m,app, and V_max,app were determined. Among various chemical compounds, Fe³⁺, Cu²⁺, and Hg²⁺ inhibited the lipase-catalyzed glycerolysis severely. However, the glycerolysis activity was partially restorable by adding histidine or glycine to the system containing these metal ions. The glycerolysis activity was dependent on water content and maximum activity was obtained at an R value of 1.21. Higher stability of the lipase was obtained in the reversed micellar system.     

A comparative study of lysozyme conformation in various reverse micellar systems

The activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane/H₂O, cetyltrioctylammoniumbromide (CTAB)/CHCl₃, isooctane/H₂O; tetraethyleneglycoldodecylether (EO₄C₁₂)/isooctane/H₂O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w₀ (= [H₂O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO₄C₁₂ or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. Biol. Chem. 256 , 837–843] dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG₃, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG₃, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w₀ using the chromophoric substrate 3,4-dinitrophenyl-tetra-N-acetyl-β-D -chitotetraoside (3,4-DNP-NAG₄). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, k_cat tends to be smaller than in aqueous solution (with quite similar K_M), whereas in the EO₄C₁₂ system (at pH 7.0) the turnover number is larger and K_M is smaller than in water. In the AOT system, the kinetic parameters at pH 7.0 are also quite comparable to those found in water.     

脂肪酶在微乳液和微乳液凝胶中催化辛酸辛醇的酯化反应

脂肪酶在合成反应中具有很高的区域选择性和立体选择性 ,已广泛用于食品工业和药物工业[1,2 ] ,在有机介质中的脂肪酶催化反应已有较多研究[3 ,4 ] 。微乳液一般由表面活性剂、助表面活性剂、油和水等组份组成 ,它是一种热力学稳定、光学透明、宏观均匀而微观不均匀的体系 ,能提供酶催化所需要的巨大油 /水界面[5] 。而将脂肪酶增溶于油包水(Ｗ /Ｏ)微乳液中的纳米级“水池”中 ,可使酶以分子水平分散[6] ,图 1(ａ) ,从而可用来模拟细胞微环境中的反应。油包水微乳液中的酶可通过加入明胶而制成固定化酶 ,含明胶的微乳液凝胶 (ＭＢＧｓ)最早…     

Solution behavior of α-chymotrypsin dissolved in nonpolar organic solvents via hydrophobic ion pairing

Dissolution of α-chymotrypsin in nonpolar organic solvents can be achieved using hydrophobic ion pairing, whereby the polar counterions are replaced by a stoichiometric number of detergent molecules. Using Aerosol OT[AOT, sodium bis(2-octyl)sulfosuccinate], it is possible to partition significant amounts of the enzyme into alkanes and chlorocarbons. Apparent solubility in isooctane is greater than 1 mg/mL (80 μM). Necessary conditions for achieving effective partitioning of α-chymotrypsin into these solvents are described. Using CD spectroscopy, it can be shown that the AOT–α-chymotrypsin (CMT) complex retains its native secondary and tertiary structure when dissolved in alkanes, and that the globular structure is stable to more than 100°C. In contrast, α-chymotrypsin unfolds at 54°C in aqueous solution. The relative solubility of the AOT–CMT complex in a variety of alkanes and chlorocarbons is also reported. The native structure of α-chymotrypsin is maintained in carbon tetrachloride, but not in methylene chloride or chloroform. © 1995 John Wiley & Sons, Inc.     

Comparison of tyrosinase activity and stability in aqueous and nearly nonaqueous environments

The activity and stability of tyrosinase were compared in aqueous and two nearly nonaqueous environments (a low-water solvent system and reversed micelles). Initial rates of oxidation of methyl- and butyl-catechols in aerosol OT, sodium di-2-ethylhexylsulfosuccinate, (AOT)/isooctane micelles were higher than in aqueous solution, showing superactivity, whereas lower rates were obtained in cetyltri-methylammonium bromide (CTAB)/hexane/chloroform micelles and in chloroform containing celite-supported enzyme. The enzyme was most stable in chloroform, whereas half-lives in aqueous buffer and in both AOT and CTAB micelles were lower. The optimal reaction temperatures were higher in both micelles than in water but lower in chloroform. Thus, tyrosinase was active in ≤3.5% v/v water with apparent K_m, V_max, and activation energies reasonably similar to those in aqueous solution.     

Characteristics of Lipase-Catalyzed Glycerolysis of Triglyceride in AOT-Isooctane Reversed Micelles

Chromobacterium viscosum lipase, solubilized in microemulsion droplets of glycerol containing small amounts of water and stabilized by a surfactant, could catalyze the glycerolysis of triolein. Kinetic analysis of the lipase-catalyzed reaction was possible in the reversed micellar system. Among surfactants and organic solvents tested, bis(2-ethylhexyl)sodiumsulfosuccinate (AOT) and isooctane were respectively most effective, for the glycerolysis of triolein in reversed micelles. Temperature effects, pH profile, K_m,app, and V_max,app were determined. Among various chemical compounds, Fe³⁺, Cu²⁺, and Hg²⁺ inhibited the lipase-catalyzed glycerolysis severely. However, the glycerolysis activity was partially restorable by adding histidine or glycine to the system containing these metal ions. The glycerolysis activity was dependent on water content and maximum activity was obtained at an R value of 1.21. Higher stability of the lipase was obtained in the reversed micellar system.