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.     

Improvement in enzyme activity and stability by addition of low molecular weight polyethylene glycol to sodium bis(2-ethyl-L-hexyl)sulfosuccinate/isooctane reverse micellar system

The activity and stability of Chromobacterium viscosum lipase (glycerolester hydrolase, EC 3.1.1.3)-catalyzed olive oil hydrolysis in sodium bis (2-ethyl-1-hexyl)sulfosuccinate (AOT)/isooctane reverse micelles is increased appreciably when low molecular weight polyethylene glycol (PEG 400) is added to the reverse micelles. To understand the effect of PEG 400 on the phase behavior of the reverse micellar system, the phase diagram of AOT/PEG 400/water/isooctane system was studied. The influences of relevant parameters on the catalytic activity in AOT/PEG 400 reverse micelles were investigated and compared with the results in the simple AOT reverse micelles. In the presence of PEG 400, the linear decreasing trend of the lipase activity with AOT concentration, which is observed in the simple AOT reverse micelles, disappeared. Enzyme entrapped in AOT/PEG reverse micelles was very stable, retaining>75% of its initial activity after 60 d, whereas the half-life in simple AOT reverse micelles was 38 d. The kinetics parameter maximum velocity (V _max)exhibiting the temperature dependence and the activation energy obtained by Arrhenius plot was suppressed significantly by the addition of PEG 400.     

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.     

Aerosol-OT在水和聚乙二醇-200混合溶液中有序结构转变及影响因素研究

通过电导、流变学方法等研究了温度和表面活性剂浓度对双(2-乙基己基)磺基琥珀酸钠(AOT)、水和聚乙二醇-200(PEG-200)体系有序结构形成和转变的影响。结果发现,在室温下高浓度AOT溶液中出现凝胶结构。当AOT的浓度为400mmol/kg以上时,随着温度的升高,观察到体系从透明粘稠到稍浑浊再到透明低粘度的过程,结合AOT的双链结构并通过流变实验验证了网络结构的形成,推测该体系在微观上经历了从凝胶到囊泡再到胶束的转变过程。当AOT的浓度为200~400 mmol/kg时,随着温度的升高,出现了凝胶和胶束混合体系到胶束体系的转变过程。     

Fabrication of poly(ethylene glycol)‐based hydrogels entrapping enzyme‐immobilized silica nanoparticles

In this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3‐aminopropyltriethoxysilane (APTES), and the resultant SNP‐immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000 Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent K_m of SNP‐immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long‐term stability test demonstrated that SNP‐immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non‐entrapped SNP‐immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six‐fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP‐immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright © 2009 John Wiley & Sons, Ltd.     

混合反胶束的电导与微结构研究

反胶束是两亲分子在非极性溶剂中形成的一种有序组合体，在医药、化工、采油、胶束催化及酶催化等领域中有重要应用．与胶束溶液相比，人们对反胶束的形成与结构的了解至今仍不充分．特别是对于由混合表面活性剂形成的反胶束的研究几乎无人涉及．本文采用动态光散射、电导及荧光光谱等手段对阴离子表面活性剂AOT与非离子表面活性剂形成的混合反胶束进行了研究，旨在探讨利用表面活性剂的复配来调节和控制反胶束的结构和性能．亚实验部分二异辛基磺化琉璃酸钠（AOT，Sigma公司）；Brij30为含4个氧乙烯基（EO基）的十二碳醇（AcrosOrgani…     

"Snap-shooting" the interface of AOT reverse micelles: use of chemical trapping

The first use of the phenyl cation trapping technique in "snap-shooting" the local molar concentrations of water and sulfosuccinate head-groups in the interfacial region of AOT-2,2,4-trimethylpentane-water reverse micelles has been accomplished. Our results demonstrate that the interfacial concentrations of the sulfosuccinate head-groups in AOT (0.1 M)-2,2,4-trimethylpentane-water reverse micelles are remarkably high (2.75-2.34 M) across the W0 (the molar ratio of water to surfactant) range 12 to 44. However, the interfacial concentrations of water in AOT- 2,2,4-trimethylpentane-water reverse micelles across the same range of solution compositions are significantly lower (27.9-32.0 M) than the molar concentration of bulk water (55.5 M). The present results provide new insight on the microenvironments of interfacially located enzymes such as lipases entrapped in AOT-2,2,4-trimethylpentane-water reverse micelles, the most extensively exploited reverse-micellar system in micellar biotechnology.     

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     

Calorimetric study of the alpha-tocopherol solubility in reversed AOT micelles

The experimental data of heat of mixing (Q) for heterogeneous system alpha-tocopherol/AOT/n-heptane with and without water at 25 degrees C are presented. The Q dependence on AOT (sodium bis (2-ethylhexyl) sulfosuccinate) concentration, and R parameter defined as R=[H2O]/[AOT] with flow calorimetric method were investigated. Using the D'Aprano model (which is formally identical to that used earlier by Magid et al.) the binding constant (K), the distribution constant of alpha-tocopherol (K distr) between hydrocarbon and the micellar phase, and the standard enthalpy of transfer (DeltaH tr 0) of alpha-tocopherol from the hydrocarbon to AOT reversed micelles were calculated. The solubility of alpha-tocopherol in AOT reversed micelles explored with the calorimetric technique was compared to the literature data obtained respectively with UV spectrophotometry for reversed micelles and by other techniques for the phospholipid bilayer.     

DNA hybridization in reverse micelles and its application to mutation detection

DNA hybridization was investigated in AOT (sodium di-2-ethylhexyl sulfosuccinate)/isooctane reverse micelles. The single-stranded DNA molecules were encapsulated in the nanoscale water pools formed in the reverse micelles, reducing the hybridization rate. The DNA hybridization can be monitored by simply measuring the UV absorbance of the reverse micellar solution at 260 nm. We found that the DNA hybridization occurred only at the restricted water content (Wo = [H2O]/[AOT] = 20) and below room temperature. We applied this DNA hybridization behavior in reverse micelles to mutation detection in a model gene p53 and successfully detected the single nucleotide mutations in 20-mer. 30-mer and 50-mer nucleotides without a DNA labeling.     

Solvent effects on AOT reverse micelles in liquid and compressed alkanes investigated by neutron spin-echo spectroscopy

Neutron Spin-Echo (NSE) spectroscopy has been employed to study the interfacial properties of reverse micelles formed with the common surfactant sodium bis-2-ethylhexyl-sulfosuccinate (AOT) in liquid alkane solvents and compressed propane. NSE spectroscopy provides a means to measure small energy transfers for incident neutrons that correspond to thermal fluctuations on the nanosecond time scale and has been applied to the study of colloidal systems. NSE offers the unique ability to perform dynamic measurements of thermally induced shape fluctuation in the AOT surfactant monolayer. This study investigates the effects of the bulk solvent properties, water content, and the addition of octanol cosurfactant on the bending elasticity of AOT reverse micelles and the reverse micelle dynamics. By altering these solvent properties, specific trends in the bending elasticity constant, k, are observed where increasing k corresponds to an increase in micelle rigidity and a decrease in intermicellar exchange rate, k(ex). The observed corresponding trends in k and k(ex) are significant in relating the dynamics of microemulsions and their application as a reaction media. Compressed propane was also examined for the first time with a high-pressure, compressible bulk solvent where variations in temperature and pressure are used to tune the properties of the bulk phase. A decrease in the bending elasticity is observed for the d-propane/AOT/W = 8 reverse micelle system by simultaneously increasing the temperature and pressure, maintaining constant density. With isopycnic conditions, a constant translational diffusion of the reverse micelles through the bulk phase is observed, conforming to the Stokes-Einstein relationship.     

Oxidative refolding of reduced, denatured lysozyme in AOT reverse micelles

The refolding kinetics of the reduced, denatured hen egg white lysozyme in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles at different water-to-surfactant molar ratios has been investigated by fluorescence spectroscopy and UV spectroscopy. The oxidative refolding of the confined lysozyme is biphasic in AOT reverse micelles. When the water-to-surfactant molar ratio (omega 0) is 12.6, the relative activity of encapsulated lysozyme after refolding for 24 h in AOT reverse micelles increases 46% compared with that in bulk water. Furthermore, aggregation of lysozyme at a higher concentration (0.2 mM) in AOT reverse micelles at omega 0 of 6.3 or 12.6 is not observed; in contrast, the oxidative refolding of lysozyme in bulk water must be at a lower protein concentration (5 microM) in order to avoid a serious aggregation of the protein. For comparison, we have also investigated the effect of AOT on lysozyme activity and found that the residual activity of lysozyme decreases with increasing the concentration of AOT from 1 to 5 mM. When AOT concentration is larger than 2 mM, lysozyme is almost completely inactivated by AOT and most of lysozyme activity is lost. Together, our data demonstrate that AOT reverse micelles with suitable water-to-surfactant molar ratios are favorable to the oxidative refolding of reduced, denatured lysozyme at a higher concentration, compared with bulk water.     

Specifics of the formation of J-aggregates of cyanine dyes in solutions of AOT/water/hexane reverse micelles

The behavior of a cyanine dye (3,3′-di-(gamma-sulfopropyl)-4,5,4′,5′-dibenzo-9-ethylthiacarbocyanine betaine pyridinium salt) was studied in AOT/water/hexane reverse micelles over a wide range of W at various concentrations of the dye, AOT, and reverse micelles. The processes occurring during the formation of the AOT/water/hexane micellar solution were studied in detail. It has been shown that, before the formation of the stable microemulsion, the dye aggregation processes occur by virtue of the interaction of the dye with the AOT anion. The amount of J-aggregates is proportional to the logarithm of the ratio of the amount of AOT molecules to the amount of dye molecules. The time behavior of J-aggregates after the formation of a micellar structure depends on the concentration of reverse micelles, thereby indicating an important role of intermicellar exchange.     

Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles

Yeast alcohol dehydrogenase (YADH) solubilized in reverse micelles of aerosol OT (i.e., AOT or sodium bis (2-ethyl hexyl) sulfosuccinate) in isooctane has been shown to be catalytically more active than that in aqueous buffer under optimum conditions of pH, temperature, and water content in reverse micelles. Studies of the secondary structure conformational changes of the enzyme in reverse micelles have been made from circular dichroism spectroscopy. It has been seen that the conformation of YADH in reverse micelles is extremely sensitive to pH, temperature, and water content. A comparison has been made between the catalytic activity of the enzyme and the α-helix content in the conformation and it has been observed that the enzyme is most active at the maximum α-helix content. While the β-sheet content in the conformation of the entrapped enzyme was found to be dependent on the enzyme–micelle interface interaction, the α-helix and random coil conformations are governed by the degree of entrapment and the extent of rigidity provided by the micelle core to the enzyme structure.     

反相胶束中辣根过氧化物酶的停流光谱研究

用停流光谱法研究了HRP在AOT、CTAB和SDS反相胶束中的吸收光谱和反应动力学，实验结果显示在AOT反相胶束中，HRP的吸收峰位置与水相中相同；而另外两种反相胶束对HRP的分子结构产生了较大影响，快速反应动力学研究显示在反相胶束中HRP形成化合物Ⅰ的速率常数远远高于化合物Ⅰ形成HRP—Ⅱ的反应速率常数，推测这是反相胶束的特殊性质造成的结果。     

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。     

反相胶束对辣根过氧化物酶催化反应的影响

胶束体系是酶学研究比较理想的体系,因为它所具有的诸如热力学稳定、光学透明及能增溶亲水分子、亲油分子或两性分子等性质,使许多酶在胶束体系中的反应速率远远高于在水相中,即人们发现的所谓“超活性”［‘j．辣根过氧化物酶（HRP）是一种比较稳定的酶,且价廉易得,具备一般过氧化物酶的典型反应．在研究中人们发现,HRP在反相胶束体系中同样具有“超活性”,由于HRP能够催化大量底物进行反应,因此“超活性”对HRP的催化反应具有重要意义．已有研究者［’、’j对CTAB反相胶束体系中HRP的性质进行了探讨,但反相胶束对HRP的…     

Investigation on the basic hydrolysis of crystal violet in mixed reverse micelles formed with AOT and nonionic surfactants

 The kinetics and thermodynamics of the basic hydrolysis of crystal violet (CV) in mixed reverse micelles formed with anionic surfactant AOT and nonionic surfactants have been investigated. It was found that the mixed reverse micelles had inhibitory effects on CV hydrolysis compared with the normal aqueous solution, and the equilibrium constant K of the reaction in mixed reverse micellar systems is smaller than that in pure water. The influence of water content and surfactant composition in reverse micelles on the second-order rate constant k ₁ of the positive reaction, on the first-order rate constant k _-1 of the reverse reaction, as well as on the equilibrium constant K of the reaction has been studied, and the results obtained were interpreted in terms of the nature of surfactants and the properties of microenvironment where the reaction took place. Received: 24 October 1997 Accepted: 18 March 1998     

Phase behaviors of AOT/longer chain n-alkanes reverse micelles in the presence of compressed ethylene

It was found that, in a suitable pressure range, ethylene could increase the amount of solubilized water in reverse micelles of sodium bis-2-ethylhexylsulfosuccinate (AOT) in longer chain n-alkanes considerably, where the phase separation was induced by a micelle-micelle interaction mechanism. The microenvironments in the ethylene-stabilized reverse micelles were investigated by the UV-vis adsorption spectra using methyl orange (MO) as a probe. The maximum absorption of MO decreased with the increase of ethylene pressure at constant W0 value. Conductivity measurements demonstrated that the percolation temperature of the reverse micellar system increased considerably after compressed ethylene was added. The results of this work confirm that some small-molecule gases have the function of cosurfactants to stabilize reverse micelles.     

Effect of the Cationic Surfactant Moiety on the Structure of Water Entrapped in Two Catanionic Reverse Micelles Created from Ionic Liquid‐Like Surfactants

The behavior of water entrapped in reverse micelles (RMs) formed by two catanionic ionic liquid‐like surfactants, benzyl‐n‐hexadecyldimethylammonium 1,4‐bis‐2‐ethylhexylsulfosuccinate (AOT‐BHD) and cetyltrimethylammonium 1,4‐bis‐2‐ethylhexylsulfosuccinate (AOT‐CTA), was investigated by using dynamic (DLS) and static (SLS) light scattering, FTIR, and ¹H NMR spectroscopy techniques. To the best of our knowledge, this is the first report in which AOT‐CTA has been used to create RMs and encapsulate water. DLS and SLS results revealed the formation of RMs in benzene and the interaction of water with the RM interface. From FTIR and ¹H NMR spectroscopy data, a difference in the magnitude of the water–catanionic surfactant interaction at the interface is observed. For the AOT‐BHD RMs, a strong water–surfactant interaction can be invoked whereas for AOT‐CTA this interaction seems to be weaker. Consequently, more water molecules interact with the interface in AOT‐BHD RMs with a completely disrupted hydrogen‐bond network, than in AOT‐CTA RMs in which the water structure is partially preserved. We suggest that the benzyl group present in the BHD⁺ moiety in AOT‐BHD is responsible for the behavior of the catanionic interface in comparison with the interface created in AOT‐CTA. These results show that a simple change in the cationic component in the catanionic surfactant promotes remarkable changes in the RMs interface with interesting consequences, in particular when using the systems as nanoreactors.