Reactivity of trypsin in reverse micelles: neglected role of aggregate size compared to water-pool components

The catalytic efficiency of trypsin was estimated in cationic reverse micelles as a function of the concentration of water-pool components and aggregate size to determine their independent influence on enzyme activity. The variation in the aggregate size/water-pool size was achieved by changing both the W0 (mole ratio of water to surfactant) and the headgroup area of surfactant through introduction of hydroxyethyl groups at the polar head. The local molar concentrations of water present inside the water-pool ([H2O]wp) of different cationic reverse micelles across varying W0 was estimated using a modified phenyl cation-trapping protocol. The [H2O]wp in cationic reverse micelles (surfactant/isooctane/n-hexanol/water) increases with W0 and attains the molarity of normal water beyond W0=40 irrespective of the nature of headgroup. Concurrently, the catalytic activity of trypsin compartmentalized within the water-pool increases with the increase in [H2O]wp upto an optimal W0=40 in organized solutions of any surfactant. The aggregate size (determined by static light scattering) also increases expectedly with W0 and noticeably with the area of the surfactant headgroup at similar W0. Since the enzyme activity rises both with the increase in water-pool size and [H2O]wp, trypsin's efficiency was compared with these two parameters across reverse micelles of varying surfactant headgroup size at similar W0 to determine their probable independent influence in regulating the enzyme activity. Noticeably, the efficiency of trypsin rises two to ninefold in spite of the [H2O]wp being distinctly lower in case of hydroxyethyl group substituted surfactants compared to cetyltrimethylammonium bromide w/o microemulsions at similar W0. Thus, the influence of the aggregate size possibly plays an important role alongwith the [H2O]wp in modulating the enzyme activity.     

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.     

Characteristics of tyrosinase in AOT-isooctane reverse micelles

Isooctane-AOT-H(2)O is a suitable system for studying enzyme behavior in organic solvents. Tyrosinase was able to catalyze a well-known reaction in aqueous medium: oxidation of 4-methylcatechol to yield 4-methyl-o-benzoquinone. This reaction was studied using the preceding ternary system with adequate amounts of each component to make up reverse micelles. 4-Methyl-o-benzoquinone stability was demonstrated in isooctane even at alkaline pH values. Apparent K(m) and V(max) were similar to those in water, but substrate inhibition was more evident. The pH and temperature appear to be shifted toward high and low values, respectively. Characteristic parameters of reverse micelles, omega(0) (= H(2)O/AOT) and percentage of H(2)O (v/v), were investigated. The results obtained showed that the steady-state rate varies either with omega(0) or with percentage of H(2)O. The variation observed with omega(0) showed an optimal value while an increase in percentage of H(2)O can lead to decreased or increased activity depending on substrate concentration.     

Reverse micelles in organic solvents: a medium for the biotechnological use of extreme halophilic enzymes at low salt concentration

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeobacterium Halobacterium salinarum (previously halobium) was solubilized at low salt concentration in reverse micelles of hexadecyltrimethyl-ammoniumbromide in cyclohexane with 1-butanol as co-surfactant. The enzyme maintained its catalytic properties under these conditions. The thermodynamic "solvation-stabilization hypothesis" has been used to explain the bell-shaped dependence of pNPPase activity on the water content of reverse micelles, in terms of protein-solvent interactions. According to this model, the stability of the folded protein depends on a network of hydrated ions associated with acidic residues at the protein surface. At low salt concentration and low water content (the ratio of water concentration to surfactant concentration; w0), the network of hydrated ions within the reverse micelles may involve the cationic heads of the surfactant. The bell-shaped profile of the relationship between enzyme activity and w0 varied depending on the concentrations of NaCl and Mn2+.     

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.     

Trypsin-SBTI interaction in reverse micelles. A slow intermicellar exchange-dependent binding

Solubilisate exchange between reverse micelles must take place before any reaction inside reverse micelles occurs if the reactants are confined to the aqueous micellar core. When the interacting species are 2 small molecules or one small molecule and one macromolecule, it has been shown that the exchange is faster than the typical turnover of an enzymatic reaction. The study of the interaction between 2 macromolecules (trypsin and soybean trypsin inhibitor) in reverse micelles carried out in this work reveals that the exchange between these macromolecule-containing reverse micelles slows down by a thousand times and the limiting-step in the exchange, the fusion, by 10(6) times. Both reverse micellar size (omega 0 = [water]/[surfactant]) and temperature affected the rate of the fusion process. A hypothesis for the proposed active role of macromolecules in the exchange process is also given.     

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.     

Dimerization and reactivation of triosephosphate isomerase in reverse micelles.

The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n-octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3-chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5-7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes.     

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.     

Thermobarostability of alpha-chymotrypsin in reversed micelles of aerosol OT in octane solvated by water-glycerol mixtures

Thermostability of alpha-chymotrypsin at normal pressure in reversed micelles depends on both an effective surfactant solvation degree and glycerol content in the system. The difference in alpha-chymotrypsin stability in reversed micelles at various glycerol concentrations [up to 60% (v/v)] was more pronounced at high surfactant degrees of solvation, R >/= 16. After a 1-h incubation at 40 degrees C in "aqueous" reversed micelles (in the absence of glycerol), alpha-chymotrypsin retained only 1% of initial catalytic activity and 10, 22, 59, and 48% residual activity in glycerol-solvated micelles with 20, 30, 50, and 60% (v/v) glycerol, respectively. The explanation of the observed effects is given in the frames of micellar matrix structural order increasing in the presence of glycerol as a water-miscible cosolvent that leads to the decreasing mobility of the alpha-chymotrypsin molecule and, thus the increase of its stability. It was found that glycerol or hydrostatic pressure could be used to stabilize alpha-chymotrypsin in reversed micelles; a lower pressure is necessary to reach a given level of enzyme stability in the presence of glycerol.     

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.     

A new way in homogeneous immunoassay: reversed micellar systems as a medium for analysis

Possibilities of a new principle for the homogeneous enzyme immunoassay utilizing the systems of surfactant reversed micelles in organic solvents have been demonstrated taking thyroxine determination as an example. The catalytic activity of an enzyme, solubilized in such systems, is determined by the ratio of geometric dimensions of the micellar matrice and the enzyme molecule. The addition of antibodies against thyroxine to the peroxidase-thyroxine conjugate, solubilized in the system of reversed micelles of aerosol OT in octane, leads to the formation of the immune complex whose size differs substantially from that of the initial enzyme-antigen conjugate. This induces changes in the peroxidase catalytic activity. The addition of free thyroxine to the system stimulates the conjugate release from the immune complex and, consequently, the reduction of the peroxidase activity to the initial level. Sensitivity of the analysis in reversed micellar systems can be regulated by changing the surfactant hydration degree. Substances of different nature (both hydrophobic and hydrophylic) can be solubilized in reverse micellar systems under standard conditions, which allows determination of water insoluble antigens.     

Reactivity of trypsin in reverse micelles: pH-effects on the W0 versus enzyme activity profiles

pH-Dependence of hydrolytic activity of trypsin has been studied in cationic reverse micellar system of cetyltrimethylammonium bromide (CTAB) in (50% v/v) chloroform/isooctane using a positively charged substrate N_α-benzoyl-L-arginine ethyl ester (BAEE). The pH of the medium was varied from 4.0 to 8.5 with addition of 0.025 M citrate-phosphate buffer containing 1 mM CaCl₂. Optimum pH for maximum enzyme activity, pH_opt in reverse micelles is found to be similar to that observed in bulk aqueous solution (8.0–8.5). However, changes in activity of trypsin (k_cat) as a function of water content W₀ (W₀ = [H₂O]/[CTAB]) in reverse micelles are found to be pH dependent. At low pH (4.0) and low water content (W₀ = 5) the enzyme is more active in reverse micelles than in bulk aqueous solution by a factor of 2. This ‘superactivity’ is lost at higher W₀ values and the k_cat in reverse micelles is found to be similar to that observed in aqueous bulk. At pH 5, the enzyme activity is found to be independent of W₀ while at pH 6.0–6.5 the enzyme activity is low at W₀ 5 and increases with water content to a constant value which is still 50% lower than that in aqueous buffer. Above pH 7, the W_o-activity profile becomes distinctly bell shaped with W₀ optimum around 10–15. The enzyme activity at optimum W₀ is close to that observed in aqueous bulk.     

Reversed micelles of polymeric surfactants in nonpolar organic solvents. A new microheterogeneous medium for enzymatic reactions.

A new microheterogeneous non-aqueous medium for enzymatic reactions, based on reversed micelles of a polymeric surfactant, was suggested. The surfactant termed CEPEI, was synthesized by successive alkylation of poly(ethyleneimine) with cetyl bromide and ethyl bromide and was found to be able to solubilize considerable amounts of water in benzene/n-butanol mixtures. The hydrodynamic radius of polymeric-reversed micelles was estimated to be in the range 22-51 nm, depending on the water content of the system, as determined by means of the quasi-elastic laser-light scattering. Polymeric reversed micelles were capable of solubilizing enzymes (alpha-chymotrypsin and laccase) in nonpolar solvents with retention of catalytic activity. Due to the strong buffering properties of CEPEI over a wide pH range, it could maintain any adjusted pH inside hydrated reversed micelles. It was found that catalytic behavior of enzymes entrapped in polymeric reversed micelles was rather insensitive to the pH of the buffer solution introduced into the system as an aqueous component, but determined mostly by acid-base properties of the polymeric surfactant itself. Both catalytic activity and stability of entrapped alpha-chymotrypsin and laccase were found to increase with increasing water content of the system. Under certain conditions, the entrapment of alpha-chymotrypsin into CEPEI reversed micelles resulted in a considerable increase in catalytic activity and stability as compared to aqueous solution. CEPEI reversed micelles were demonstrated to be promising enzyme carriers for use in membrane reactors. Owing to the large dimensions of CEPEI reversed micelles, they are effectively kept back by a semipermeable membrane, thus allowing an easy separation of the reaction product and convenient recovery of the enzyme.     

Reverse micelles as life-mimicking systems

In this review, we attempt to demonstrate that reverse micelles are simple artificial systems that mimic many life systems from cell division to the creation of an enzyme catalytic mechanism. For a membranous enzyme like placental alkaline phosphatase, the kinetic properties observed in reverse micelles might represent those found under physiological conditions. The reverse micellar system, consisting of a positively charged surfactant, mimics a detoxification enzyme glutathione transferase. We propose a novel island-in-oil-lake reverse micellar model for the glutathione transferase that can account for almost all the catalytic properties of this enzyme. Reverse micelles may provide an excellent model system in investigating the reaction mechanism of other detoxification enzymes.     

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.     

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.     

Increased stability of malate dehydrogenase from Halobacterium salinarum at low salt concentration in reverse micelles

The stability of malate dehydrogenase (hMDH) from Halobacterium salinarum in aqueous medium at low salt concentrations (1 and 0.5 M NaCl) was studied at 4 degrees and 25 degrees C. The results showed that hMDH was more stable at the higher salt concentration and the low temperature. hMDH was introduced into reverse micelles of hexadecyltrimethylammonium bromide in cyclohexane with 1-butanol as co-surfactant. The hMDH stability in this system was studied at two omega(0) ([H(2)O]/[surfactant]) values and the effects of salt concentration, presence of substrate and dilution before or after its introduction into reverse micelles were examined. The results showed that the half-life of hMDH dissolved in buffer with 1 M NaCl was 12-50 days in reverse micelles (depending on the various conditions), in contrast to only about 1 day in aqueous medium at 25 degrees C. These observations indicate that reverse micelles provide a microenvironment that allows a much greater stability of this enzyme compared with an aqueous medium.     

Regulation of the catalytic activity and supermolecular structure of penicillin acylase from Escherichia coli in an aerosol OT reversed micelle system in octane]

The properties of penicillin acylase from E. coli solubilized by hydrated reversed micelles of Aerozol OT (AOT) in octane were studied. The catalytic activity dependence on the hydration degree, a parameter which determines the size of the micelle inner cavity, represents a curve with three optima, each corresponding to the enzyme functioning either in a dimer form (omega 0 = 23) or in the form of separate subunits--heavy, beta, and light, alpha, at omega 0 = 20 and 14, respectively. Reversible dissociation of the enzyme was confirmed by ultracentrifugation followed by electrophoresis. Preparative isolation of penicillin acylase subunits, their catalytic activity being retained, was shown to be possible.     

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.