非离子反胶束中木素过氧化物酶催化性能研究

木素过氧化物酶（LiP）在环己烷/Brij30/水反胶束体系中可体现催化活力,然而在水/醇/TritonX100/环己烷反胶束体系中却没有催化活力。对影响Brij30反胶束中LiP催化活力各主要因素进行了优化并测定了LiP在其中的时间稳定性;结果表明,20℃下,使LiP体现最佳活力的Brij30反胶束介质条件为：ω₀＝8.5,pH＝2.2,［Brij30］＝600mmol/L;在此条件下,LiP的半衰期可达到50h;与水介质相比,酶活力下降了,但稳定性却提高了。直链醇是TritonX100形成反胶束的必要组分,为揭示醇的作用,还考察了戊醇对Brij30 反胶束中LiP催化活力的影响,发现高浓度戊醇对LiP有失活作用。据此推测助表面活性剂醇可能是LiP在环己烷/TritonX100/戊醇/水反胶束中不能体现催化活力的主要原因。     

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.     

Cutinase-AOT interactions in reverse micelles: the effect of 1-hexanol

Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution.Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.     

Variations in the activity of microsomal palmitoyl-CoA hydrolase in mixed micelle solutions of palmitoyl-CoA and non-ionic detergents of the triton X series

The kinetics of palmitoyl-CoA hydrolase were influenced by both the availability of the substrate and formation of micelles. At palmitoyl-CoA concentrations below the critical micelle concentration, addition of non-ionic detergent increased the activity until the critical micelle concentration of the mixed micelles was reached. At palmitoyl-CoA concentrations above the critical micelle concentration, inhibitor of the activity was observed, but addition of detergents of the Triton X series reversed the inhibition. Maximum palmitoyl-CoA hydrolase activity was found when the ratios (w/v) of palmitoyl-CoA: Triton X-100 and palmitoyl-CoA: Triton X-405 were approximately 0.35 and 0.05, respectively. At these above the mixed critical micelle concentration. The results indicate that monomer palmitoyl-CoA is the substrate and that monomer forms of the non-ionic detergents of the Triton X series activate the enzyme. Isolated microsomal lipids activated the microsomal palmitoyl-CoA hydrolase, suggesting that a hydrophobic environment is advantageous for interaction between enzyme and substrate in vivo. The maximum activity in the presence of mixed micelles is discussed in relation to a model where mixed micelles are regarded as artificial membranes to which the enzyme may adhere in an equilibrium with the monomer substrate and detergent in the monomer form. It is suggested that intracellular membranes may resemble mixed micelles in equilibrium with detergent-active substrates such as palmitoyl-CoA.     

Spectroscopic properties of horse liver alcohol dehydrogenase in reversed micellar solutions

Catalytic and spectroscopic properties of alcohol dehydrogenase from horse liver, incorporated in reversed micellar media, have been studied. Two different reversed micellar systems have been used, one containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT], the other containing a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. With 1-hexanol as substrate the turnover number of the enzyme in AOT-reversed micelles is strongly dependent on the water content of the system. At low wo ([H2O]/[surfactant]) (wo less than 20) no enzymatic activity can be detected whereas at high wo (wo = 40) the turnover is only slightly lower than in aqueous solution. In CTAB-reversed micelles the dependence of the turnover number on wo is much less. The enzymatic activity is in this case significantly lower than in aqueous solution and increases only slightly with an increasing water content of the reversed micelles. Possible interactions of the protein with the surfactant interfaces in the reversed micellar media were studied via circular dichroism and fluorescence measurements. From the circular dichroism of the protein backbone it is observed that the protein secondary structure is not significantly affected upon incorporation in the reversed micelles since the far-ultraviolet spectrum is not altered. Results from time-resolved fluorescence anisotropy experiments indicate that, especially in AOT-reversed micelles, interactions between the protein and the surfactant interface are largely electrostatic in nature, as evident from the dependence on the pH of the buffer used. In CTAB-reversed micellar solutions such interactions appear to be much less pronounced than in AOT.     

Characterization of lipase in reversed micelles formulated with cibacron blue F-3GA modified span 85

Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil as a model reaction. The micellar hydrodynamic radius results reflected, to some extent, the redistribution of surfactant and water after enzyme addition, and the correlation between surfactant formulation, water content (W0), micellar size, and enzyme activity. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited a different activity behavior versus W0. The optimal pH and temperature of the encapsulated lipase remained unchanged, but the apparent activity was significantly higher than that of the native enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant was found to decrease with increasing surfactant concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.     

Kinetic behavior of Desulfovibrio gigas aldehyde oxidoreductase encapsulated in reverse micelles

We report the kinetic behavior of the enzyme aldehyde oxidoreductase (AOR) from the sulfate reducing bacterium Desulfovibrio gigas (Dg) encapsulated in reverse micelles of sodium bis-(2-ethylhexyl) sulfosuccinate in isooctane using benzaldehyde, octaldehyde, and decylaldehyde as substrates. Dg AOR is a 200-kDa homodimeric protein that catalyzes the conversion of aldehydes to carboxylic acids. Ultrasedimentation analysis of Dg AOR-containing micelles showed the presence of 100-kDa molecular weight species, confirming that the Dg AOR subunits can be dissociated. UV-visible spectra of encapsulated Dg AOR are indistinguishable from the enzyme spectrum in solution, suggesting that both protein fold and metal cofactor are kept intact upon encapsulation. The catalytic constant (k(cat)) profile as a function of the micelle size W(0) (W(0)=[H(2)O]/[AOT]) using benzaldehyde as substrate showed two bell-shaped activity peaks at W(0)=20 and 26. Furthermore, enzymatic activity for octaldehyde and decylaldehyde was detected only in reverse micelles. Like for the benzaldehyde kinetics, two peaks with both similar k(cat) values and W(0) positions were obtained. EPR studies using spin-labeled reverse micelles indicated that octaldehyde and benzaldehyde are intercalated in the micelle membrane. This suggests that, though Dg AOR is found in the cytoplasm of bacterial cells, the enzyme may catalyze the reaction of substrates incorporated into a cell membrane.     

Engineering of functional supramacromolecular complexes of proteins (enzymes) using reversed micelles as matrix microreactors.

The size of the inner water cavity of reversed micelles formed in a triple system 'water-surfactant-organic solvent' can be widely varied by changing the degree of surfactant hydration. This gives grounds to use reversed micelles as matrix microreactors for the design of supramolecular complexes of proteins. Using ultracentrifugation analysis, it has been demonstrated that the oligomeric composition of various enzymes (ketoglutarate dehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) solubilized in reversed micelles of Aerosol OT [sodium bis(2-ethylehexyl)sulfosuccinate] in octane changes upon variation of the degree of hydration. An oligomeric complex forms under conditions when the radius of the micelle inner cavity is big enough to incorporate this complex as a whole. At lower degrees of hydration the micelles 'uncouple' such complexes to their components. The catalytic properties of various oligomeric complexes have been studied. Possibilities of using reversed micelles for the separation of subunits of oligomeric enzymes under non-denaturating conditions have been demonstrated. In particular, the isolated subunits of alkaline phosphatase, lactic dehydrogenase and glyceraldehyde-3-phosphate have been found to be active in Aerosol OT reversed micelles. The dependences of the catalytic activity of oligomeric enzymes represent saw-like curves. The maxima of the catalytic activity observed at these curves relate to the functioning of various oligomeric forms of an enzyme. The radii of the micelle inner cavity under conditions when these maxima are observed correlate with the linear dimensions of the enzyme oligomeric forms. Correlation of the position of a maximum with the shape of an oligomeric complex is discussed.     

Sedimentation analysis of muscle glycogen phosphorylase b entrapped in hydrated reversed micelles of aerosol OT in octane

The oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscles have been studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. Sedimentation studies show that the oligomeric state of the enzyme is controlled by the degree of hydration of micelles. Monomeric, dimeric, trimeric, tetrameric, hexameric, or octameric forms of the enzyme were observed depending on the degree of micelle hydration.     

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.     

Biodegradation of pesticides in nonionic water-in-oil microemulsions of tween 85: Relationship between micelle structure and activity

Water-in-oil microemulsion systems have been studied in recent years for a number of applications in protein separation and enzymology. Although it is well established that reversed micelle systems provide an excellent medium for nonaqueous biocatalytic studies, there is still much speculation as to the interaction of the enzyme with the surfactant interface. Polyoxyethylene sorbitan trioleate (Tween 85) is a nonionic surfactant which has some interesting properties for microemulsion formation and protein solubilization. In conjunction with a separate article describing the structural features of Tween 85 reversed micelles in hexane with isopropanol as a cosurfactant, this work describes the activity of an enzyme, organophosphorus hydrolase, for degrading organophosphorus pesticides in this microemulsion system. Ternary phase diagrams were constructed to outline the phase boundaries at different temperatures and isopropanol concentrations, which elucidate the role of the cosurfactant alcohol, as well as some features of micelle structure. Kinetic and stability studies with organophosphorus hydrolase show the effect of enzyme partitioning between the micelle surfactant layer and aqueous core. (c) 1994 John Wiley & Sons, Inc.     

Enzymes incorporated into reversed micelles of surfactants in organic solvents. Study of the protein-aerosol OT-H2O-octane system by sedimentation analysis

Using ultracentrifugation, the systems of reversed micelles of aerosol OT in octane containing solubilized protein (alpha-chymotrypsin, lysozyme, trypsin, egg albumin, alcohol dehydrogenase from horse liver and gamma-globulin) were studied. The changes in the sedimentation coefficients of reversed micelles during incorporation of the protein are correlated (within a wide range of experimental conditions, e. g. degree of surfactant hydration or protein concentration) exclusively with the molecular weight of the solubilized protein. The simplest solubilization model, according to which the protein molecule is incorporated into the inner cavity of the reversed micelle at the stoichiometric ratio of 1 : 1, which does not affect the external sizes of the reversed micelle, has been proposed. Using alpha-chymotrypsin as an example, the conditions, under which the sedimentation properties of the systems deviate from this model, have been found. These deviations occurred at sufficiently low degrees of the surfactant hydration, when the inner cavity of the reversed micelle is smaller than the effective size of the solubilized protein molecule. In the latter case the protein forms a new micelle of necessary (i. e. larger) size. Since the hydrated micelle can be regarded as an elementary (30-100 A) fragment of biomembranes, the results obtained should be taken into consideration when analyzing the structural organization and functioning of the latter.     

Protein refolding in reversed micelles

A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant-free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.     

Activation of lignin peroxidase in organic media by reversed micelles

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media.     

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.     

Characteristics of Lipase Catalysis During Ester Synthesis in Reversed Micellar Systems

The ability of lipase from Candida cylindracea to catalyze ester synthesis from a long chain fatty acid (palmitic acid) and alcohols of varying chain length, is examined. The enzyme is located in the minimal-water environment of reversed micelles. Lipase activity is a strong function of the mode of encapsulation. Direct solid lipase addition to reversed micelles leads to encapsulation in an inactive state unless the enzyme is contacted with the acyl substrate. The alcohol inhibits activity, with low molecular weight alcohols tending to denature the enzyme. Implications to reversed micelle based biocatalyst preparation are briefly discussed.     

Activity of lipoxygenase incorporated into reversed micelles of aerosol OT in octane

Soybean lipoxygenase (EC 1.13.11.12) incorporated into the reversed micelles of aerosol OT in octane has been studied for its catalytic properties. The enzyme is shown to preserve up to 10% activity as compared with the activity in the aqueous solution. In this case Km of lipoxygenase for linoleic acid increases from 10(-5) M to 5 X 10(-4) M. The activity of lipoxygenase is maximal, the aerosol OT concentration being 0.03 M and a degree of reversed micelle hydratation 40. Cationic detergents of the cetyltrimethyl ammonium bromide type are not good to form reversed micelles of lipoxygenase, since they inhibit the latter with IC50 = (4 divided by 6) x 10(-4) M. The lipoxygenase preparations in reversed micelles of aerosol OT in octane may be used to synthesize natural metabolites of polyunsaturated fatty acids, for instance of eicosanoids.     

Molecular Dynamics Simulations of Micelle Formation around Dimeric Glycophorin A Transmembrane Helices

Insertion and formation of membrane proteins involves the interaction of protein helices with one another in lipid environments. Researchers have studied glycophorin A (GpA) transmembrane helices embedded in sodium dodecyl sulfate (SDS) micelles to identify contacts significant for helix dimerization. However, a detailed picture of the conformation and dynamics of the GpA-SDS system cannot be obtained solely through experiment. Molecular dynamics simulations of SDS and a GpA dimer can provide an atomic-level picture of SDS aggregation and helix association. We report 2.5-ns simulations of GpA wild-type and mutants in a preformed micelle as well as a 32-ns simulation showing the formation of a complete micelle around wild-type GpA from an initially random placement of SDS molecules in an aqueous environment. In the latter case, an initial instability of GpA helices in water is reversed after the helices become surrounded by SDS. The properties of the spontaneously formed micelle surrounding the GpA are indistinguishable from those of the preformed micelle surrounding the GpA dimer.     

Protein recovery from reversed micellar solutions through contact with a pressurized gas phase.

We describe a new process for the recovery of encapsulated protein from reversed micellar solution in concentrated form. The method involves desolubilization of the protein by decreasing solvent density through gas dissolution. Under appropriate thermodynamic conditions, the micellar water pool can be converted to clathrate hydrates. Protein recovery is facilitated by clathrate hydrate formation, which causes the desolubilized protein to exist in a solid phase, distinct from the micellar supernatant. The process is carried out without any ionic strength or pH modification.     

The influence of heptane-1,2,3-triol on the size and shape of LDAO micelles. Implications for the crystallisation of membrane proteins

The presence of small amphiphiles has been found to be necessary in the crystallization of several membrane-protein/surfactant complexes. It has been suggested that the role of the small amphiphile may be to reduce the size of the surfactant belt around the protein, making the formation of crystals easier. Thus far it was not known if this would involve changes in micellar size in general or whether the small amphiphile would merely replace LDAO during crystal growth. In the present study we have used small angle neutron scattering to study mixed micelles of lauryldimethyl amine oxide (LDAO; hydrogenated and deuterated) and heptane-1,2,3-triol (HP). Our results show that with increasing overall HP concentrations mixed LDAO/HP micelles of decreasing mass and radius are formed. The composition of these micelles has been determined. HP thus may decrease the size of the surfactant belt around a protein before crystallisation by insertion into a host micelle. As HP is a 'small amphiphile' compared to the surfactants used for solubilization of membrane proteins, the curvature of the host micelle will be increased by its insertion.