Regulation and Prediction of Enzyme Activity in Reversed Micelles

The rate of cholesterol oxidation has been studied in cholesterol oxidase containing reversed micellar media consisting of the surfactant cetyltrimethylammonium bromide (CTAB), the surfactant octanol, a buffered aqueous solution, and a variety of organic solvents. By varying the composition of the medium systematically it could be deduced that the rate of cholesterol oxidation obeys the same rules as described earlier for the conversion of apolar steroids by 20β-hydroxysteroid dehydrogenase in CTAB-hexanol-organic solvent reversed micelles (Hilhorst et al. 1984). The general applicability of these rules in optimizing biocatalysis in reversed micelles is discussed.     

Comparative study of the regulation of catalytic activity of soluble and membrane forms of enzymes in reverse micellar systems. Gamma-glutamyltransferase and aminopeptidase]

The regulations of functioning of water soluble and membrane forms of enzymes in the systems of reversed micelles of surfactants in organic solvents are compared. By an examples of gamma-glutamyltransferase (in AOT reversed micelles in octane) and amino-peptidase (in Brij 96 reversed micelles in cyclohexane) the principal difference in the catalytic activity regulation of water soluble and membrane forms is demonstrated. The catalytic activity of the membrane form depends largely on the surfactant concentration at the constant hydration degree, whereas the activity of the water soluble form is constant under these conditions. The catalytic activity dependence on the surfactant concentration is regarded as a "test for the enzyme's membrane activity".     

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.     

Solubilization capacity of nonionic surfactant micelles exhibiting strong influence on export of intracellular pigments in Monascus fermentation

In this study, perstractive fermentation of intracellular Monascus pigments in nonionic surfactant micelle aqueous solution had been studied. The permeability of cell membrane modified by nonionic surfactant might have influence on the rate of export of intracellular pigments into its extracellular broth while nearly no effect on the final extracellular pigment concentration. However, the solubilization of pigments in nonionic surfactant micelles strongly affected the final extracellular pigment concentration. The solubilization capacity of micelles depended on the kind of nonionic surfactant, the super‐molecule assembly structure of nonionic surfactant in an aqueous solution, and the nonionic surfactant concentration. Elimination of pigment degradation by export of intracellular Monascus pigments and solubilizing them into nonionic surfactant micelles was also confirmed experimentally. Thus, nonionic surfactant micelle aqueous solution is potential for replacement of organic solvent for perstractive fermentation of intracellular product.     

An extreme halophilic enzyme active at low salt in reversed micelles.

Possible biotechnological applications of extreme halophilic enzymes are strongly determined by their high salt requirement of around 4 M NaCl. Consequently, the use of these in organic media seemed to be unlikely. However, we have succeeded in dissolving a halophilic enzyme, p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum, in an organic medium by creating a reverse micellar system with very low salt concentration. The enzyme retained its catalytic properties in reversed micelles made with an anionic surfactant (dioctyl sodium sulphosuccinate) or with a cationic surfactant (hexadecyltrimethylammonium bromide) in cyclohexane plus 1-butanol as co-surfactant. The dependence of the rate of hydrolysis of p-nitrophenylphosphate phosphate on the molar water/surfactant ratio (w(0) value) showed a bell-shaped curve for each surfactant system. Kinetic parameters were determined in each system. The enzymatic reaction appeared to follow Michaelis-Menten kinetics with the anionic surfactant only. The kinetic behaviour was determined at different concentrations of Mn(2+) in reversed micelles of dioctyl sodium sulphosuccinate as surfactant.     

Stabilisation of tyrosinase by reversed micelles for bioelectrocatalysis in dry organic media

The enzymatic and bioelectrocatalytic activity of tyrosinase from mushrooms was studied in a system of reversed micelles formed by Aerosol OT (AOT) in hexane. The optimal catechol oxidising activity of tyrosinase incorporated in reversed micelles was found at a hydration degree of w(0)=25. The catalytic activity was comparable with tyrosinase activity in aqueous media. When immobilized at an Au electrode, either directly or in reversed micelles, tyrosinase exhibited a similar efficiency of the bioelectrocatalytic reduction of O(2) mediated by catechol; however, a rapid decrease in the activity correlated with the destruction of reversed micelles and/or the removal of tyrosinase from the electrode surface. The system containing tyrosinase in reversed micelles with caoutchouk, spread on the surface of the Au electrode and successively covered with a Nafion membrane layer, was found to result in stable tyrosinase-modified electrodes, which were resistant to inactivation in dry acetonitrile. The proposed technique offers possibilities for further development of highly active and stable surfactant/enzyme-modified electrodes for measurements carried out in organic solvents.     

反胶束萃取胰蛋白酶的研究

本文以含有反胶束的有机溶剂作为萃取剂,进行了将胰蛋白酶从水相传入有机相,再从有机相传入另一水相的研究。结果表明：影响萃取率的主要因素为水相ｐＨ值、离子强度和种类,以及反胶束溶液中表面活性剂浓度等;在适宜的条件下,酶的单级萃取和反萃取率都很高,显示了良好的工业应用前景。     

Characteristics of lipase-catalyzed hydrolysis of olive oil in AOT-isooctane reversed micelles

Candida rugosa lipase solubilized in organic solvents in the presence of both surfactant and water could catalyze the hydrolysis of triglycerides, and kinetic analysis of the lipase-catalyzed reaction was found to be possible in this system. Among eight organic solvents tested, isooctane was most effective for the hydrolysis of olive oil in reversed micelles. Temperature effect, pH profile, K(m,app) and V(max,app) were determined. Among various chemical compounds, Cu(2+), Hg(2+), and Fe(3+) inhibited lipase severely. But the enzyme activity was restorable partially by adding histidine or glycine to the system containing these metal ions. The enzyme activity was dependent on R (molar ratio of water to surfactant) and maximum activity was obtained at R = 10.5. Upon addition of glycerol to the reversed micelles, lipase activity was affected in a different fashion depending on the R values. Stability of the lipase in reversed micelles was also dependent on R, and it was most stable at R = 5.5.     

The principal difference in regulation of the catalytic activity of water-soluble and membrane forms of enzymes in reversed micelles. Gamma-glutamyltransferase and aminopeptidase

The regularities of their functioning of enzyme, water-soluble and membrane forms, in the systems of the reversed micelles of surfactants in organic solvents are compared. Using as examples gamma-glutamyltransferase (in AOT reversed micelles in octane) and aminopeptidase (in Brij 96 reversed micelles in cyclohexane), the principal difference in the catalytic activity regulation of water-soluble and membrane forms is demonstrated. The catalytic activity of the membrane form depends considerably on the surfactant concentration at the constant degree of hydration, whereas the activity of the water-soluble form is constant under these conditions. The catalytic activity dependence on the surfactant concentration is regarded as a test for enzyme membrane activity.     

Catalysis by Laccase (From Coriolus uersicolor) in Microheterogeneous Media of the Water/Organic Solvent/Surfactant Type

Catalysis by laccase from Coriolus uersicolor solubilized in the ternary systems of surfactant/water/organic solvent type, namely, Aerosol OT/water/octane, Brij 56/water/cyclohexane and egg lecithin/water/octane + pentanol + methanol mixture, has been studied. The laccase activity is found to depend, in principle, not only on the water/surfactant molar ratio, but on the surfactant concentration (with its hydration degree being constant) as well. The following inferences should be emphasized. Firstly, in all the systems under study, the catalytic activity (k_cat) of laccase entrapped into surfactant reversed micelles increases more than 50 times (when the surfactant concentration is extrapolated to zero) compared with the k_cat value in aqueous solution. Secondly, the catalytic activity (k_cat) of laccase entrapped in hydrated Aerosol OT aggregates, having lamellar, reversed cylindrical (hexagonal) and reversed micellar structure, depends greatly on the aggregate type. In other words, the phase transitions, i.e. an alteration in the packing of hydrated Aerosol OT molecules, evokes a sharp reversible change in the enzymatic activity. Thirdly, in the same phase, the catalytic activity of the solubilized enzyme depends on the linear dimensions of water cavities inside the surfactant aggregates (i.e. on the water content in the system under study). All these effects, regulating enzymatic activity, are probably caused by an alteration of the conformational mobility of laccase molecules incorporated into the inner polar cavities inside the surfactant aggregates.     

Fluorescence dynamics of green fluorescent protein in AOT reversed micelles

We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the organic solvents were isooctane and (the more viscous) dodecane, respectively. The water content of the water pools could be controlled through the parameter w0, which is the water-to-surfactant molar ratio. With steady-state fluorescence, it was observed that subtle fluorescence changes could be noted in reversed micelles of different water contents. EGFP can be used as a pH-indicator of the water droplets in reversed micelles. Time-resolved fluorescence methods also revealed subtle changes in fluorescence decay times when the results in bulk water were compared with those in reversed micelles. The average fluorescence lifetimes of EGFP scaled with the relative fluorescence intensities. Time-resolved fluorescence anisotropy of EGFP in aqueous solution and reversed micelles yielded single rotational correlation times. Geometrical considerations could assign the observed correlation times to dehydrated protein at low w0 and internal EGFP rotation within the droplet at the highest w0.     

The effect of reversed micelles of cetyltrimethylammonium bromide on equilibrium constants and kinetics of oxidation-reduction reactions with the participation of cytochrome c

To model the effect of membrane environment on the electron transfer reactions we studied the thermodynamics and kinetics of the reactions of cytochrome c and 2,6-dichlorophenolindophenol with ferri- and ferrocyanide in the reversed micelles cetyltrimethylammonium bromide in chloroform/octan mixture. Incorporation of the protein in micelles leads to increasing the equilibrium constant (K1) up to 300 times. This effect is mainly due to a decrease in the ferrocytochrome c oxidation rate constant in the reaction with ferricyanide. Micellar solubilization of the dye also leads to a marked increase in the equilibrium constant K2. The estimations of the values K1 and K2 in water-alcohol mixtures and in aqueous micellar solutions of surfactant together with kinetical and spectral data show that the increase of K1 and K2 in reversed micelles is caused generally by redox potential changes of low-molecular reagents. The latter change their environment after adsorption on the micellar surface.     

Protein extration from an aqueous phase into a reversed micellar phase: Effect of water content and reversed micellar composition

In the system composed of the cationic surfactant TOMAC (10 mM), the nonionic (co)surfactant Rewopal HV5 (2 mM), and octanol (0.1% v/v) in isooctane, reversed micelles are formed upon contact with an aqueous phase containing 50 mM ethylene diamine. alpha-Amylase can be transferred from the aqueous phase into reversed micelles in the pH range 9.5 to 10.5 and re-extracted into a second aqueous phase of different composition. The size of the reversed micelles (as reflected in the water content of the organic phase) can be varied by changes in percentage of octanol, type of counterion in the aqueous phase, or in the number of ethoxylate head groups of the nonionic surfactant. An increase in size results in transfer at lower pH values. Experiments in which the charge density in the reversed micellar interface was changed by incorporation of charged derivatives of the nonionic surfactant, without influencing the water content, revealed that an increased charge density facilitated transfer, resulting in a broader transfer profile. Replacement of TOMAC by other quaternary ammonium surfactants differing in number and length of tails revealed that, of the 14 surfactants tested, only 2 gave appreciable amounts of transfer. The amount of transfer is related to the dynamics of phase separation of the surfactants: those giving a poor phase separation inactivate the enzyme. This inactivation is caused by electrostatic interactions between the charged surfactant head groups and charged groups on the enzyme. Electrostatic interactions are the first step of transfer, and can result in either incorporation in a reversed micelle, or, if reversed micelle formation is slow, in enzyme inactivation. (c) 1995 John Wiley & Sons, Inc.     

Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ([H(2)O]/[surfactant]), salt concentration, with and without Mn(+2). At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.     

Enzyme kinetics in reversed micelles. 3. Behaviour of 20 beta-hydroxysteroid dehydrogenase

The kinetic parameters of 20 beta-hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine 'apparent' Michaelis constants and V values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium-di(ethylhexyl)sulphosuccinate-reversed micelles and the observed low activity in Triton-reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles.     

Ester Synthesis by a Recombinant Cutinase in Reversed Micelles of a Natural Phospholipid

Fusarium solani pisi recombinant cutinase solubilized in reversed micelles of a nonionic surfactant (phosphatidylcholine) in isooctane was used to catalyze the esterification of fatty acids with 2-butanol. Various parameters affecting the catalytic activity of the microencapsulated cutinase, such as pH, w_o (molar ratio water/surfactant), temperature and substrate concentration were investigated. Maximal specific activity were obtained with w_o=13, at pH 10.7 and 35d`C. The cutinase showed a higher specific activity for short length fatty acids, namely butyric acid. Calculation of the apparent kinetic parameters (k_m and V_max) for the synthesis of butyl butyrate, showed a low apparent affinity of the cutinase in phosphatidylcholine reversed micelles for both substrates.     

A novel approach to study of action of water-insoluble inhibitors of enzymic reactions

The effect of water-insoluble compounds on enzyme catalytic properties was studied using a colloidal solution of water in organic solvent as reaction medium. In this microheterogeneous medium enzyme is entrapped into hydrated reversed micelles of a surfactant, the dimensions of the internal hole of the micelles being dependent on the ratio of water to surfactant. At sufficiently low values of this ratio the molecule of entrapped enzyme has limited mobility in the micelle. Because of this the interaction of the enzyme with water-insoluble compound which is added in assay solution and intercalated in the surface layer of the micelle may be manifested. The suggested method was used to study the inhibitory action of dihydroriboflavin esters on D-amino acid oxidase from pig kidney and soybean lipoxygenase. The reaction medium was hydrated reversed micelles of Aerosol OT in octane. The method of sedimentation in an analytical ultracentrifuge has shown the dihydroriboflavin esters to be completely included into reversed micelles.     

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.     

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.     

Dynamic light scattering of cutinase in AOT reverse micelles

The fungal lipolytic enzyme cutinase, incorporated into sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles has been investigated using dynamic light scattering. The reversed micelles form spontaneously when water is added to a solution of sodium bis-(2ethylhexyl) sulfosuccinate in isooctane. When an enzyme is previously dissolved in the water before its addition to the organic phase, the enzyme will be incorporated into the micelles. Enzyme encapsulation in reversed micelles can be advantageous namely to the conversion of water insoluble substrates and to carry out synthesis reactions. However protein unfolding occurs in several systems as for cutinase in sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles. Dynamic light scattering measurements of sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles with and without cutinase were taken at different water to surfactant ratios. The results indicate that cutinase was attached to the micellar wall and that might cause cutinase unfolding. The interactions between cutinase and the bis-(2ethylhexyl) sulfosuccinate interface are probably the driving force for cutinase unfolding at room temperature. Twenty-four hours after encapsulation, when cutinase is unfolded, a bimodal distribution was clearly observed. The radii of reversed micelles with unfolded cutinase were determined and found to be considerable larger than the radii of the empty reversed micelles. The majority of the reversed micelles were empty (90-96% of mass) and the remainder (4-10%) containing unfolded cutinase were larger by 26-89 A.