Marco Maestro

Application of reverse micelles for the extraction of proteins

The solubilization of proteins in hydrocarbon solutions of reverse micelles is examined at the aim of establishing whether this process can be utilized for the separation and extraction of proteins. Two techniques of solubilization are considered, the phase transfer of proteins from an aqueous solution into a supernatant micellar solution and the direct extraction of the protein powder into the micellar solution. Basic questions concerning the influence of structural parameters of the proteins, as well as the influence of external parameters (pH, salt concentration) on the specificity of the solubilization process are discussed.

Research report on proteins in reverse micelles. Structural aspects and enzymology

Abstract In this research report we present various aspects of our basic investigations on proteins solubilized in AOT reverse micelles. In a first section, we present data on the rate of formation of water pools of reverse micelles: the time course of water solubilization is biphasic and it is often accelerated by the presence of proteins. Upon the protein uptake, the various components of the micellar system redistribute themselves completely into a new thermodynamic equilibrium. A detailed insight of these changes has been obtained by the ‘double-dye ultracentrifugation technique’ by which filled (i.e. containing proteins) and unfilled micelles can be simultaneously monitored in a sedimentation run. The nature of the final equilibrium is also approached theoretically on the basis of a thermodynamic treatment which deals in particular with the nature of the driving forces which are responsible for the protein solubilization in reverse micelles. The stability of the solubilized proteins has been studied with differential scanning calorimetry and circular dichroism spectroscopy from − 16 up to + 80°C. The temperature induced unfolding of the proteins within the reverse micelles is a two-state process. In an other section, kinetic studies of trypsin in reverse micelles are presented with the aim of a comparison with α-chymotrypsin. In contrast to the latter protease, trypsin solubilized in AOT/isooctane did not show ‘superactivity’ at the pH optimum of the reaction. Finally, preliminary solubilization studies with lecithin as the surfactant in organic solvents are presented. In formic acid butyl ester for example, proteins up to a molecular weight of 2500 D could be solubilized in the presence of 100 inM soybean lecithin.

Bell-shaped curves of the enzyme activity in reverse micelles: A simplified model for hydrolytic reactions

Abstract A general characteristic of enzymes entrapped in reverse micelles (or water-in-oil microemulsions) is the bell-shape dependence of the reaction rate constant with increasing water content in the system. A simple model for a possible explanation of this bell-shape phenomenon in the particular case of hydrolytic reactions is presented. The model is based mainly on the diffusion theory and utilizes a minimum of adjustable parameters.

A two-state stochastic model for the dynamics of constrained water in reversed micelles

Abstract Translational diffusion leads the tagged water molecule to explore regions of the reversed micelle with a varying degree of space hindrance, thereby modulating the rotational relaxation process. A two-state modulation is shown to result in satisfactory agreement with the NMR experimental data on the rotational correlation time as a function of the micelle radius.

Stochastic simulations of micellization kinetics

The Monte Carlo method introduced by Gillespie (cf. D. T. Gillespie, J. Phys. Chem. 81, 2340 1977), was applied to the kinetics of micelle formation according to a mechanism that allows associations and dissociations among ennamers of whatever aggregation number. A careful choice of thermodynamic and dissociation kinetic constants made it possible to reproduce both equilibrium and kinetic properties of hypothetical surfactant solutions. The results obtained by stochastic simulations are shown and compared to the accepted theory and to the experimental evidence available in literature.

A simplified kinetic model for an autopoietic synthesis of micelles

Abstract Recently, some chemical systems have been implemented which have an autopoietic behaviour according to Varela and Maturanas definition. They consist of micelles or reversed micelles which are able to produce their building material inside themselves, and, in such a way reproduce themselves. For one of these systems we have elaborated a kinetic model to rationalize the long lag phase and the abrupt rate increase displayed during the autopietic process of micellar self-replication. The theoretical predictions are in good agreement with the experimental observations available to date.

Application of a simple diffusion model for the enzymatic activity of α-chymotrypsin in reverse micelles

Abstract In reverse micellar systems, solubilized enzymes often display their highest activity at a particular w0 value ( w 0 = [H 2 O] [surfactant]) ; below as well as above this value, the enzyme activity is lower. Recently, we have presented a theoretical model which describes this characteristic behavior in the case of hydrolytic enzymes, mainly on the basis of the diffusion theory [ M. Bianucci, M. Maestro, and P. Walde, Chem. Phys. 141, 273 (1990) ]. In this note, we report on some improvements of the model and we compare the theoretically calculated activity behavior with experimental data obtained for α-chymotrypsin in reverse micelles formed by the surfactant bis(2-ethylhexyl) sodium sulfosuccinate in isooctane. Although our theoretical calculations are not superimposable with the experimental values, the comparison indicates that inter- and/or intramicellar diffusion processes may affect the catalytic behavior of enzymes in reverse micellar systems.

Ab initio calculations for ground states of CH2Li− and CH2Be

The ground state of CH2Li− and CH2Be molecules has been investigated by an SCF calculation using a contracted Gaussian basis set. Only for the second system a bound state with respect to the ground states of the molecular fragments has been found.

A Simplified Thermodynamic Model For Protein Uptake by Reverse Micelles

A simplified thermodynamic treatment is presented for describing the phase transfer of proteins from a water solution into a supernatant organic solution containing water-in-oil microemulsions or reverse micelles. The equilibrium physical observables (like the percentage of protein and water transfer, the radius of the protein-containing micelles) are analytically expressed by imposing the minimization of the free energy of the system together with a series of geometrical constraints. The model assumes that the total free energy change is the sum of four main contributions: an electrostatic interaction term, an electrostatic entropy term, a dilution entropy change, and a mixing free energy change. Some rather drastic assumptions must be introduced in order to obtain manageable equations. Eventually, these lead to reasonable calculations of the size and distribution of reverse micelles after protein uptake, as shown on the basis of a numerical example.

PCILO calculation on the ansa conformation of hexahydrorifamycin S

Abstract PCILO calculation on the conformation of the 16, 17, 18, 19, 28, 29 hexahydrorifamycin S points to an ansa chain conformation of the C(12)–C(28) fragment from the alternatives given by a previous 1H NMR spectroscopic study. The energetically favoured conformation of the C(19)–N fragment has also been determined.