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.     

Enzymatic activity of an extremely halophilic phosphatase from the Archaea Halobacterium salinarum in reversed micelles

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeon Halobacterium salinarum (previously halobium) was solubilized in reversed micelles of cetyltrimethylammonium bromide (CTAB) in cyclohexane with 1-butanol as cosurfactant. The hydrolysis reaction appears to follow Michaelis–Menten kinetics. The dependency of the maximum reaction rate (V_max) on the water content θ (% v/v) (or ω₀ value: molar ratio of water to surfactant concentrations) showed a bell-shaped curve for 0.3 M CTAB, but not for 0.2 M CTAB. The enzyme activity increased with the surfactant concentration at a constant ω₀ value (10.27). When the surfactant concentration was increased at a constant θ, the enzyme activity decreased. The enzyme was more stable in reversed micelles than in aqueous media.     

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.     

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.     

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.     

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%.     

Enzyme-Catalyzed oxidation of cholesterol in physically characterized water-in-oil microemulsions

The enzymatic conversion of cholesterol to cholestenone by cholesterol oxidase (Brevibacterium sp.)in reversed micelles in a system composed of AOT/isooctane/water/cholesterol has been examined. The catalytic activity of the enzyme was correlated with the physicochemical properties of water in water-in-oil (w/o) microemulsion systems. In a system consisting of 3 wt % AOT in isooctane, reversed micelles started to form as the [H(2)O]/[AOT] (e.g., the w(0)) ratio increased above 4-5. The formation of reversed micelles with a core of neat (bulk) water was verified from determinations of both the partial molar volume of water and the scissors vibration of water [with Fourier transform infrared (FTIR) spectroscopy] in the w/o microemulsion systems. A plot of enzyme activity vs. w(0) indicated that the hydration of enzyme molecules per se was not sufficient to give rise to catalytic activity. Instead, it appeared that the formation of an aqueous micellar core was necessary for full activation of the enzyme. Based on micelle size distribution analysis, it was estimated that about one micelle per one thousand contained an enzyme molecule. Since the apparent reaction rate could be markedly enhanced by increasing the enzyme/water ratio, we conclude that the number of enzyme-containing micelles was an important rate-limiting factor in the system.     

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 Chromobacterium viscosum 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.     

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 new purification procedure and molecular properties of Pseudomonas cytochrome oxidase

A procedure has been developed for purification of the cytochrome oxidase from Pseudomonas aeruginosa (EC 1.9.3.2) using DEAE- and CM-cellulose chromatography, gel filtration and crystallization. The final preparation was found to be homogeneous according to ultracentrifugal and disc electrophoretic criteria. The crystalline preparation also exhibited nitrite reductase activity. The spectrum of the enzyme characterizes it as cytochrome cd. At 280 nm E^{1 %}_{1 cm} was 18.5 after dry weight analysis.

The molecular weight of the cytochrome oxidase was calculated to be 119000 based on a sedimentation coefficient s° _20,w = 7.36 S, diffusion coefficient D _20,w = 5.36×10⁻⁷ cm²×s⁻¹ and partial specific volume of 0.72 ml/g. The iron content of the enzyme (0.166 %) indicates that this entity contains four iron atoms per molecule. Succinylation of the enzyme produced two probably identical subunits containing both hemes c and d, having a sedimentation coefficient s° _20,w = 4.30 S and an approximate molecular weight of 65000. In dodecylsulphate-acrylamide gel electrophoresis the cytochrome oxidase also dissociates into two subunits with molecular weight of 63000.     

Dependence of Lipase Activity on Water Content and Enzyme Concentration in Reverse Micelles

The activity of Candida rugosa lipase (EC 3.1.1.3) in reverse micelles has been measured at various concentrations of water and enzyme with the aim of answering the question, why is the enzyme activity affected by the molar ratio of water to surfactant (w₀ = [H₂O]/[Surfactant])? In the low range of water content (below w₀ ≈ 6), the activity increases with increasing water content, indicating the requirement of a minimum amount of water for the full expression of enzymatic activity. The minimal w₀-value for obtaining maximal activity depends on the enzyme concentration: The higher the enzyme concentration, the higher w_{0, max}. In addition, it was found that, at least for the case of Candida rugosa lipase, the measured dependence of enzyme activity on w₀ does not represent a true chemical equilibrium. Changing the w₀-value during the reaction does not change the activity as expected on the basis of the w₀-activity profile obtained for single w₀ point measurements. All these observations, however, cannot be directly generalized to all enzymes in reverse micelles, due to the peculiarity of lipase. In particular, the enzyme seems to inactivate irreversibly during the solubilization process.     

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.     

Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate

The initial rate and enantioselectivity of enzymatic asymmetric hydrolysis of amino acid esters were examined in methylimidazolium-based ionic liquids with anions including tetrafluoroborate, chloride, bromide and bisulfate and in typical organic solvents. Papain displayed much higher enantioselectivity but lower activity in phosphate buffer solution of 1-butyl-3-methylimidazolium tetrafluoroborate BMIM·BF₄ than in other media tested (i.e. E=100, V ₀=0.21 mM min^-1 in BMIM·BF₄, E=2, V ₀=0.43 mM min^-1 in phosphate buffer, E=14-92, V ₀=0.22-0.25 mM min^-1 in organic solvents for D,L-phenylglycine methyl ester). The influence of BMIM·BF₄ on enzyme activity and enantioselectivity also varied with the substrate and the enzyme used. All of the enzymes assayed showed no activity or low enantioselectivity in the ILs with anions including chloride, bromide and bisulfate.     

Stability and Enzymatic Studies of Glucose Dehydrogenase from the Archaeon Haloferax mediterranei in reverse micelles

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w₀=[H₂O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP⁺ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.     

Application of the hydro(solvo)thermal technique to the synthesis of metal carbonyl chalcogenide clusters Part 3. Synthesis, structural characterization, and spectroscopic studies of the clusters [{M(CO)4}n(MS4)] (M = Mo, W; N=1, 2)

The methanothermal reactions of M(CO)₆ (M = Mo, W) with Na₂S₂ gave a series of homonuclear clusters [{M(CO)₄}_n(MS₄)]²⁻ (M=Mo, W; N=1, 2), i.e. (Ph₄P)₂[(CO)₄Mo(MoS₄)] (I), (Ph₄P)₂[(CO)₄W(WS₄)] (II), (Ph₄P)₂[(CO)₄Mo(MoS₄)Mo(CO)₄] (III) and (Ph₄P)₂[(CO)₄W(WS₄)W(CO)₄] (IV). The two dimers, I and II, as well as the two trimers, III and IV, are isostructural to each other, respectively. All compounds crystallize in the triclinic space group with Z=2. The cell dimensions are: a=12.393(8), b=19.303(9), c=11.909(6) Å, =102.39(5), β=111.54(5), γ=73.61(5)°, V=2522(3) ų at T=23 °C for I; a=12.390(3), b=19.314(4), c=11.866(2) Å, =102.66(2), β=111.49(1), γ=73.40(2)°, V=2511(1) ų at T=23 °C for II; a=11.416(3), b=22.524(4), c=10.815(4) Å, =91.03(2), β=100.57(3), γ=88.96(2)°, V=2733(1) ų at T=−100 °C for III, a=11.498(1), b=22.600(4), c=10.864(3) Å, =90.92(2), β=100.85(1), γ=88.58(1)°, V=2771(2) ų at T=23 °C for IV. The dimers are each formed by the coordination of the tetrathiometalate as a bidentate chelating ligand to an M(CO)₄ fragment while addition of another M(CO)₄ fragment to the dimers results in the trimers. All compounds contain both tetrahedral and octahedral metal centers with the formal 6+ and 0 oxidation states, respectively.     

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.     

Characterization of cholesterol oxidase activity in AOT-isooctane reverse micelles and its dependence on micelle size

Summary Characterization of cholesterol oxidase in AOT reverse micelles was performed. pH and temperature profiles show that the entrapment of the enzyme does not change its characteristics appreciably. The enzyme tends to behave as it does in water when micelle size increases and does not maximum rate at some intermediate micelle size. Km was 55–60 fold that in waterAbbreviations and parameters AOT Dioctyl sodium sulfosuccinate - CTAB Cetyl trimethylamonium bromide - K1^E Equilibrium constant of the enzyme between free and bound water - K2^E Equilibrium constant of the enzyme between bound water and surfactant - kf Catalytic constant in free water - kb Catalytic constant in bound water - ks Catalytic constant in surfactant - n Number of water molecules strongly bound to one surfactant molecule     

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.     

Influence of Igepal reverse micellar and micellar systems on activity and stability of heme peroxidases

The activities of horseradish peroxidase (HRP) and lactoperoxidase (LPO) entrapped in reverse micelles of Igepal CO-520 in cyclohexane were studied. When the molar ratio of water to surfactant, w ₀ was ≥13, the activity of HRP encapsulated in the water pool of the reverse micelle was comparable with that measured in buffer. For LPO, however, lower activity was observed after its incorporation into the same system.

The activity of the investigated peroxidases was also measured in an aqueous solution of Igepal CO-720 or after incubation with this surfactant. The enzymes became inactivated in an aqueous micellar solution of Igepal CO-720, although this process was reversible.

The stability of HRP and LPO at 37 or 50°C was lower in the micellar systems than in buffer with the exception for HRP in reverse micelles at 50°C.     

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.