The influence of composition and structure of water-in-oil microemulsions on activities of Iraqi Turnip peroxidase

The activity of the enzyme Iraqi Turnip peroxidase (ITP) is studied in a reverse microemulsion composed of chloroform, aqueous buffer, sodium dodecylsulfate (SDS) and alcohols of the homologous series 1-propanol to 1-hexanol through the measurements of absorbancy of the product of oxidation at the wavelength of 470 nm in the course of reactions. The ITP catalyzed reaction is the oxidation of guaiacol by hydrogen peroxide. Maximum enzyme activity was obtained at ω₀ (molar ratio of water to surfactant) = 8. It was found that the oxidation reaction obeyed Michaelis–Menten kinetics in the investigated concentration rang (0.08–0.8 mM) of the substrate, and the Michaelis constant K_m and maximal reaction rate V_m were determined. The enzyme inhibition caused by the alcohols in microemulsions is a consequence of both the solubility of the alcohols in the buffer and the flexibility of the interfacial film.     

Horseradish peroxidase activity in a reverse catanionic microemulsion

In this paper we present the first results of enzymatic activities in a reverse microemulsion medium based on a mixture of an anionic and a cationic surfactant, called catanionic microemulsion. The studied system is composed of sodium dodecyl sulfate (SDS)/dodecyltrimethylammonium bromide (DTAB)/n-hexanol/citrate buffer/n-dodecane, with high SDS/(SDS + DTAB) weight fractions. It turns out that the results are similar to those obtained in classical reverse microemulsions, except that the presence of DTAB exerts an inhibiting effect on the enzyme. Nevertheless, enzymatic superactivities are found even at a DTAB to total surfactant ratio of 15%, corresponding to 3% weight fraction of cationic surfactant in the microemulsion. The influence of pH and hexanol content on the enzymatic activities is also studied.     

Tailoring of horseradish peroxidase activity in cationic water-in-oil microemulsions

Horseradish peroxidase (HRP) in cationic water-in-oil (W/O) microemulsions has always been ignored in reverse micellar enzymology, mainly because cationic surfactants are inhibitors of enzyme peroxidase. In the present study, for the first time, we have successfully introduced the cationic W/O microemulsion as an attractive host for efficient HRP activity. To this notion, much improved activity of HRP was observed in the W/O microemulsion of cetyltrimethylammonium bromide (CTAB) with an increase in n-hexanol concentration and W0 ([water]/[surfactant]), presumably due to the increased interfacial area of the microemulsions. In support of our above observation, six surfactants were synthesized with an increased headgroup size where the methyl groups of CTAB were subsequently replaced by the n-propyl and 2-hydroxyethyl groups, respectively, to prepare mono-, di-, and tripropylated/hydroxyethylated n-hexadecylammonium bromide. The peroxidase activity enhanced with headgroup size and also followed an overall trend similar to that found in the case of CTAB. Possibly, the reduced positive charge density at the augmented interfacial area by means of increase, either in headgroup size, cosurfactant concentration, and/or W0, is not capable of inactivating HRP. Also, the larger space at the interface may facilitate easier solubilization of the enzyme and increase the local concentration of enzyme and substrate, leading to the higher activity of HRP. The best activity was obtained with surfactant N-hexadecyl-N,N,N-tripropylammonium bromide, the highest ever found in any cationic W/O microemulsions, being almost 3 times higher than that found in water. Strikingly, this observed highest activity is comparable with that observed in an anionic bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT)-based system, the best W/O microemulsions used for HRP.     

Tween80/BmimPF6/醇/甲苯体系的相行为

以环境友好型的Tween80为表面活性剂, 以醇(乙醇、正丁醇、正己醇、正辛醇、正癸醇和异戊醇)为助表面活性剂, 对离子液体1-丁基-3-甲基咪唑六氟磷酸盐(bmimPF6)和甲苯进行了微乳化实验, 绘制了不同条件下Tween80离子液体的微乳体系的拟三元相图, 考察了醇的种类、含量对单相微乳区的影响, 并用电导法研究了在乙醇为助表面活性剂情况下, 单相微乳区的结构转变. 结果表明, 当醇(异戊醇)固定时, 随着表面活性剂/醇的质量比增加, 单相微乳区的面积逐渐增大; 不同链长的直链醇对单相微乳区的面积影响与该醇在离子液体中的溶解情况有关, 单相微乳区的面积随着直链醇链长的增加而越小; 当乙醇作助表面活性剂时, 所得到的单相微乳区的面积最大, 且单相微乳区存在着O/IL(oil-in-ionic liquid)、双连续相和IL/O(ionic liquid-in-oil)三种微结构. 尤其对离子液体微乳体系的电导随油的含量的增加而最初增大的现象进行了解释, 这一现象是由于油主要起到减少离子液体中离子对或离子的积聚, 提高带电离子淌度的作用.     

The effect of changing the microstructure of a microemulsion on chemical reactivity

A kinetic study was carried out on various solvolytic reactions in water/ NH4OT /isooctane microemulsions. The NH4OT surfactant is a derivative of the sodium salt of bis(2-ethylhexyl) sulfosuccinate (NaOT or AOT), where the Na+ counterion has been replaced by NH4+. The counterion substitution effects the phase diagram of the system, and therefore, NH4OT-based microemulsions with high water content reaching values of W = 350 (W = [H2O]/[NH4OT]) can be obtained. The presence of high W values suggests a transition in the microemulsion microstructure from water-in-oil (w/o) to oil-in-water (o/w), as was confirmed by conductivity and 1H NMR self-diffusion measurements. The interpretation of the kinetic studies in terms of pseudophase formalism allows us to analyze the effect of the microemulsion on chemical reactivity, regardless of its microstructure. It has been confirmed that the values of the solvolytic rate constants at the interphase of oil-in-water microemulsions are similar to those obtained for aqueous SDS systems, showing that the hydration degree of the interphase of the oil-in-water microemulsions is independent of W. The influence of the surfactant counterion on the solvolytic rate constants was analyzed by comparing HOT-, NaOT-, and NH4OT-based microemulsions. An important influence on the rate constants caused by the changes in the structural properties of water has been observed as was confirmed by the water 1H NMR signals.     

THE HYDROLYSIS OF PHOSPHATIDYLCHOLINE BY PHOSPHOLIPASE A2 IN MICROEMULSION AS MICROREACTOR

Phosphotipase A₂ (PLA₂) was used to hydrolyze phosphatidylcholine (PC) in microemulsions. Phase diagrams were constructed for mixtures of alcohols (C₂-C₆), medium chain triglycerides (tricaprylin, TC) or vegetable oils, PC and water, and areas corresponding to isotropic systems were identified. The PC hydrolysis was carried out with high yields at various PC/(TC+alcohol) compositions within the areas of isotropic systems at increasing amounts of solubilized water.

The initial reaction rates depended on the aggregation state of the PC (size and nature of microemulsion structures), as well as on temperature and mode of calcium addition.

At low enzyme concentrations, hydrolysis was preceded by a “lag phase” followed by an abrupt increase in rate. By contrast, no latency was observed at higher PLA₂ PC ratios and the rate was significantly higher.

The easy access of the enzyme to the substrate in the curved phospholipid-containing microemulsion particles facilitate activation of the enzyme and “pushes” the reaction to completion. Hence, these microemulsions can serve as microreactors for the enzymatic high yield hydrolysis.     

Enzyme Activity and Structural Dynamics Linked to Micelle Formation: A Fluorescence Anisotropy and ESR Study

Activities of the enzymes, protease subtilisin and horse radish peroxidase (HRP) have been increased 50 and 40%, respectively, in the presence of the nonionic surfactant, alkyl polyglucoside, compared with the activities in buffer alone. This enzyme hyperactivity reaches a peak at 3.0 mm of surfactant. Investigation into the structure of surfactant aggregates indicates “giant” micelle superstructures at this range of surfactant concentration of 1.7 μm in diameter—dramatically decreasing to 60 and 70 nm at higher surfactant concentrations, while surface tension measurements indicate two critical micelle concentration inflection points at 0.2 and 5.0 mm , which suggests transitions in micelle structure with respect to concentration. Furthermore, electron spin resonance (ESR) indicates that the micelles in first critical micelle concentration regime are loosely packed relative to the second aggregate phase. We hypothesize that this loose packing results in diminished hydration shell repulsion between the micelles, leading to the large, micrometer‐sized aggregates. We further hypothesize that it is the interaction with these loosely packed micelles that affects the flexibility of the HRP and protease enzyme structure. Time‐resolved fluorescence anisotropy of subtilisin in Brij‐30 indicates increasing flexibility of catalytic active site with surfactant concentration. This is correlated with an increase in enzymatic activity.     

Structural studies of 1-butyl-3-methylimidazolium tetrafluoroborate/TX-100/ p-xylene ionic liquid microemulsions.

The ionic liquid (IL) 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF4) forms nonaqueous microemulsions with p-xylene, with the aid of the nonionic surfactant TX-100. The phase behavior of the ternary system is investigated, and three microregions of the microemulsions-ionic liquid-in-oil (IL/O), bicontinuous, and oil-in-ionic liquid (O/IL)-are identified by conductivity measurements, according to percolation theory. On the basis of a phase diagram, a series of IL/O microemulsions are chosen and characterized by dynamic light scattering (DLS). The size of aggregates increases on increasing the amount of added polar component (bmimBF(4)), which is a similar phenomenon to that observed for typical water-in-oil (W/O) microemulsions, suggesting the formation of IL/O microemulsions. The microstructural characteristics of the microemulsions are investigated by FTIR and 1H NMR spectroscopy. The results indicate that the interaction between the electronegative oxygen atoms of the oxyethylene (OE) units in TX-100 and the electropositive imidazolium ring may be the driving force for the solubilization of bmimBF4 into the core of the TX-100 aggregates. In addition, the micropolarity of the microemulsions is investigated by using methyl orange (MO) as a UV/Vis spectroscopic probe. A relatively constant polarity of the microemulsion droplets is obtained in the IL microemulsion. Finally, a plausible structure for the IL/O microemulsion is presented.     

Studies on the micropolarities of bmimBF4/TX-100/toluene ionic liquid microemulsions and their behaviors characterized by UV-visible spectroscopy

Ionic liquids (ILs), 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF4), were substituted for polar water and formed nonaqueous microemulsions with toluene by the aid of nonionic surfactant TX-100. The phase behavior of the ternary system was investigated, and microregions of bmimBF4-in-toluene (IL/O), bicontinuous, and toluene-in-bmimBF4 (O/IL) were identified by traditional electrical conductivity measurements. Dynamic light scattering (DLS) revealed the formation of the IL microemulsions. The micropolarities of the IL/O microemulsions were investigated by the UV-vis spectroscopy using the methyl orange (MO) and methylene blue (MB) as absorption probes. The results indicated that the polarity of the IL/O microemulsion increased only before the IL pools were formed, whereas a relatively fixed polar microenvironment was obtained in the IL pools of the microemulsions. Moreover, UV-vis spectroscopy has also shown that ionic salt compounds such as Ni(NO3)2, CoCl2, CuCl2, and biochemical reagent riboflavin could be solubilized into the IL/O microemulsion droplets, indicating that the IL/O microemulsions have potential application in the production of metallic or semiconductor nanomaterials, and in biological extractions or as solvents for enzymatic reactions. The IL/O microemulsions may have some expected effects due to the unique features of ILs and microemulsions.     

Enhanced permeation, leaf retention, and plant protease inhibitor activity with bicontinuous microemulsions

Bicontinuous microemulsions (BCMEs) have excellent solubulizing properties along with low interfacial tension and aqueous content that can be controlled. In this work, water soluble plant protease inhibitor (PI), well characterized for its activity against insect pests, was incorporated into a BCME system and explored for permeation on hydrophobic leaf surfaces and protease inhibition activity. The bicontinuous nature of the microemulsion containing water:2-propanol:1-butanol (55:35:10 w/w) was characterized using conductivity and self-diffusion coefficient measurements. The PI was soluble in the water-rich bicontinuous domains, stable in the microemulsions, and protease inhibition activity was retained for a prolonged duration. The microemulsions ensured greater wettability and a wider spread of the PI on hydrophobic leaf surfaces as revealed by contact angle measurements. Significantly, trypsin inhibition activity assays of the PI recovered from the leaves after delivery from the microemulsion indicated a significant increase in the PI retention on the leaf. This BCME enabled greater leaf permeation and retention of the PI can be attributed to a temporary disruption of the waxy leaf surface followed by self-repair without causing any long term damage to the plant.     

THE EFFECT OF ALCOHOLS WITH DIFFERENT STRUCTURES ON THE FORMATION OF WARM O/W MICROEMULSIONS

The influence of twenty five different alcohols on the formation of warm oil-in-water (O/W) microemulsions was investigated. Selected concentrations of each alcohol were added to fixed amounts of stearic acid, Tween 20 and water at 65 ° C. Fifteen alcohols formed microemulsions, at least at one of the concentrations. A pattern recognition study was performed to elucidate the activities of the alcohols by Principal Component Analysis (PCA). Linear Discriminant Analysis (LDA) was used to classify them. Two classification functions, obtained for alcohols forming / not forming microemulsions, suggest that the formation of warm O/W microemulsion is linked to the nature and the dimension/lipophilicity of the alcohol.     

Biocompatible microemulsions based on limonene: formulation, structure, and applications

The preparation of biocompatible (w/o) microemulsions based on R-(+)-limonene, water, and a mixture of lecithin and either 1-propanol or 1,2-propanediol as emulsifiers was considered. The choice of the compositions of the microemulsions used was based on the pseudo-ternary phase diagrams of the four-component system determined at 30 degrees C for different weight ratios of the components. When 1-propanol was considered as co-surfactant, the area of the microemulsion zone was remarkably increased. Interfacial properties and the dynamic structure of the emulsifier's monolayer were studied by electron paramagnetic resonance (EPR) spectroscopy using the spin-labeling technique. The rigidity and polarity of the interface were affected by the nature of the alcohol used as co-surfactant. When 1-propanol was used, the emulsifier's interface was much more flexible, indicating a less tight packing of lecithin molecules than in the case of 1,2-propanediol. In addition, the membrane's polarity was decreased when the diol was added as co-surfactant in the microemulsion system. To evaluate the size of the dispersed aqueous domains as a function of water content and other additives concentration, dynamic light scattering (DLS) measurements were carried out. Radii in the range from 60 to 180 nm were observed when 1-propanol was used as co-surfactant, and the water content varied from 0 to 12% w/w. Electrical conductivity measurements of R-(+)-limonene/lecithin/1-propanol/water microemulsions with increasing weight fractions of water indicated the appearance of a percolation threshold at water content above 4% w/w. Lipase from Rhizomucor miehei was solubilized in the aqueous domains of the biocompatible microemulsions, and the esterification of octanoic, dodecanoic, and hexadecanoic acids with the short-chained alcohols used as co-surfactants for the formulation of microemulsions was studied. The enzyme efficiency was affected by the chain length of the carboxylic acids and the nature of the alcohol. In the case of 1-propanol, a preference for the long-chain carboxylic acids was observed. On the contrary, when 1,2-propanediol was used formulation of the corresponding esters was not observed. This behavior could be possibly attributed to either the specificity of the lipase toward the alcohol employed for the esterification of the acids or the structural changes induced in the system when 1-propanol was replaced by 1,2-propanediol.     

Microstructure and structural transition in palm-kernel oil microemulsion using 1.5-order differential electroanalysis

The microemulsification in palm-kernel oil/cetyltrimethylammonium bromide/iso-pentanol/water system is investigated. The effect of iso-pentanol concentration in microemulsions on the size of single-phase microemulsion region is also discussed. It is found that the maximum microemulsion domain is obtained when iso-pentanol-to-cetyltrimethylammonium bromide mass ratio is 1.75. The diffusion coefficients of electroactive probe (ferrocene) in microemulsion microenvironment are measured by 1.5-order differential electroanalysis. The microstructure and structural transition from water-in-oil to oil-in-water microemulsions through a bicontinuous structure is examined. The results are found to be in agreement with that of conductivity measurements.     

Oil-continuous microemulsions composed of hexane, water, and 2-propanol

On the basis of conductivity and centrifugation data and visual examination, ternary phase diagrams were prepared for systems composed of hexane, water, and 2-propanol. Certain ternary compositions were shown to be oil-continuous microemulsions, even though a soap or detergent was not present. Of a number of short-chain alcohols tested, only 2-propanol and 1-propanol could stabilize a microemulsified ternary solution. Addition of a small amount of hexadecyltrimethylammonium perchlorate to the ternary solutions produced only modest changes in the ternary phase diagram. However, in the presence of hexadecyltrimethylammonium salts, most short-chain alcohols containing less than eight carbons could stabilize an oil-continuous microemulsion.     

Percolation in alkyl polyglycoside microemulsions

Mixtures of oil, water, alkyl polyglycosides and long-chain alcohols form almost-temperature-invariant microemulsions. The phase behaviour depends on the content of cosurfactant, usually long-chain alcohols. We show that the system C_8/10G_1.5/octane/water/octanol exhibits cosurfactant-induced percolation phenomena. The percolation transition from an electrically conducting oil-in-water microemulsion to an electrically non-conducting water-in-oil microemulsion with increasing cosurfactant content is observed by measurements of electrical conductivity and time-resolved electric birefringence. The field-off relaxation time yields information on the internal length scale. The scaling behaviour of field-off relaxation times and Kerr constants with respect to the percolation point leads to insight into the influence of cosurfactant on phase behaviour. Received: 27 July 1999/Accepted: 8 February 2000     

Microemulsion structure in the lenticular monophasic areas of brine/SDS/pentanol/dodecane or hexane systems: electrochemical and fluorescent studies

The properties of pseudo-ternary systems involving brine, sodium dodecylsulfate (SDS), pentanol and dodecane or hexane have been investigated. When the hydrocarbon is dodecane, the microemulsion system includes in addition to the main monophasic area a small lens inserted in an oil-rich zone. With hexane, the microemulsion domain splits into two parts, one of which consists of a narrow scythe-blade shaped area which spreads from the water top up to compositions very rich in hydrocarbon. The properties of these uncommon microemulsions were compared with those of microemulsions belonging to the main monophasic area in order to gain additional understanding of their structure. Results of experiments including electrical conductivity and viscosity measurements, electrochemical and fluorescent probe studies lead to the following conclusions: Microemulsions of the small lens of the brine/SDS/pentanol/dodecane system exhibit properties which are reminiscent of structures with at once water and oil countinuous phases. Microemulsions of the lenticular area of the brine/SDS/pentanol/ hexane system seem to undergo the same structural transitions as microemulsions of systems which present a single monophasic area: water-continuous, bicontinuous and oilcontinuous structures.     

Gelled polymerizable microemulsions. 2. Microstructure

Using bicontinuous microemulsions as templates opens a new field for the design of novel structures and thus novel materials, but has significant challenges due to the very small composition and temperature windows in which microemulsions are bicontinuous. In previous work we had shown that we can take a ternary base system (water-n-dodecane--C 13/15E 5), add monomer and cross-linker ( N-isopropylacrylamide and N, N'-methylenebisacrylamide) to the water phase, and add a gelator (12-hydroxyoctadecanoic acid) to the oil phase while remaining in the one-phase region of the phase diagram. It was also possible to allow the gelator to form an organogel by changing the temperature such that we crossed the sol--gel line, which fell within the one-phase region. In this work, we show conclusively that addition of the monomers and the gelator does not affect the microemulsion microstructure and that, even in the gelled state, the polymerizable microemulsion is indeed bicontinuous. 1H NMR self-diffusion, conductivity, and small-angle neutron scattering measurements all confirm the bicontinuous nature of the gelled polymerizable microemulsion.     

Spectroscopic and catalytic studies of lipases in ternary hexane-1-propanol-water surfactantless microemulsion systems

A series of water-in-oil microemulsion systems formulated without surfactant were used to solubilize lipases from Rhizomucor miehei and Candida antarctica B. The effect of the system's composition on the velocity of enzymic reactions was investigated following a model esterification reaction. The interaction between enzymes and the microemulsion environment was studied by steady state fluorescence spectroscopy. The site of localization of the enzyme within the different microdomains of the dispersed phase was investigated by applying the fluorescence energy transfer technique. To determine the properties of the interface between water and organic solvent of the surfactantless microemulsion systems the Electron Paramagnetic Resonance (EPR) spectroscopic technique was applied. The results indicated that even at low water content, water-rich structures are formed. This was confirmed by conductivity measurements. By the addition of enzyme it was observed that when the aqueous phase of the surfactantless microemulsion systems exceeds 2% (v/v) the enzyme retains its catalytic activity, as it is located within the water pools that protect it from the organic solvent. These confined water phases show a propanol rich interface with hexane and their structure depends on the system's composition.     

微乳液结构的研究

测定了十二烷基磺酸钠／正丁醇／２０％苯乙烯／水体系的相平衡，用冷冻刻蚀、ＥＳＲ、ＦＴ－ＩＲ研究了上述体系微乳液的结构，研究表明，苯乙烯含量恒定时，随着体系中水含量增加，电导确定的双连续结构的微乳液经历着从油包水到以连续再到水包油变化，ＦＴ－ＩＲ测定表明，Ｗ／Ｏ微乳兴较Ｏ／Ｗ微乳液的ＯＨ伸缩振动和弯曲振动频率有显著减小，说明Ｗ／Ｏ微乳兴中氢键缔合要比Ｏ／Ｗ强得多。ＥＳＲ测定表明Ｏ／Ｗ微乳液的旋转相关     

Microstructure and structural transition in coconut oil microemulsion using semidifferential electroanalysis

The microemulsification in coconut oil/octadecyltrimethylammonium bromide/iso-pentanol/water system is investigated. The effect of iso-pentanol concentrations on the size of single-phase microemulsion region is discussed. It is found that the maximum microemulsion domain is obtained when cosurfactant (iso-pentanol)-to-surfactant (octadecyltrimethylammonium bromide) mass ratio is 1.5. The diffusion coefficients of ferrocene (electroactive probe) in microemulsion microenvironment are measured by semidifferential electroanalysis. The microstructure and structural transition from water-in-oil to oil-in-water microemulsions through a bicontinuous structure is examined. The results are found to be in agreement with that of conductivity measurements.