Photosynthetic activity of cyanobacteria in water-in-oil microemulsions

The solubilization and the photosynthetic activity of cyanobacteria (Anabaena variabilis) in water-in-oil microemulsions consisting of (Tween85/Span80)/hexadecane/water is investigated. Transparent and stable solutions containing up to 10(8) cells/mL could be obtained. The physical state and stability of the cells in the microemulsion, as evidenced from optical and electron microscopy, is dependent upon the physical parameters of the system, and in particular on the hydrophylic-lypophilic balance (HLB) of the surfactant system. Conditions could be found, under which the cells in the microemulsion system display photosynthetic activity This was judged by measuring polarographically the oxygen evolution and by studying the photosynthetic activity in the presence of specific inhinbitors.     

Solubilization and growth of Candida pseudotropicalis in water-in-oil microemulsions

Some new aspects of microbiology in water-in-oil microemulsions are investigated using Candida pseudotropicalis in a hexadecane solution containing Tween85/Span80 (each 5% wt:wt) as surfactant, and limited amount of water (up to 3%, vol:vol), Microemulsion solutions containing cells up to 10 mg fresh weight per milliliter can be prepared, which display a greater time stability and a much smaller light scattering than aqueous suspensions having the same cell concentration. This is ascribed to a lower aggregation tendency of the cells in the microemulsion environment. It is also shown that C. pseudotropicalis cells are able to grow (up to a factor of approximately 6-7 within a few days) in the microemulsion system containing nutrient medium in the aqueous microphase; but they are also able to grow at the expense of the hexadecane. This is proved with radioactive-labeled hexadecane by measuring the increase of radioactivity in the cells as well as the emission of (14)CO(2). The growth rate of the cells is then compared with the growth rate of cellular proteins during cell reproduction in the microemulsion system. Two regimes are observed: a first one, in which cells growth rate and protein growth rate proceed parallel to each other; and a second one (established after 0.5-1 day) characterized by depletion of proteins in the microemulsion system. The implications of these findings for cell metabolism in microemulsion and for possible biotechnological applications are discussed.     

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.     

Photosynthetic activity of plant cells solubilized in water-in-oil microemulsions

With the aim of possibly extending plant microbiology and photosynthesis beyond the usual applicability in aqueous solution, we investigated the solubilization of plant cells inorganic media with the help of water-in-oil microemulsions. Cells isolated from leaves of Rumex obtusifolius were solubilized in a water/2-ethyl-hexyl-sodiumsulfosuccinate/isooctane system, containing 20% water (v:v) and 240 mM surfactant, and the oxygen evolution/consumption was measured polarographically. Although no oxygen evolution was detectable in the organic medium, the cells were able to carry out photosynthetic oxygen consumption at the expense of ascorbate. To a lesser extent, photosynthetic oxygen consumption was measured using N, N, N', N'-tetramethyl-p-phenylenediamine as electron donor. The rate of ascorbate photooxidation was linearly related to the concentration of cells.     

Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

The activity and the kinetic properties of horse liver alcohol dehydrogenase have been studied in water-in-oil microemulsions containing sodium dodecyl sulfate (SDS) or hexadecyl trimethylammonium bromide (CTAB), 1-butanol or 1-pentanol or 1-hexanol or t-butanol, water and cyclohexane alone or with octane. In the anionic microemulsions (i.e. containing sodium dodecyl sulfate), the enzyme quickly lost its activity, but was efficiently protected by the coenzyme and some adenine nucleotides. In the cationic microemulsions (i.e. containing hexadecyl trimethylammonium bromide), the enzyme activity was more stable and with higher alcohols was stable for at least 20 min. The Michaelis constant of NAD+ calculated with respect to the water content was nearly constant and higher than in water. The maximum velocity in anionic microemulsions depends on the water content whereas in cationic microemulsions, the maximum velocity did not show a clear dependence on the water content and was close to the maximum velocity found in water. The pH dependence of Km and Vmax in these microemulsions was similar to that observed in water. The kinetic data for a hydrophobic substrate, cinnamyl alcohol, showed that this alcohol partitions between the pseudo-phases and thus the apparent Michaelis constant and the concentration at which substrate-excess inhibition appeared were increased. The catalytic properties of the enzyme in microemulsions were illustrated by the preparative reduction of cinnamaldehyde with cofactor recycling. The rate determination of NAD+ reduction and of 1-butanol/cinnamaldehyde redox reaction showed that at low water content (2.8%), the NAD+ reduction rate was close to zero whereas the redox reaction rate was about half of the rate at higher water content. Probably at low water content the coenzyme binding-dissociation rates are reduced much more than the binding-dissociation rates of the substrates and the rates of the ternary complex interconversion. The cationic microemulsions seemed to be very favorable medium for enzyme activity, the tetraalkyl ammonium surfactant causing less denaturation than the anionic detergent dodecyl sulfate.     

Kinetic study of lipase catalyzed esterification reactions in water-in-oil microemulsions

The kinetics of the esterification of lauric acid by (-)menthol, catalyzed by Penicillium simplicissimum lipase, was studied in water/bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping-Pong Bi--Bi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59-72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio, W(o) = [H(2)O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern. (c) 1993 John Wiley & Sons, Inc.     

A near infrared assay for lipolysis in lecithin-stabilised water-in-oil microemulsions

Summary A near infrared (N-IR) spectroscopic assay has been developed for in situ monitoring of lipolysis in water-in-oil (w/o) microemulsions stabilised by soybean lecithin. Water, fatty acid and partial glyceride concentrations may be individually determined. N-IR has general utility in the study of hydrolysis/condensation reactions in low-water organic media.     

Solubilization of soybean mitochondria in AOT/isooctane water-in-oil microemulsions

The water-in-oil microemulsion system bis(2 ethyl-hexyl-sodium-succinate (AOT)/isooctane/water is able to solubilize soybean nodules mitrochrondria. Transparent and thermodynamically stable hydrocarbon solutions are obtained, which can be assayed for mitochondrial activity just as aqueous solutions. Malate dehydrogenase (MDH) activity was measured in vivo and gave in reverse micelles very similar results as in water. However the kinetic behavior of this reaction in AOT/isooctane reverse micelles shows some differences with respect to water. Mitochondria in reverse AOT micelles are able to retain about 70% of their initial MDH activity after three days. Mitochondria can be back-transferred from reverse micelles to water and show respiratory activity almost identical to the native organelles. Electron microscopy studies show that the dimensions of mitochondria back-transferred into water from AOT micelles are comparable to the dimensions of the native organelles.     

Interfacial charge transport in water-in-oil microemulsions stabilized by ionic-non-ionic surfactant mixtures

Interfacial charge transport and mobility have been studied in system-spanning nanodroplet clusters formed in water-in-oil microemulsions stabilized by non-ionic-ionic surfactant mixtures. An unexpectedly large reduction in the counterion mobility was derived from a two-step adsorption process which described the Langmuir-plot-like conductivity isotherm remarkably well. It also conformed very satisfactorily to mobility data of the sodium bis-2-ethylhexylsulfosuccinate (AOT) counterion derived from frequency-dependent dielectric dispersion measurements with singly dispersed nanodroplets of the same system as used with the cluster studies. The activation energy attributed to the counterion mobility allowed one to calculate approximately the minimum distance between the (positive) counterion in the bulk and the surfactant anion in the interface.     

Recovery of proteins from water-in-oil microemulsions in highly concentrated form through dilution techniques

Summary Bovine serum albumin (BSA) was recovered from water/Aerosol-OT (Sodium bis [2-ethylhexyl] sulfosuccinate)/isooctane water-in-oil microemulsion solution as a solid precipitate that was virtually free of surfactant by the addition of isooctane or 0.01 v/v 1-butanol. The recovery was greater when the fractional occupancy of BSA was higher and the extent of dilution greater. Proteins which interact with the microemulsion interface are more likely to be recovered.     

Refolding of denatured lysozyme by water-in-oil microemulsions of sucrose fatty acid esters

Water-in-oil (w/o) microemulsion of sucrose fatty acid ester was used to renature denatured hen egg white lysozyme without aggregation. After lysozyme was denatured in 5 M guanidine hydrochloride for 24 h, the resultant denatured lysozyme was held in the microemulsion, overnight at 25°C. Renatured lysozyme was transferred from the microemulsion phase to the recovery aqueous phase by conventional liquid-liquid extraction. The enzymatic activity of the recovered lysozyme was 93%.     

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.     

Solubilization of enzymes in water-in-oil microemulsions and their rapid and efficient release through use of a pH-degradable surfactant

α-Chymotrypsin and lysozyme were solubilized in a water/O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl)methoxy]–O′-methoxy poly(ethylene glycol) (CK-2,13 surfactant)/isooctane water-in-oil microemulsion solution at 1.5–2 and 10 g l⁻¹ for 0.15 and 1.2 M CK-2,13, respectively. Upon contact with an equal volume of 0.1 M NaH₂PO₄/Na₂HPO₄ buffer, pH 5, a three-phase system (Winsor-III system) was formed, consisting of a surfactant-rich middle phase and aqueous and isooctane-rich “excess” phases. Both enzymes were rapidly released into the aqueous excess phase, with 70% recovery of each in 30 and 60 min for microemulsion solutions containing 0.15 and 1.2 M surfactant, respectively. The recovered enzymes retained >90% of their original specific activity.     

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: I. Effect of pH and water content on reaction kinetics

Lipolase and Lipozyme are produced in large quantities (as a result of genetic engineering and overexpression) for the detergents market and provide a cheap source of highly active biocatalysts. Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RmL) have been isolated in partially purified form from commercial preparations of Lipolase and Lipozyme, respectively. These lipases were solubilized in Aerosol-OT (AOT)-stabilized water-in-oil (w/o) microemulsions in n-heptane. HIL and RmL activity in these microemulsions was assayed by spectrophotometric measurement of the initial rate of p-nitophenyl butyrate hydrolysis, and by chromatographic determination of the initial rate of octyl decanoate synthesis from 1-octanol and decanoic acid. The hydrolytic activity of HIL in microemulsions measured as a function of buffer pH prior to dispersal, followed a sigmoidal profile with the highest activities observed at alkaline pHs. This broadly matches the pH-activity profile for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. The hydrolytic activity of RmL in the same microemulsions, measured as a function of pH, gave a bell-shaped profile with a maximum activity at pH 7.5. Again, the observed pH-activity profile was similar to that reported for a purified RmL in a tributyrin-based aqueous emulsion assay. In contrast, the esterification activity exhibited by both HIL and RmL in AOT microemulsions over the available range pH 6.1 to 10.4, decreases as the pH increases, most likely reflecting the effect of substrate ionization. The dependence of the hydrolytic and condensation activity of HIL on R, the mole ratio of water to surfactant, were similar with both profiles exhibiting a maximum at R = 5. The hydrolytic and esterification activities of RmL followed similar R-dependent profiles, but the profiles in this case exhibited a maximum at R = 10. The water activities at these R values were directly measured as 0.78 and 0.9, respectively. Measured water activities were unperturbed by the presence of lipase at the concentrations used in these studies. (c) 1995 John Wiley & Sons, Inc.     

Fundamental study on a gene transfection methodology for mammalian cells using water-in-oil droplet deformation in a DC electric field

We have developed a gene transfection method called water-in-oil droplet electroporation (EP) that uses a dielectric oil and a liquid droplet containing live cells and exogenous DNA. When a cell suspension droplet is placed between a pair of electrodes, an intense DC electric field can induce droplet deformation, resulting in an instantaneous short circuit caused by the droplet elongating and contacting the two electrodes simultaneously. Small transient pores are generated in the cell membrane during the short, allowing the introduction of exogenous DNA into the cells. The droplet EP was characterized by varying the following experimental parameters: applied voltage, number of short circuits, type of medium (electric conductivity), concentration of exogenous DNA, and size of the droplet. In addition, the formation of transient pores in the cell membrane during droplet EP and the transfection efficiency were evaluated.     

Early epidemic spread,percolation and Covid-19

Journal of Mathematical Biology - Human to human transmissible infectious diseases spread in a population using human interactions as its transmission vector. The early stages of such an outbreak...     

Wetting, percolation and morphogenesis in a model tissue system

Artificial tissues constructed of cells or polystyrene beads suspended in a solution of type I collagen will, under appropriate conditions, protrude into regions of similar matrices lacking particles, but containing the extracellular glycoprotein fibronectin. This phenomenon has been termed "matrix-driven translocation". Conditions required for the effect include the presence of heparin-like molecules on the cell or bead surfaces, appropriate concentrations of particles and collagen, and physiological ionic strength and pH. Here we consider the idea that the driving force for the concerted movement of matrix and suspended particles is the thermodynamically spontaneous spreading or wetting behavior of two immiscible fluids bounded by common substrata. Wetting theory is shown to be capable of accounting for the behavior of this model system, but this analysis requires that the two matrix regions constitute separate phases at thermodynamic coexistence. We show that one plausible mechanism for the generation of separate phases is the formation of a percolation network of collagen fibers on a lattice of cells or beads. It is argued that the concepts of wetting and percolation apply to properties in common between the model system and living tissues, and may therefore be used to provide a physical account of aspects of tissue morphogenesis.     

Temperature- and dose-dependent internalization of concanavalin A in monolayer culture

When rat hepatoma cells (R117-21B) were incubated for 20 h at 37 degrees C with 125I-labeled concanavalin A at low concentrations (0.5-10 micrograms/ml), only 20-30% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside, but at high concentrations (50-500 micrograms/ml), 60-80% of the cell-associated radioactivity was released. At 4 degrees C, the cell-associated radioactivity decreased with the increase in concentration of concanavalin A, and more than 80% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside. These results suggest that the amount of cell-associated concanavalin A is related to the physicochemical state of the plasma membrane, which can be altered by the incubation temperature or by the concentration of concanavalin A, the transitional concentration being 5-10 micrograms/ml.     

Solubilization and activity of yeast cells in water-in-oil microemulsion

Baker yeast cells are solubilized in organic solvents by the use of surfactants and small amounts of water. Data are reported for three different systems, Tween/Isopropylpalmitat, Asolectin/IPP, Asolectin/Hexadecane. The viability (life-capability) can remain as high as 80% for 10 Days, the asolectin systems being more efficient. The viability is significantly higher for yeast cells derived from cultures which had been previously solubilized in the microemulsion system. The implications of the finding for microbiology in organic solvents, and some general mechanistic aspects, are briefly discussed.