Studies on the catalytic behaviour of a cholinesterase-like abzyme in an AOT microemulsion system

The hydrolytic activity of a monoclonal catalytic antibody (9A8) (abzyme) with acetylcholinesterase-like activity was investigated in water-in-oil (w/o) microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane, using p- and o-nitrophenylacetate (p-and o-NPA) as substrates. The dependence of the abzyme hydrolytic activity on the molar ratio of water to surfactant (w(o)) showed a bell-shaped curve, presenting a maximum at w(o)=11.1. An increase of the AOT concentration at constant w(o), resulted in a decrease of the catalytic activity suggesting a possible inhibition effect of the surfactant. The incorporation of the abzyme into the reverse micelle system caused a blue shift of the fluorescence emission maximum by a magnitude of 7-10 nm depending on the w(o) value. This result indicates that the antibody molecule, or a large part of it, is located in the aqueous microphase of the system. Kinetic studies showed that the hydrolysis of p-and o-NPA in microemulsion system as well as in aqueous solution follows Michaelis-Menten kinetics. The catalytic efficiency (k(cat)/K(m)) in w/o microemulsion was significant lower than in aqueous solution.     

Effect of hydration degree of aerosol OT reversed micelles and surfactant concentration in heptane on spectral and catalytic properties of catalase.

The influence of micelle hydration degree (w0) and AOT concentration on fluorescence, circular dichroism (CD), catalytic activity, and stability of catalase in Aerosol OT (AOT) reversed micelles in heptane was investigated. The quantitative parameters--differential fluorescence of catalase (DeltaI), protein molar ellipticity ([theta]lambda), initial rate of catalytic reaction, catalase efficiency (kcat/Km), and rate constant of enzyme inactivation (kin, sec-1)--decreased with increasing AOT concentration in micellar systems, reflecting the interaction of solubilized catalase with the AOT micellar aggregates in heptane. The dependences of all these parameters on increasing hydration degree of micelles (w0) were characterized by the appearance of maxima at w0 of 8, 15-18, and 26-30. These maxima are suggested to reflect three different states of catalase in the micellar system, distinguished by their conformations and catalytic activity, which is determined by the micellar microenvironment of the enzyme.     

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.     

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.     

Solubilization of soybean mitochondria in AOT/isooctane water-in-oil microemulsions

The water-in-oil microemulsion system bis(2 ethyl-hexyl-sodium-succinate (AOT)/isooctane/water is able to solubilize soybean nodules mitrochrondria. Transparent and thermodynamically stable hydrocarbon solutions are obtained, which can be assayed for mitochondrial activity just as aqueous solutions. Malate dehydrogenase (MDH) activity was measured in vivo and gave in reverse micelles very similar results as in water. However the kinetic behavior of this reaction in AOT/isooctane reverse micelles shows some differences with respect to water. Mitochondria in reverse AOT micelles are able to retain about 70% of their initial MDH activity after three days. Mitochondria can be back-transferred from reverse micelles to water and show respiratory activity almost identical to the native organelles. Electron microscopy studies show that the dimensions of mitochondria back-transferred into water from AOT micelles are comparable to the dimensions of the native organelles.     

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.     

Effect of Chemical Modification of Lipase on the Regulation of Its Lipolytic Activity in Reversed Micelles

Hydrophilized and hydrophobized forms of the lipase from Mucor miehei were obtained by its chemical modification with cellobiose and N-succinimidyl palmitate with a modification degree of 4 in both cases. A comparative analysis of the regulation of the catalytic activities of the native and modified lipases was carried out in the system of reversed micelles of OT aerosol (AOT) in isooctane. The level of catalytic activity of all the lipase preparations in the micellar medium was found to be higher than that in aqueous solution. The chemical modification of lipase did not result in a change in the regulation of the oligomeric composition of the enzyme controlled by the degree of micelle hydration Ω₀ (micelle size). The k _cat dependences on Ω₀ for each lipase preparation exhibit two maxima, corresponding to the functioning of lipase monomers and tetramers. The changes in the hydrophilic-lipophilic balance of the lipase surface significantly affect the character of the regulation of enzyme activity due to changes in the surfactant concentration (the number of micelles). The lipase hydrophobization results in a decrease in the enzyme activation effect with an increase in the AOT concentration in comparison with the native lipase. The lipase hydrophilization dramatically decreases the activity of lipase tetramer when the AOT concentration is increased. The catalytic activity of the monomer of hydrophilized lipase is practically independent of the AOT concentration. Kinetic data indicate a mixed type of activation of both oligomeric forms of the native and the hydrophobized lipase by AOT molecules and the noncompetitive type of the activation and AOT inhibition of the monomer and the tetramer of the hydrophilized lipase, respectively.     

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.     

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.     

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.     

Effect of chemical modification of lipase on the regulation of its lipolytic activity in reversed micelles

Hydrophilized and hydrophobized forms of the lipase from Mucor miehei were obtained by its chemical modification with cellobiose and N-hydroxysuccinimidyl palmitate with a modification degree of 4 in both cases. A comparative analysis of the regulation of the catalytic activities of the native and modified lipases was carried out in the system of reversed micelles of OT aerosol (AOT) in isooctane. The level of catalytic activity of all the lipase preparations in the micellar medium was found to be higher than that in aqueous solution. The chemical modification of lipase did not result in a change in the regulation of the oligomeric composition of the enzyme controlled by the degree of micelle hydration omega0 (micelle size). The kcat dependences on omega0 for each lipase preparation exhibit two maxima, corresponding to the functioning of lipase monomers and tetramers. The changes in the hydrophilic-lipophilic balance of the lipase surface significantly affect the character of the regulation of enzyme activity due to changes in the surfactant concentration (the number of micelles). The lipase hydrophobization results in a decrease in the enzyme activation effect with an increase in the AOT concentration in comparison with the native lipase. The lipase hydrophilization dramatically decreases the activity of lipase tetramer when the AOT concentration is increased. The catalytic activity of the monomer of hydrophilized lipase is practically independent of the AOT concentration. Kinetic data indicate a mixed type of activation of both oligomeric forms of the native and the hydrophobized lipase by AOT molecules and the noncompetitive type of the activation and AOT inhibition of the monomer and the tetramer of the hydrophilized lipase, respectively. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

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.     

BEHAVIOUR OF HORSERADISH PEROXIDASE IN AOT REVERSED MICELLES

The activity and stability of horseradish peroxidase (HRP) solubilised in AOT reversed micelles in isooctane and decalin was studied using guaiacol (2-methoxyphenol) as the electron donor.

The activity of the enzyme in both reversed micellar systems increases with the water content until reaching a maximum value that remains fairly constant for water contents higher than 3.05% (v/v) in isooctane and 2.20% in decalin. The effect of pH on the activity profile was studied in the system AOT/isooctane. The enzyme is fully active at pH 7 and 8 for water contents higher than 3.05% (v/v) but it was completely deactivated at pH 9. The effect of surfactant concentration on HRP activity was also investigated. At low water contents a strong dependence was observed, whilst no further activity increase was observed for water content values higher than 2.7% (v/v).

The stability of HRP was found to be strongly dependent on the water content of the system with higher levels of stability obtained for higher values of water content. HRP stability is also affected by the presence of substrates. Whilst the stability increases markedly when the enzyme is incubated with guaiacol, it does not appear to be so strongly affected by the presence of hydrogen peroxide, at the concentrations studied.     

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.     

Improvement in extraction and catalytic activity of Mucor javanicus lipase by modification of AOT reverse micelle

Reverse micelles are formed in apolar solvents by spontaneous aggregation of surfactants. Surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) is most often used for the reverse micellar extraction of enzymes. However, the inactivation of enzyme due to strong interaction with AOT molecules is a severe problem. To overcome this problem, the AOT/water/isooctane reverse micellar system was modified by adding short chain polyethylene glycol 400 (PEG 400). The modified AOT reverse micellar system was used to extract Mucor javanicus lipase from the aqueous phase to the reverse micellar phase. The extraction efficiency (E) increased with the increase in PEG 400 addition and the maximum E in PEG 400 modified system was twofold higher than that in the PEG 400-free system. Upon addition of PEG 400, the water activity (a(w)) of aqueous phase decreased, whereas a(w) of reverse micellar phase increased. The circular dichroism spectroscopy analysis revealed that PEG 400 changes the secondary and tertiary structure of lipase. The maximum specific activity of lipase extracted in PEG 400-modified reverse micellar system was threefold higher than that in the PEG-free system.     

Stability and activity modulation of chymotrypsins in AOT reversed micelles by protein-interface interaction: interaction of alpha-chymotrypsin with a negative interface leads to a cooperative breakage of a salt bridge that keeps the catalytic active conformation (Ile16-Asp194)

The stability of alpha-chymotrypsin and delta-chymotrypsin was studied in reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. alpha-Chymotrypsin is inactivated at the interface and at the water pool, while delta-chymotrypsin is inactivated only at the water pool. The mechanism of inactivation at the interface is related to the interaction of N-terminal group alanine 149 (absent in delta-chymotrypsin) with the negative interface. The dependence of enzyme activity on water content of these two enzymes in reversed micelles of AOT is also related with the interface interaction, since delta-chymotrypsin does not have a bell-shaped curve as observed for alpha-chymotrypsin.     

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.     

Esterification of Short Chain Organic Acids with Alcohols by a Lipase Microencapsulated in Reversed Micelles

A Mucor miehei lipase was used to catalyse the esterification reaction between propionic acid and oleyl alcohol in reversed micelles of AOT in isooctane. Small-scale model studies were performed to study the influence of various parameters on the formation of oleyl propionate by this lipase. The maximum synthetic activity was obtained at w₀ = 4.0. At high temperatures (65°C) the enzyme displays a better stability for a low water content (w₀ = 3.3). The specificity of lipase was influenced by the solubilized water in the reversed micelles.     

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.     

Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.