Thermostability of <Emphasis Type="Italic">Cromobacterium viscosum</Emphasis> lipase in AOT/isooctane reverse micelle

The thermostability of Cromobacterium viscosum lip ase (EC 3.1.1.3) entrapped in AOT (sodium bis-[2-ethylhexyl] sulfosuccinate) reverse micelles was in creased by the addition of short-chain polyethylene glycol (PEG 400). Two different approaches were considered: (1) the determination of half-life time and (2) the mechanistic analysis of deactivation kinetics. The half-life of lipase entrapped in AOT/isooctane reverse micelles with PEG 400 at 60°C was 28h, ninefold higher than that in reverse micelles without PEG 400. The lip ase entrapped in both reverse micellar systems followed a series-type deactivation mechanism involving two first-order steps. The deactivation constant for the first step at 60°C in PEG containing reverse micelles was 0.055 h¹¹, 11-fold lower than that in reverse micelles without PEG, whereas it remained almost constant for the second step. The inactivation energy of the lip ase entrapped in reverse micelles with and without PEG 400 was 88.12 and 21.97 kJ/mol, respectively.     

Thermostability of Cromobacterium viscosum lipase in AOT/isooctane reverse micelle

The thermostability of Cromobacterium viscosum lipase (EC 3.1.1.3) entrapped in AOT (sodium bis-[2-ethylhexyl] sulfosuccinate) reverse micelles was increased by the addition of short-chain polyethylene glycol (PEG 400). Two different approaches were considered: (1) the determination of half-life time and (2) the mechanistic analysis of deactivation kinetics. The half-life of lipase entrapped in AOT/isooctane reverse micelles with PEG 400 at 60 degrees C was 28 h, ninefold higher than that in reverse micelles without PEG 400. The lipase entrapped in both reverse micellar systems followed a series-type deactivation mechanism involving two first-order steps. The deactivation constant for the first step at 60 degrees C in PEG containing reverse micelles was 0.055 h!1, 11-fold lower than that in reverse micelles without PEG, whereas it remained almost constant for the second step. The inactivation energy of the lipase entrapped in reverse micelles with and without PEG 400 was 88.12 and 21.97 kJ/mol, respectively.     

AEOT反胶束中脂肪酶的催化活性

反胶束已广泛应用于膜模拟化学和蛋白质的液 液萃取中[1～ 3] ,反胶束酶反应作为实现有机相酶催化的方法之一 ,具有许多独特的优点 ,反胶束独特的结构特征使表面活性剂分子组成的膜将油水相隔开 ,从而有利于保持酶的活性和稳定性。酶在反胶束的微水环境中比在水溶液中更接近天然的细胞内环境 ,在这里酶和底物分子均可得到有效的分散 ,接触几率大大提高 ,因而催化效率也得到很大提高。反胶束可以适用于各种类型的 (亲水的、疏水的和双亲的 )底物[4] ,已逐步形成“胶束酶学”的研究分支 ,研究胶束酶学的Martinek等[3] 曾预言 :反胶束体系有可…     

Effect of the Pretreatment of Lipase with Organic Solvents on its Conformation and Activity in Reverse Micelles

The activity and conformation of Chromobacterium viscosum lipase-pretreated with various organic solvents were investigated. The pretreatment of lipase led to a substantial increase of enzyme activity in AOT (sodium bis [2-ethyl -1-hexyl] sulfosuccinate)/isooctane/water reverse micelles. Among the organic solvents used, n-hexane was found to be most effective. It was observed that higher hexane content with shorter agitation time and vice versa had almost the same effect on the initial activity of lipase. The kinetic study showed that the Michaelis constant (K _m) and the substrate adsorption equilibrium constant (K _ad) were reduced by the pretreatment of lipase with hexane, whereas the change in the maximum reaction rate (V _max) was insignificant. The two spectroscopic techniques (Fluorescence spectra of lipase encapsulated in RMs and Fourier transform infrared [FTIR] spectra of lipase powders) were performed to detect possible conformational changes in the enzyme caused by the pretreatment. A correlation between the maximum fluorescence intensity and the activity of treated lipase was found as a function of agitation time. The FTIR spectrum of lipase showed a new shape peak corresponding to 1,500 cm⁻¹ as a result of pretreatment with organic solvents.     

二—（2—乙基己基聚氧化烯烃）琥珀酸双酯磺酸钠的表面活性及应用

二－（２－乙基己基聚氧化烯烃）琥珀酸双酯磺酸钠的表面活性及应用姚传义吴金川何志敏＊（天津大学化学工程研究所天津３０００７２）关键词二－（２－乙基己基聚氧化烯烃）琥珀酸双酯磺酸钠，表面活性，反胶团酶反应，橄榄油水解１９９７－０９－１９收稿，１９９７－１...     

Influence of Gold Nanoparticles of Varying Size in Improving the Lipase Activity within Cationic Reverse Micelles

Herein, we report the effect of gold nanoparticles (GNPs) in enhancing lipase activity in reverse micelles of cetyltrimethylammonium bromide (CTAB)/water/isooctane/n‐hexanol. The size and concentration of the nanoparticles were varied and their specific roles were assessed in detail. An overall enhancement of activity was observed in the GNP‐doped CTAB reverse micelles. The improvement in activity becomes more prominent with increasing concentration and size of the GNPs (0–52 μM and ca. 3–30 nm, respectively). The observed highest lipase activity (k₂=1070±12 cm³ g^?1 s^?1) in GNP‐doped CTAB reverse micelles ([GNP]: 52 μm, ca. 20 nm) is 2.5‐fold higher than in CTAB reverse micelles without GNPs. Improvement in the lipase activity is only specific to the GNP‐doped reverse micellar media, whereas GNP deactivates and structurally deforms the enzyme in aqueous media. The reason for this activation is probably due to the formation of larger‐sized reverse micelles in which the GNP acts as a polar core and the surfactants aggregate around the nanoparticle (‘GNP pool’) instead of only water. Lipase at the augmented interface of the GNP‐doped reverse micelle showed improved activity because of enhancement in both the substrate and enzyme concentrations and increased flexibility in the lipase conformation. The extent of the activation is greater in the case of the larger‐sized GNPs. A correlation has been established between the activity of lipase and its secondary structure by using circular dichroism and FTIR spectroscopic analysis. The generalized influence of GNP is verified in the reverse micelles of another surfactant, namely, cetyltripropylammonium bromide (CTPAB). TEM, dynamic light scattering (DLS), and UV/Vis spectroscopic analysis were utilized to characterize the GNPs and the organized aggregates. For the first time, CTAB‐based reverse micelles have been found to be an excellent host for lipase simply by doping with appropriately sized GNPs.     

Nonionic surfactants: a key to enhance the enzyme activity at cationic reverse micellar interface

The primary objective of the present study is to understand how the different nonionic surfactants modify the anisotropic interface of cationic water-in-oil (W/O) microemulsions and thus influences the catalytic efficiency of surface-active enzymes. Activity of Chromobacterium viscosum lipase (CV-lipase) was estimated in several mixed reverse micelles prepared from CTAB and four different nonionic surfactants, Brij-30, Brij-92, Tween-20, and Tween-80/water/isooctane/n-hexanol at different z ([cosurfactant]/[surfactants]) values, pH 6 (20 mM phosphate), 25 degrees C across a varying range of W0 ([water]/[surfactants]) using p-nitrophenyl-n-octanoate as the substrate. Lipase activity in mixed reverse micelles improved maximum up to approximately 200% with increasing content of non-ionic surfactants compared to that in CTAB probably due to the reduced positive charge density as well as plummeted n-hexanol (competitive inhibitor of lipase) content at the interfacial region of cationic W/O microemulsions. The highest activity of lipase was observed in CTAB (10 mM) + Brij-30 (40 mM)/isooctane/n-hexanol)/water system, k2 = 913 +/- 5 cm3 g-1 s-1. Interestingly, this observed activity is even higher than that obtained in sodium bis (2-ethyl-1-hexyl) sulfosuccinate (AOT)/n-heptane reverse micelles, the most popular W/O microemulsion in micellar enzymology. To ascertain the influence of non-ionic surfactants in improving the activity of surface-active enzymes is not limited to lipase only, we have also investigated the catalytic activity of Horseradish peroxidase (HRP) in different mixed W/O microemulsions. Here also following the similar trend as observed for lipase, HRP activity enhanced up to 2.5 fold with increasing concentration of nonionic surfactants. Finally, the enzyme activity was correlated with the change in the microenvironment of mixed reverse micelles by steady-state fluorescence study using 8-anilino-1-napthalenesulphonic acid (ANS) as probe.     

反胶团相转移法提纯酵母脂肪酶

反胶团相转移法是80年代兴起的一种新型分离技术，它利用表面活性剂分子在有机溶剂中自发形成的反向胶团（反胶团），在一定条件下将水溶性蛋白质分子增溶进反胶团的极性核（水池）中，再创造条件将蛋白质抽提至另一水相，实现蛋白质的相转移，达到分离和提纯蛋白质的目的[1]．反胶团中的蛋白质分子受到周围水分子和表面活性剂极性头的保护，仍保持一定的活性，甚至表现出超活性[2]．由于蛋白质增溶于反胶团与蛋白质所带电荷及反胶团内表面电荷间的静电作用及反胶团的大小有关[3～5]，因而表面活性剂的种类、水溶液的PH值及离子强度等因素…     

Effects of Nonpolar Solvents on the Solubilization of Pepsin into Bis(2-Ethylhexyl) Sodium Sulfosuccinate and Cetyltrimethylammonium Bromide Reverse Micelles

Solubilization of pepsin by bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and cetyltrimethylammonium bromide (CTAB) reverse micelles has been studied at 20C. Isooctane, cyclohexane and hexane were used as solvents, and n-butanol, amyl alcohol and hexanol were used as cosurfactants for CTAB. AOT concentrations were varied from 50 to 500 mM and pepsin concentrations were varied from 2 to 10 mg-mL^–1. At 250 mM, AOT can solubilize more than 85% of the Pepsin in each solvent. The effect of aqueous-phase pH on the solubilization of Pepsin has been studied from pH 1 to 8. The maximum solubilization of pepsin was observed below the isoelectric point (pI = 1.5) of the protein at pH 1.0 with 300 mM of AOT. The CTAB solutions were prepared by dissolving CTAB in isooctane with varying concentrations (0–100% v/v) of n-butanol, amyl alcohol or hexanol cosurfactants. It was found that 5% cosurfactant with 100 mM of CTAB was sufficient to solubilize more than 90% of the total pepsin. Pepsin solubilization by AOT reverse micelles increases with increasing polarizability and molar volume of the solvents.     

Structural evolution of a two-component organogel

Dry reverse micelles of AOT in isooctane spontaneously undergo a microstructural transition to an organogel upon the addition of a phenolic dopant, p-chlorophenol. This microstructural evolution has been studied through a combination of light scattering, small-angle neutron scattering (SANS), NMR, and rheology. Several equilibrium stages between the system of dry reverse micelles of AOT and a 1:1 AOT/p-chlorophenol (molar ratio) gel in isooctane have been examined. To achieve this, p-chlorophenol is added progressively to the dilute solutions of AOT in isooctane, and this concentration series is then analyzed. The dry micelles of AOT in isooctane do not undergo any detectable structural change up to a certain p-chlorophenol concentration. Upon a very small increment in the concentration of p-chlorophenol beyond this "threshold" concentration, large strandlike aggregates are observed which then evolve to the three-dimensional gel network.     

Influence of Confined Water on the Photophysics of Dissolved Solutes in Reverse Micelles

The photophysical parameters of two probes with largely different hydrophobic character, namely, coumarin 1 and coumarin 343, are investigated in sodium bis‐(2‐ethylhexyl)sulfosuccinate (AOT)/hexane/water reverse micelles at various water/AOT molar ratio w₀. Correlation of photophysical parameters such as fluorescence quantum yield, fluorescence lifetime, and emission maxima with w₀ indicate distinctly different trends below and above w₀≈7 for both probes. The variation of the average rotational correlation times obtained from fluorescence anisotropy decays for both probes in reverse micelles further corroborate the above observation. Similar studies were also performed in nonaqueous reverse micelles with acetonitrile as polar solvent. Similar to aqueous reverse micelles, breaks in the photophysical parameters with increasing acetonitrile/AOT molar ratios w′₀ were also observed in these cases, although at a much lower w′₀ value of 3. The present results indicate that around w₀≈7 for aqueous reverse micelles (and around w′₀≈3 for nonaqueous reverse micelles) a distinct change occurs in the probe microenvironment, which is rationalized on the basis of the relative populations of interfacial and core water. We propose that until the ionic head groups and counterions are fully solvated by polar solvents, that is, up to w₀≈7 (or w′₀≈3), the interfacial water population dominates. Above these molar ratios coalescence of excess water molecules with each other to form truncated H‐bonded water clusters leads to a sizable population of core water. This is further substantiated by changes in the IR absorption spectra for the O? D stretching mode of diluted D₂O in reverse micelles with varying w₀. Critical comparison of the present results with relevant literature reports provide clear support for the proposals made on water structure in reverse micelles. The role of relative size of the probe and the reverse micelles for differences in polar solvent to AOT ratios (w₀=7 and w′₀=3) in the observed breaks in the two types of reverse micelles is also discussed.     

Preparation and properties of surfactant-bacillolysin ion-pair in organic solvents

The transesterification-active enzyme bacillolysin was extracted into organic solvents such as isooctane by enzyme-AOT (bis (2-ethylhexyl) sulfosuccinate) ion-pairing preserving its natural second structure and catalytic activity. Extraction efficiency was affected by the interaction mode of the two phases, ionic strength, and pH of aqueous phase, surfactant and enzyme concentration. Magnetic stirring with phase mixing was favorable for the enzyme extraction. Optimal ionic strength and pH were 8 mM CaCl₂ and 5.0, respectively. Critical number of AOT molecule for an enzyme molecule to be extracted into isooctane was 89. Optimal initial enzyme concentration in the aqueous phase was 7 mg mL⁻¹ while the initial AOT concentration in isooctane was 3 mM. Within CMC (critical micellar concentration) of AOT in isooctane, the increase of initial AOT concentration enhanced the extraction efficiency.     

Effects of Surfactant and Salt Species in Reverse Micellar Forward Extraction Efficiency of Isoflavones with Enriched Protein from Soy Flour

Suitability of reverse micelles of anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT) and sodium dodecyl sulfate (SDS), cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) and nonionic surfactant polyoxyethylene p-t-octylphenol (TritonX-100) in organic solvent isooctane for extraction of soy isoflavone-enriching proteins was investigated. The results showed that the order of combined isoflavone contents was SDS>CTAB>Triton X-100>AOT, while the order of protein recovery was SDS>AOT>TritonX-100>CTAB. As compared with ACN-HCl extraction, the total amount of isoflavones was lower than reverse micellar extraction. Ion strength was one of the important conditions to control extraction of isoflavone-enriching proteins with AOT reversed micelles. For the six salt systems, KNO₃, KCl, MgCl₂, CaCl₂, NaCl, and Na₂SO₄, extracted fraction of isoflavone-enriching proteins was measured. Salt solutions greatly influenced the extraction efficiency of isoflavones in an order of KNO₃>MgCl₂>CaCl₂>KCl>NaCl>Na₂SO₄, while protein in an order of MgCl₂>CaCl₂>NaCl>KNO₃>Na₂SO₄>KCl.     

SOMATOSTATIN IN A WATER-RESTRICTED ENVIRONMENT: FLUORESCENCE AND CIRCULAR DICHROISM STUDY IN AOT REVERSE MICELLES

Abstract— Steady-state and time-resolved fluorescence emission from the single tryptophan residue of somatostatin, and the kinetics of quenching of this emission, were studied in aqueous solution and in reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT)/water/isooctane, a system that mimics the water-membrane interface well- Incorporation into micelles caused blue shifts and reduced band-widths of the emission peaks and altered the quantum yields with respect to emission from bulk water. Steady-state anisotropy values also increased considerably on micellization. These observations point to reduced polarity of the environment around the Tip residue of the peptide, as well as restricted freedom of its rotational motions, due to transfer from the aqueous to the micellar phase. Fluorescence emission kinetics of the Tip moiety followed biexponential decay laws in both aqueous and micellar media. Static and dynamic quenching constants were measured for acrylamide and CC1₄ quenchers localized in the micellar and organic pseudophases, respectively, using both steady-state and time-resolved experiments. The efficiency of dynamic quenching by acrylamide became vanishingly small in going from water to reverse micelles, in sharp contrast to the comparable quenching efficiencies exhibited by CC1₄ in micelles and acrylamide in water. The circular dichroic (CD) spectrum of the native peptide in water indicated the possibility of some amount of P-type secondary structure being present. Conformational analysis of CD spectra in micelles showed that the relative amount of this structural feature was enhanced for the micellized peptide but was insensitive to the water content of micelles. The above results, put together, indicate that the Trp-8 residue in somatostatin is likely to be located in the close neighborhood of the water-AOT molecular interface, where the water molecules are strongly immobilized. This work also demonstrates the role of reverse micelles as a convenient membrane-mimetic medium for the study of membrane interactions of bioactive peptides.     

高压CO~2对反胶束溶解蛋白质性质的影响

在308.15K下,研究了表面活性剂琥珀酸二(2-乙基己基)酯磺酸钠(Aerosol-Ot,简称AOT)的浓度和水的含量不同时,溶解的CO~2对反胶束溶解牛血清蛋白(BSA)的性质和异辛烷中AOT反胶束稳定性的影响。实验表明,在适当条件下,CO~2可以使反胶束溶液中的蛋白质全部析出。本研究对有关机理进行了初步分析。     

Micropolarity of sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles prepared in supercritical ethane and near-critical propane

The micropolarity of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles prepared in supercritical ethane and near-critical propane has been determined in terms of a solvent polarity parameter, E _T(30) values, by using absorption probes, 1-ethyl-4-methoxycarbonyl pyridinium iodide and 2,6-diphenyl-4-(2,4,6-triphenylpyridinio)-phenolate as a functions of pressure and the molar ratio of water to AOT, W ₀, at a constant temperature of 310 K. The micropolarity comparable to that of ethanol was observed for reverse micelles containing water of W ₀ = 2. The micropolarity increased with the water content and became independent of pressure after the system changed to a one-phase reverse micelle solution. For a given W ₀ value, no difference in the micropolarity was noticed in the micelles prepared in ethane and propane. Phase behaviour investigations have revealed that complete dissolution of 50 mM AOT occurred at 20 MPa in supercritical ethane, while a much lower pressure of 1 MPa was required in near-critical propane. The amount of water solubilized in reverse micelles formed in supercritical ethane was relatively low, reaching a W ₀ value of 7 at 36 MPa. In contrast, the amount of water solubilized in near-critical propane reverse micelles was W ₀ = 11 at a much lower pressure of 6 MPa. A higher pressure was required to solubilize larger amount of water in reverse micelles prepared in both ethane and propane. Received: 9 October 1998 Accepted in revised form: 12 February 1999     

Intermicellar material exchange in reverse micelles formed by ionic AOT and nonionic Igepal surfactants studied by means of pulse radiolysis. Influence of the temperature

The recombination of thiocyanate anion radicals, (SCN) ₂ ⁻ , formed pulse radiolytically within the water pools of reverse micelles stabilized with anionic AOT and nonionic Igepal surfactants, was proved as an indicator reaction to study intermicellar exchange. It was found that the exchange process is slower inIgepal than in AOT reverse micelles with the same water to surfactant ratio. The apparent activation enthalpy and entropy of the exchange process were determined in different alkanes. For the AOT and Igepal reverse micelles the activation parameters increase with the droplet size, but for the AOT systems they do not significantly change with the increase of droplet concentration. For non-percolated systems the activation parameters for Igepal reverse micelles approach those for AOT reverse micelles. This result supports existing suggestions that the mechanism of intermicellar exchange does not differ in principle between reverse micelles stabilized with ionic and nonionic surfactants.     

Micellar Solubilization of Biopolymers in Hydrocarbon Solvents III. Empirical Definition of an Acidity Scale in Reverse Micelles

This paper deals with the problem of defining, and measuring, the pH inside the water pool (which we define as pH_wp) of reverse micelles, i.e. micelles formed by surfactants dissolved in apolar solvents in the presence of minimal amounts of water. The conceptual and experimental difficulties are discussed, and it is argued that no absolute determination of pH_wp is possible, mostly because water in the water pools of reverse micelles is a new solvent, for which no standardization of acidity is available. The problem can be approached only on the basis of an empirical acidity scale. An empirical acidity scale for water pools in reverse micelles of bis (2-ethyl-hexyl) sodium sulfosuccinate (AOT) in isooctane has been defined by measuring the ³¹P-chemical shifts of phosphate buffers. The chemical shifts in bulk water were compared to those found in reverse micelles under the assumption that the pK of phosphate ion is the same in the two systems. It was found that in most cases there was little difference (less than 0.4 pH units) between pH_wp and the pH of the starting buffer in bulk water (which we define as pH_st). However, this difference between pH_wp and pH_st may become much larger in certain cases. The difference (pH_wp–pH_st) is measured under a variety of conditions, and this permits the determination of an operational acidity in the micelle water pools as a function of the pH_st with which the aqueous micelles are prepared. The significance of such data for interpreting the behaviour of enzymes confined in the micelles water pool is discussed. Based on the pH_wp scale, the apparent pK_a of phenol-red and 4-nitrophenol were determined in reverse micelles containing different buffers and different water content. The pK_a values obtained were rather sensitive to changes of both these factors, which was taken to signify that organic dies have only a very limited applicability to measure the acidity of the water pools of reverse micelles.     

Activity and kinetics studies of yeast alcohol dehydrogenase in a reverse micelle formulated from functional surfactants

Yeast alcohol dehydrogenase (YADH) showed substantial decrease in its catalytic activity due to the strong electrostatic interaction between the head groups of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and YADH in AOT reverse micelles. However, the catalytic activity of YADH in a nonionic reverse micellar interface (GGDE/TX-100) obtained from a functional nonionic surfactant N-gluconyl glutamic acid didecyl ester (GGDE) and Triton X-100 (TX-100) was higher than that in AOT reverse micelle under the respective optimum conditions. A comparison of the kinetic parameters showed that the turnover number k_cat in GGDE/TX-100 reverse micelle was 1.4 times as large as that in AOT reverse micelle, but the Michaelis constants in AOT reverse micelle for ethanol K_m^B was twice and for coenzyme NAD⁺ K_m^A was 5 times higher than their counterparts in GGDE/TX-100 reverse micelle. For the conversion of ethanol, the smaller K_m^B and larger k_cat in GGDE/TX-100 reverse micelle resulted in higher catalytic efficiency k_cat/K_m^B. The stability of YADH in GGDE/TX-100 reverse micelle was also found to be better than that in AOT reverse micelle. They were mainly attributed to the absence of electric charge on the head groups of GGDE and TX-100 in the GGDE/TX-100 reverse micelle.      

Specific Behavior of Hydrogen Peroxide in a System of AOT Reverse Micelles

It is revealed that, in contrast to organic hydroperoxides, hydrogen peroxide (H₂O₂) is rapidly decomposed in a system of reverse micelles of sodium bis(2-ehylhexyl)sulfosuccinate (AOT) in n-decane. The yield of free radicals upon the decomposition of H₂O₂ in a system of reverse micelles upon the interaction between AOT and cobalt acetyl acetonate (Co(acac)₂) is studied by the inhibitor method using an original spin trap. It is established that the interaction between H₂O₂ and AOT proceeds with no radical formation. Co(acac)₂ catalyses the radical decomposition of H₂O₂ in an aqueous solution. In micellar AOT solutions in n-decane, H₂O₂ and Co(acac)₂ in practice do not react, because H₂O₂ is localized in a micelle water pool, Co(acac)₂ and the spin trap, in the organic phase. In this case, the generation of radicals is not observed.