Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio (W₀), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters (V_max, K_m and K_ad), and the values of K_m and K_ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.     

Stability of a Pseudomonas Sp. Lipase:Comparison Between Solubilized Enzyme in Reverse Micelles and Suspended Lipase in Dry Solvents

The stability of a relatively hydrophobic lipase from Pseudomonas sp., solubilized in reverse micellar media or suspended in dry solvents, was studied and compared. Factors such as the enzyme-solvent interaction, enzyme environment, hydration degree of the system, interphase quality, droplet size, and water activity were studied. A mixed micellar system which stabilized the lipase is reported. In the case of simple AOT micelles, lipase destabilization with respect to water in small droplet sizes and stabilization in the biggest micelles was observed. These effects resulted from lipase penetration into the interphase of the smaller nanodroplets, and the restriction of its conformational mobility in the region of structured water of the largest micelles, respectively. Mixed micelles increased lipase stability, which was mainly related to increased droplet size. Modification with polyethylene glycol decreased lipase stability in reverse micelles, due to the greater interaction with the micellar interphase. The preparation of nanodroplets, in which native and modified lipases were 5.4 and 9.4 times, respectively, more stable than in water, is reported. In contrast to the micellar media, low water contents (low A_w values) stabilized the solid lipase suspended in organic solvent systems. Under the hydration conditions studied here, lipase stability increased when more polar solvents were used. Two alternatives were necessary to obtain similar stabilities in n-heptane as compared with polar solvents: reduction of the water content or use of a low aquaphilic support.     

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.     

Kinetics of lipase deactivation in AOT/isooctane reversed micelles

The stability of lipase in AOT/isooctane reversed micellar solution was investigated. It was found that the lipase deactivated to a stable state that was not completely inactivated. The lipase residual activity after achieving the stable state in AOT/isooctane reversed micelles at 30 °C, pH 7.0, W₀=8.0 was found to be 0.15, and the first-order deactivation rate coefficient of lipase at the same conditions was regressed to be 0.75 h⁻¹. The stability of lipase was increased while oleic acid was added. Assuming the protection of oleic acid to lipase stability is due to the lipase–oleic acid complex does not decay, the kinetic model of lipase deactivation in AOT/isooctane reversed micellar solution including the influence of oleic acid was established. It was shown with the model equation that the increase in stability of the enzyme by oleic acid could be quantitatively estimated by the dissociation constant of lipase–oleic acid complex which was determined by product inhibition experiments. The model equation fit the experimental data well with an average relative deviation of 3.40%.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

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.     

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.     

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.     

Measurements of hydrodynamic diameter of AOT reverse micelles containing lipase in supercritical ethane and its enzymatic reaction

The enzymatic reaction by aerosol-OT (AOT)reverse micelles containing lipase in supercritical ethane was examined and is the focus of this paper. The reverse micelles were formed under various conditions at which their hydrodynamic diameters were measured by using the dynamic light scattering spectrophotometer. The reverse micelles in supercritical ethane were formed in the range of Wo (water/surfactant) less than six. The hydrodynamic diameter of the reverse micelles ranged from 2 to 5 microm. The hydrolysis reaction of triolein by the lipase in reverse micelles was also examined. The observations indicate that lipase in AOT reverse micelles in supercritical ethane showed activity. The conversion of triolein increased with the increase in size of reverse micelles and Wo, and reached its maximum near the critical temperature. Moreover, although the conversion of triolein increased with pressure, it was independent of pressure near the critical temperature.     

Surfactant Effects on Lipase-Catalyzed Hydrolysis of Olive Oil in AOT/ISOOCTANE Reverse Micelles

The effects of surfactant concentration on the hydrolytic activity of Candida rugosa lipase in AOT/isooctane reverse micelles with olive oil as the substrate has been investigated. A noncompetitive inhibition by the surfactant on the enzyme was observed. Strong dependences of the kinetic constants k_cat and k_M, but not k_I on the water-to-surfactant ratio (R value) have been identified. The benefits of carrying out the hydrolysis at higher surfactant and water concentrations were demonstrated from the improvement of the initial rate and time course of conversion.     

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.     

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ₀ (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C₁₂E₄), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C₁₂E₄ addition. The stabilizing effect of C₁₂E₄ resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C₁₂E₄ may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.     

Micellar catalysis of polyphenol oxidase in AOT/cyclohexane

The catalytic behaviour of mushroom polyphenol oxidase has been studied in dioctylsulphosuccinate (AOT)/cyclohexane reverse micelles. The steady-state conditions were accomplished up to 20 min and 17 μg protein in the assay towards 4-methylcatechol and no loss of specific activity was observed relative to aqueous medium. The pH activity profile of the enzyme was kept in reverse micelles as in water, showing a plateau between 5 and 6.5. The stability of polyphenol oxidase to pH was also studied and about 20% inactivation was found in reverse micelles relative to aqueous medium at neutral pHs. Moreover there was a decrease of stability at acidic pHs. The optimum W_o obtained was 20 and the enzyme was nearly independent of the surfactant concentration at constant W_o.

Kinetic studies of polyphenol oxidase towards several substrates showed that the substrate inhibition by p-cresol and 4-methylcatechol observed in buffer was not kept in AOT/cyclohexane reverse micelles. Moreover, the K_m increased and the catalytic efficiency (V/K_m) of the enzyme decreased as the hydrophobicity of substrates was increased.     

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.     

Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles

The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent media, respectively. The influences of the molar ratio of water to surfactant omega0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show "superactivity" in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of omega0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at omega0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.     

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.     

Stability of free and immobilised peroxidase in aqueous–organic solvents mixtures

The activity and stability of horseradish (Amoracia rusticana) peroxidase (HRP) free in solution and immobilised onto silica microparticles was studied in the presence of organic co-solvents.

The effect of several hydrophilic organic solvents, namely dimethyl sulfoxide, dimethylformamide, dioxan, acetonitrile and tetrahydrofuran, in the activity and stability of free HRP was studied. From the solvents tested, DMSO led to the highest activities and stabilities. After 2 h of incubation at 35°C, the remaining activity of the enzyme in the presence of 30% of each solvent was less than 30%, with exception of DMSO for which the enzyme remained fully active.

In order to increase stability, HRP was covalently immobilised onto silica microparticles. The half-life of the enzyme in buffer at 50°C increased from 2 to 52 h when the enzyme was immobilised. The stability of both free and immobilised HRP was also studied at 50°C in aqueous mixtures of 3.5, 20, 35 and 50% (v/v) DMSO. Free HRP stability was not affected by the presence of 3.5 and 20% DMSO, but higher contents lead to a more pronounced deactivation. Immobilised HRP stability increased with DMSO content up to 20%, decreasing for higher contents. The enzyme half-life increased more than 300% when changing from buffer to 20% DMSO.

The deactivation of free HRP was modelled using the simple exponential decay, and the deactivation of immobilised HRP was described by a two-step inactivation model.     

Kinetic study of lipase catalyzed esterification reactions in water-in-oil microemulsions

The kinetics of the esterification of lauric acid by (-)menthol, catalyzed by Penicillium simplicissimum lipase, was studied in water/bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping-Pong Bi--Bi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59-72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio, W(o) = [H(2)O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern. (c) 1993 John Wiley & Sons, Inc.     

Extraction of flexibly structured protein in AOT reverse micelles: the flexible structure of protein is the dominant factor for its incorporation into reverse micelles

The extraction of flexibly-structured protein in Aerosol-OT (AOT)/isooctane reverse micelles was investigated. A flexibly-structured lysozyme was prepared by reduction and carboxymethylation of the disulfide bonds in the lysozyme molecule. For a comparison, lysozymes whose surface hydrophobicity was modified by monoacylation of the amino groups were also used. The extraction rate of the flexibly-structured lysozyme into the micellar phase was greater than that of the native and monoacylated lysozymes, although the free energy change of the lysozyme prepared by breaking the disulfide bonds was smaller than that of the lysozymes whose surfaces were monoacylated. Viscosity measurement of the micellar organic phase containing the modified lysozymes indicated that extraction of the flexibly-structured lysozyme changed the micelle–micelle interaction, while measurement of the interfacial tension between the AOT/isooctane and protein aqueous systems showed the flexibly-structured lysozyme to be the most amphiphilic in character. These results indicated that the flexible structure of a protein was more dominant than its surface hydrophobicity for its incorporation into reverse micelles, and that it leads to greater micelle–micelle interaction.