Reverse micelles, protein extraction

Keywords. Downstream processing. Reverse micelles, Protein/enzyme extraction. Modeling, Recent developments... 

Reverse micelles are self-organized aggregates of amphiphilic molecules that provide a hydrophilic nano-scale droplet in apolar solvents. This polar core accommodates some hydrophilic biomolecules stabilized by a surfactant shell layer. Furthermore, reverse micellar solutions can extract proteins from aqueous bulk solutions through a water-oil interface. Such a liquid-liquid extraction technique is easy to scale up without a loss in resolution capability, complex equipment design, economic limitations and the impossibility of a continuous mode of operation. Therefore, reverse micellar protein extraction has great potential in facilitating large-scale protein recovery processes from fermentation broths for effective protein production. 

To obtain the high extraction efficiency of a target protein, we need to design reverse micelles having sufficient hydrophobicity to extract the protein and prepare the conditions for enhancing the protein-surfactant interaction to form a protein-surfactant complex, which is a crucial intermediate for reverse micellar protein extraction. 

Novel aspects of protein extraction with reversed-micelles include both fundamental studies and process design studies/approaches. Fundamental studies are essential in order to design a reversed-micelles based extraction process in a rational manner. Such theoretical programs have been initiated and are providing a better understanding of the partitioning and transport phenomena in such systems (31). In this book, Jolivalt tal. (32) review the modeling aspects and the applications of reversed micelles for protein separations. 

Hayadii, Y., Yoshioka, S., Aso, Y., Po, A. L. W., and Terao, T., 1994, Entrapment of proteins in poly(L-lactide) microspheres using reversed micelle solvent extraction, Pharm. Res. 11 337-340. 

Recent development of the use of reversed micelles (aqueous surfactant aggregates in organic solvents) to solubilize significant quantities of nonpolar materials within their polar cores can be exploited in the development of new concepts for the continuous selective concentration and recovery of heavy metal ions from dilute aqueous streams. The ability of reversed micelle solutions to extract proteins and amino acids selectively from aqueous media has been recently demonstrated the results indicate that strong electrostatic interactions are the primary basis for selectivity. The high charge-to-surface ratio of the valuable heavy metal ions suggests that they too should be extractable from dilute aqueous solutions. 

Using a solution of water-containing reversed micelles of di(2-ethylhexyl)phospho-rothioic acid in isooctane, hemoglobin was extracted and concentrated. Desolubilization of the protein entrapped in the reversed micelles by weak alkahne solution was realized by adding small amounts of n-octanol [167]. 

Salt type and concentration For back-extraction, increases in pH are not enough to strip the protein out from reverse micelles this is also due to the size exclusion elfect resulting from a decrease in the reverse micelle size [31,32]. This means that high salt concentration and salts that form small reverse micelles favor back transfer. Most of the work reported in the literature used KCl solution, normally 1.0 mol dm KCl coupled with a pH around 7.5. Marcozzi et al. [23] also showed that the back transfer efficiency of a-chymotrypsin depends on the salt type and concentration used in the forward transfer. 

Counterion extraction Due to the relative slowness of back extraction based on the methods above, the back-extraction of proteins encapsulated in AOT reverse micelles was evaluated by adding a counterionic surfactant, either TOMAC or DTAB, to the reverse micelles [33]. This novel backward transfer method gave higher backward extraction yields compared to the conventional method. The back-extraction process with TOMAC was found to be 100 times faster than back-extraction with the conventional method, and as much as three times faster than forward extraction. The 1 1 complexes of AOT and TOMAC in the solvent phase could be efficiently removed using adsorption onto montmorillonite so that the organic solvent could be reused. 

For the scale-up of reverse micelle extractions, it is important to know which factors determine the mass transfer rate to or from the reverse micelle phase. So far most work has concentrated on the kinetics of solubilization of water molecules [34,35], protons [36], metal ions [20,35,37,38 0], amino acids [41], and proteins [8,35,42,43]. There are two separate processes forward transfer, which is transfer of solute from the aqueous to the reverse micelle phase, and back transfer, which is the antithesis of the first one. 

The ease that certain protein mixtures can be separated using reverse micelle extraction was clearly demonstrated by Goklen and Hatton [46], Goklen [31], and Jarudilokkul et al. [25], who investigated a series of binary and ternary protein mixtures. In two cases, they were able to quantitatively extract cytochrome c and lysozyme from a ternary mixture of these proteins with ribonuclease A. Woll and Hatton [24] investigated the separation of a mixture of ribonuclease A and concanavalin A, and showed that the system behaved ideally and that there was no interaction between the proteins. 

Reverse micelles of CTAB in octane with hexanol as cosurfactant were reported to be able to lyse whole cells quickly and accommodate the liberated enzyme rapidly into the water pool of surfactant aggregates [50,51]. In another case a periplasmic enzyme, cytochrome c553, was extracted from the periplasmic fraction using reverse micelles [52]. The purity achieved in one separation step was very close to that achieved with extensive column chromatography. These results show that reverse micelles can be used for the extraction of intracellular proteins. 

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

Application of reverse micelles for the extraction of amino acids and proteins. Chimia, 44, 270-82. 

The most commonly used anionic surfactant in reversed micelles for the extraction of proteins is AOT [sodium bis(2-ethylhexyl)sulphosuccinate]... 

Pires, M. J., Aires-Barros, M. R., and Cabral, J. M. S. (1996). Liquid-liquid extraction of proteins with reversed micelles. Biotechnol. Prog. 12, 290-301. 

Goklen, K. E., and Hatton, T. A. (1987). Liquid-liquid extraction of low molecular-weight proteins by selective solubilization in reversed micelles. Sep. Sci. Technol. 22, 831-841. 

Novel and effective bioseparation techniques must be continuously researched and developed for profitable removal of proteins and other bioproducts of interest from very dilute solutions (A. Ramakrishnan and A. Sadana, personal communication, 1999). There appear to be two techniques that have tremendous potential for commercial applications the reverse micelle technique and aqueous two-phase extraction. Before these techniques achieve their potential, it will be necessary to further delineate the effect of mass transfer, interactions at the interfaces, and other parameters that affect both the quality and quantity of proteins separated by these techniques. It is also necessary to have a large data bank of a wide variety of proteins and bioproducts with regard to their characteristics and stability to assist future improvements in bioseparations. 

Most systems examined to date have employed the AOT anionic reversed micellar system (366-370). In one case, amylase was extracted using trioctylmethylammonium chloride (cationic surfactant) in isooctane (375) while in another, catalase was extracted using a cationic DTAB/octane/hexanol reversed micelle (377). In our own research, we have successfully employed nonionic Igepal CO-530 -CCl, cationic CTAB - hexanol, and zwitterionic lecithin - CC1, reversed micellar systems in the extraction of some amino acids and proteins (379). The availability of such a pool of different charge-type micellar systems allows one flexibility in the development of such extraction schemes. In fact, preliminary results seem to indicate that better extractions are obtainable in some instances via use of zwitterionic reversed micellar media (379). 

Extraction of Proteins and Amino Acids Using Reversed Micelles... 

The solubilisation of proteins and amino acids in organic solvents by reversed micelles provides a new method for the selective recovery, separation and concentration of bioproducts using liquid->liquid extraction techniques. Selectivity is affected by electrostatic interactions between the charged residues or moieties of the solute and the surfactant headgroups. These interactions are mediated by electrostatic screening as affected by solution ionic strength. The more hydrophobic the amino acid residue, the more favourable is the solubilisation of this residue in the partially structured water pool of the reversed micelle relative to the bulk, unstructured water phase. 

Here, we review the key roles of the interaction between proteins and surfactants in liquid-liquid protein extraction. These assist in the understanding of the protein transfer mechanism through a water/oil interface and in the design of reverse micelles for protein separation processes. 

For separating and purifying proteins, a forward extraction operation facilitates the selective transfer of a target protein from an aqueous solution containing some kinds of proteins into an organic phase, and the extracted proteins are quantitatively recovered into a fresh aqueous solution by the subsequent back extraction. The transfer selectivity is based on the interaction between surfactants, which form reverse micelles, and the protein surface. On the other hand, the quantitative recovery of an objective protein from reverse micelles is accomplished by severing proteins from the enclosure with a surfactant layer. 

Aqueous pH alters the protein charge property and affects the extraction efficiency. Haemoglobin (Mw 64,500, pi 6.8) is a difficult protein in terms of being able to completely extract it into reverse micelles. The representative anionic surfactant, di-2-ethylhexyl sulfosuccinate (AOT), cannot extract it, and gives rise to an interfacial precipitate. In contrast, we succeeded in the complete extraction of haemoglobin using synthetic anionic surfactants, dioleyl phosphoric acid (DOLPA), as seen in... 

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