Separation of Proteins by Using Reversed Micelles - ACS Publications

Chapter 5

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch005

Separation of Proteins by Using Reversed Micelles Claude Jolivalt, Michel Minier, and Henri Renon Centre Réacteurs et Processus, Ecole Nationale Supérieure des Mines de Paris, 60 Boulevard Saint-Michel, 75006 Paris, France

S i n c e it appears possible to use active biomolecules outside a natural environment, and even more so since genetic engineering enables the production by recombinant microorganisms or animal cell cultures of more and more various proteins, a great deal of investigation has been carried out in order to separate and purify these compounds from complex medium broths. Due to the continually renewed diversity and complexity of proteins and media, a simple scheme of extraction and purification process does not exist, each example being a specific case for which, usually, a sequence of several techniques must be developed, depending on the properties of the protein, the nature of the impurities and the final purity demanded. Extraction of proteins using reversed micelles emerges as a new and attractive technique (1-4) in terms of selectivity and concentration. It presents a close similarity with liquid-liquid extraction since both are diphasic processes which consist of partitioning a targeted solute between an aqueous feed phase and an organic phase and then operate the back transfer to a second aqueous stripping phase. The yield and selectivity of the separation is determined by thermodynamic properties at equilibrium. However, classical organic solvents are not suitable to achieve the extraction of most proteins which are hydrophilic molecules and, therefore, insoluble in apolar solvents. Moreover, due to their low polarity, organic solvents modify the interactions which stabilize the native protein conformation, leading to its denaturation. In order to extract and preserve the protein, it is necessary to maintain its aqueous environment by adding a surfactant which aggregates in apolar solvent and 0097-6156/90/0419-0087$06.25/0 © 1990 American Chemical Society

In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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forms reversed micelles. The organic phase can thus be described as a t h e r m o d y n a m i c a l l y stable d i s p e r s i o n of spherical water droplets s u r r o u n d e d b y a m p h i p h i l e molecules w h i c h prevent the proteins solubilized i n the water core from direct interactions w i t h the apolar solvent (figure 1). The first interest for reversed micelles i n the biological area has been to point out that enzymes actually retained their catalytic activity (5-7) when solubilized i n organic solvents v i a reversed micelles. Catalysis by enzymes i n reversed micelles was exploited for conversion purposes, taking advantage of the micro-compartmentation of the organic phase, w h i c h permitted contacting hydrophilic and hydrophobic reactants and r e m o v i n g hydrophobic products. Yields a n d kinetics of enzymatic reactions could thus be improved or changed with regard to those obtained in b u l k aqueous phases. Related publications were reviewed i n detail recently by Shield et al. (8). It was noted that activity and conformation change w i t h the amount of water inside micelles, pointing out the importance of the aqueous environment, easily adjustable as a function of trie water content, w h i c h is i m p o s s i b l e w h e n studies are p e r f o r m e d i n aqueous s o l u t i o n s . Furthermore, because many properties (9) of the water core resemble those of water present at interfaces i n biological systems, reversed micelles p r o v i d e an excellent system for s t u d y i n g the interactions between polypeptides a n d interfacial water (10-11) or more generally their conformation when solubilized i n micelles (12-15), depending on where the biopolymer is located inside the micelle and what its conformation is. Three techniques (16) have been a p p l i e d to prepare enzymecontaining micellar solutions. The first consists of stirring solid protein with a hydrocarbon solution of surfactant, the second b y injecting a few microliters of a concentrated enzyme solution into it. The solution is then stirred until it becomes transparent, allowing spectroscopic studies. Such methods are suitable for incorporating a given amount of protein into a reversed micellar system and for controlling the water content but, because they i n v o l v e o n l y monophasic systems, they are of no interest for purification purposes. The third alternative is to transfer the protein from an aqueous phase into the reversed micellar phase. This method has been more or less successfully applied i n the above mentioned studies, focused mainly on properties of proteins after solubilization but has established the feasibility of the extraction. M a n y detailed reviews have been published o n these subjects (17-23) which are a good introduction to our present topic, extraction of biopolymers using reversed micelles. M a n y surfactants are k n o w n to form reversed micelles i n apolar media and have already provided a suitable environment for elucidating catalytic activity or conformation properties of some proteins i n n o n aqueous media. But to conduct an extraction t w o conditions must be fulfilled: reversed micelles must exist i n the organic phase i n equilibrium with an excess aqueous phase and the performance of the extraction must be significant. The nature of both surfactant and solvent, the composition


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of the aqueous phase as w e l l as the v o l u m e ratio between phases determine both the ability of the system to form such diphasic systems and the extraction and separation of the desired protein from other protein impurities. Manipulating different parameters i n order to achieve an efficient extraction has emphasized that the criteria of yield a n d selectivity are mostly controlled by two different phenomena: electrostatic interactions due to the charges born both by the protein and the surfactant and the "steric effect" related to the relative sizes of the protein and micelle.

PARAMETERS AFFECTING THE SOLUBILIZATION In this section, we shall focus on experiments dealing with the extraction of peptides, amino acids or proteins from an aqueous phase into an organic phase, following the typical procedure of liquid-liquid extraction processes. A l l the extraction experiments presented below were performed w i t h synthetic aqueous solutions, i.e. purified proteins solubilized i n a suitable buffer. A l l parameters and components are k n o w n , w h i c h is not the case with fermentation broths or culture media which w i l l be reviewed in a special paragraph. The general extraction procedure can be described as follows: the aqueous phase containing the protein to be extracted and the organic phase prepared separately are contacted a n d stirred. A f t e r the chemical equilibrium has been reached, the system is centrifuged or allowed to settle until phase separation occurs. Separation and eventually titration of both phases are performed. Back extraction follows a similar process. The laden organic phase is mixed with a suitable aqueous phase where the protein is recovered after settling. A m o n g all surfactants developed for experiments o n solubilization of biomolecules into reversed micelles, only two have, u p to now, received the greatest attention to achieve studies o n extraction. Sodium bis (2ethylhexyl) sulfosuccinate (the commercial name of which is A O T ) is an anionic surfactant. Its ability to form water i n o i l microemulsions i n ternary oil/surfactant/water systems without adding a cosurfactant reduces the set of parameters, making the system easier to control. This property can be explained by the geometric modelling of the surfactant packing at the interface, developed by Mitchell and N i n h a m (24). The key element of their theory is the packing ratio defined as the ratio of the cross-sectional area of the hydrocarbon chain v / l to that ao of the polar head of a surfactant molecule at the interface. A necessary geometric condition for the existence of reversed micelles is v / a l > 1. However, A O T is a double chain surfactant with a relatively small head. The packing ratio is thus expected to be higher compared to that of a single chain surfactant for which the incorporation of a cosurfactant i n the interface is necessary to increase the mean hydrocarbon volume without affecting either ao or l . c

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c


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The structure of the A O T micellar system, as well as the state of water entrapped inside swollen micelles, have been characterized using different techniques, such as photon correlation spectroscopy (25), positron annihilation (26), N M R (27, 28), fluorescence (29-32) and more recently small angle neutron scattering (33). The existence of reversed micelles has been demonstrated i n the domain of concentrations explored by protein extraction experiments. Their size (proportional to the molar ratio of water to surfactant k n o w n as wo), shape and aggregation number have been determined. Furthermore, the micelle size distribution is believed to be relatively monodisperse. Q u a t e r n a r y a m m o n i u m salts, i n most cases t r i a l k y l m e t h y l ammonium salts, have been the second focal point of w o r k i n this field. These molecules are cationic amphiphiles and have been revealed to be a complement to studies on the influence of the charge of the surfactant, compared to anionic A O T . In their early studies, L u i s i et aL (34, 35) used t r i m e t h y l o c t y l a m m o n i u m c h l o r i d e ( T O M A C ) at l o w surfactant concentrations (about 12 m M ) i n cyclohexane. But at higher concentrations, this molecule cannot be solubilized i n apolar solvents, such as isooctane. It is necessary to add a fourth component, usually an alcohol. Investigations were undertaken to understand reversed micellar structure using quasi-elastic light scattering measurements (36). It was concluded that i n a 40 m M solution of Aliquat 336 (a mixture of trialkyl ( C - C ) methyl ammonium chloride salts) dissolved i n isooctane with 2.5 percent isotridecanol, there exist relatively monodisperse particles, the hydrodynamic radius of which is about 22 A . This size was found to be i n fairly good agreement with that calculated taking into account the amount of water i n the organic phase and assuming the hypothesis of a molecular model of spherical micelles. In this section, we shall attempt to review the most relevant observations and, assuming that reversed micelles exist i n the organic phase, explain them i n terms of both electrostatic interactions and steric effects. 8

1 0

Electrostatic Interactions Since i n a l l cases considered here both surfactant and proteins are electrically charged molecules, one of the major forces governing their interactions is expected to be of electrical origin. The most direct feature is that p H determines the rate of dissociation of the charged residues which compose the primary structure of the protein and thus the net charge of the protein. W h e n p H = p i , the protein is globally neutral, when p H < p i (respectively p H > pi), the global charge of the protein is positive (respectively negative). Depending on the p H , the species to be extracted is negatively, neutral, or positively charged, affecting electrostatic interactions with the surfactant.



5. JOLIVALT ET AL.


Since proteins are polymers of amino acids, it is interesting to test the above trend and consider the effects of p H of the aqueous phase on the extraction of amino acids or small peptides. L u i s i et aL (35) described the transfer of tryptophan and a dipeptide, L-tryptophylglycine into an organic phase composed of T O M A C i n cyclohexane. It was shown that the p H profile of the percent transfer was determined by the ionization of the amino group. The p K a values obtained from these profiles agreed closely with values found i n the literature. Thien et aL (37) reported very similar results i n A O T microemulsions, leading to a fairly good agreement between the calculated titration curves for arginine and the amount of amino acid extracted as a function of the p H . From the results obtained w i t h surfactants of opposite charge, it can be concluded that for amino acids, a direct relationship exists between the net charge of the molecule to be extracted and its transfer fraction. It w o u l d be expected that the trend described above should also be verified for proteins. Göklen and Hatton confirmed this hypothesis (38, 39), at least for l o w molecular weight proteins. They studied the solubilization of three proteins, cytochrome C , ribonuclease and lysozyme of similar size but w i t h distinct p i , from a 1 m g / m l aqueous protein solution into a 50 m M AOT/isooctane organic phase (figure 2) and showed that no extraction occurs for p H > p i , i.e. for p H values where the net charge of the protein is negative. A s soon as the p H decreases below the p i value, there is an abrupt enhancement of the protein solubilization: the surfactant a n d the protein bear opposite charges and electrostatic interactions become attractive. The decrease of protein solubilization at l o w p H values is interpreted by the authors i n terms of protein precipitation at the interface due to denaturation. A n increase of the relative amount of protein i n the organic phase versus p H w o u l d be expected to take place when a cationic surfactant is used. Hatton (2) reported results on solubilization of catalase using a cationic surfactant, dodecyltrimethylammonium bromide (DTAB) i n η-octane, w i t h hexanol as cosurfactant. A t p H values below p i = 5.3 no solubilization occurred, while there was a significant transfer for p H above pi. O t h e r results of e x t r a c t i o n of p r o t e i n s u s i n g d i f f e r e n t trialkylammonium salts differ somewhat from this tendency. Jolivalt et al. (36) studied the solubilization of oc-chymotrypsin i n the Aliquat 336 system using isotridecanol as a cosurfactant. A l p h a - c h y m o t r y p s i n was most significantly extracted for p H above its isoelectric point but as the p H decreased below the p i , the extraction yield decreased very s l o w l y and became negligible at 4 p H units b e l o w the p i . This means that α-chymotrypsin can be extracted i n significant proportion by a cationic surfactant even when positively charged overall. A completely different behavior was observed by Van't Riet and Dekker (40) with α-amylase (isoelectric point around 5.5). The surfactant used i n this work was T O M A C , 12 m M dissolved with octanol i n isooctane.


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.s^Ü^,

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Figure 1. Protein solubilization i n organic solvent by reversed micelles. (Reproduced with permission from Ref. 38. Copyright 1987 M . Dekker.)

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6

θ

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12

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pH

Figure 2. Effect of p H on solubilization of three proteins i n A O T system (o) cytochrome c (pl=10.6); (•) lysozyme ( p l = l l . l ) ; (Δ) ribonuclease (pl=7.8). (Reproduced with permission from Ref. 38. Copyright 1987 M . Dekker.)



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Separation ofProteins by Using Reversed Micelles

Figure 3 shows that solubilization of α-amylase only occurred i n a very narrow p H range around p H 10. A t this p H , α-amylase bears a negative net charge and electrostatic interactions w i t h surfactant are supposed to favor extraction, but this property cannot provide any satisfactory interpretation for the poor extraction i n the range of p H between 5.5 and 10. In an attempt to explain this shift i n the solubilization feature, it was noted that at p H = 10, each basic residue of the protein was deprotonated, so that the molecule s h o u l d not bear any positive charge. Assessing that each negative charge is a complexation site w i t h a surfactant molecule, the resulting complex is a neutral species which can be extracted i n the organic phase without the help of a reversed micelle. Extraction w o u l d involve an i o n - p a i r i n g process, as classically reported i n the literature (41) on extraction of metal ions. Implicitly, the surfactant molecule is considered as a carrier w h i c h enables the transport of the protein into the organic phase. In reversed micellar systems, the surfactant plays another role: it builds, together w i t h the cosurfactant when necessary, a spherical shell surrounding the micelle. A c c o r d i n g to the investigations of W o n g et a l . (28), at least 30 percent of the counterion sodium is dissociated, creating an electrical double layer potential i n the vicinity of the charged heads of the surfactant which affects the electrostatic interactions with the protein. This potential field depends strongly on the environment and i n particular the forces between the protein surface and the surfactant head group decrease as the inverse of the square of the ionic strength. The most general feature (36, 38, 42) of the influence of the ionic strength is the decrease i n the extraction yield when ionic strength increases, as it can be seen i n figure 4 for three different proteins. This property is used for back extraction when contacting a laden organic phase with a high concentrated salt solution. In addition, it is worth noting i n figure 4 that the "critical" K C l concentration, for w h i c h a significant decrease of solubilization occurs, depends on the protein. H o w e v e r , it can be noted that direct measurements of the concentration of every ionic species present inside the micelle generally were not achieved. L e o d i d i s and H a t t o n (43) p u b l i s h e d results on partitioning of cations i n A O T reversed micelles. Studies on the effect of the ionic strength have focused on the ionic strength of the aqueous excess phase, supposing that it reflects qualitatively that i n the organic phase. Similar results are reported using cationic surfactants. The partition coefficient of α-chymotrypsin i n A l i q u a t solutions decreased w i t h increasing N a C l concentration (36). However, i n order to interpret these results it is necessary to take into account that alkylmethyl a m m o n i u m salts are k n o w n to be i n v o l v e d i n anion exchanges. A s s u m i n g an exchange e q u i l i b r i u m between the protein and chloride counterion, an increase of the chloride ion concentration should disfavor the extraction of the protein, following a mechanism of mass action law.


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Recovery (%) of α - amylase i n W 2


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A s s u m i n g that the standard chemical potentials are equal i n bulk and inside micelles, it was deduced that: k T l n i C ^ / C i ^ P i r ) ) + k Tln( b

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Ψ(Γ) is the local electrostatic potential inside the micelle, deduced from a model of the electrostatic double layer. This m o d e l is based on the assumptions of electroneutrality, monodispersity and spherical symmetry of reversed micelles. The original contribution of Leodidis and Hatton is to assume the fluctuation of the surfactant head position between the radii R i and Ro as shown i n figure 5 and to take into account the cation penetration between surfactant polar heads. From the surfactant head distribution, the authors arrive at an analytical expression of Ψ(Γ). The last term developed i n equation 9 contains the ratio of the ion activity coefficient i n each phase. It was expressed using a sum of three contributions: , k

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The electrostatic free energy change i n this equation differs somewhat from that used by Caselli et aL. Indeed, Leodidis and Hatton introduce the excess free energy of the bulk water whereas Caselli et aL consider only the water p o o l contribution. Moreover, the m a i n difference is the m o d e l for electrostatic interactions. Leodidis and Hatton eliminated the Poisson-



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Figure 5. Schematic diagram of the assumed interfacial structure of the reversed micelle. Leodidis and Hatton (43).



5. JOLIVALT ET AL.


Boltzman equation because it does not distinguish between ions of like charge and therefore cannot account for the specificity of the distribution. In order to reflect the experimental reality, the Leodidis and Hatton model takes into account three characteristics of ions: their charge, size and electrostatic free energy of hydration. In its present form, the model is focused o n small cations but could provide a suitable starting point for further approaches including other electrolytes and, i n particular, proteins. It must be noted that Leodidis and Hatton's w o r k refers to some theoretical studies o n microemulsions. Indeed, as shown i n figure 6, reversed micelles are one of the various possible association structures of the microdomains w h i c h compose microemulsions. The most fundamental questions that the numerous works p u b l i s h e d i n this field attempt to answer are related to the mechanism of formation of microemulsions a n d their thermodynamic stability (62, 63). In particular, it was demonstrated that the systems of interest for extraction, i.e. water i n o i l microemulsions i n e q u i l i b r i u m with an excess phase, are governed by the bending stress of the interfacial film (64, 65). In spite of their often being only theoretical, these approaches give a good understanding of the factors characterizing the reversed micellar phase, such as the size of the micelles. They could provide the basic concepts of a general thermodynamics quantification of protein partitioning.

APPLICATIONS Apart from micellar enzymology (the term Martinek coined to describe enzymatic catalysis i n reversed micelles), the main challenge i n reversed micelles research is the separation of a targeted protein from a complex m e d i u m for further purification and recovery. In this case, diphasic systems are involved i n successive steps of extraction and stripping. The relevant parameters of the recovery of proteins from aqueous solutions are mass transfer characteristics, i.e. the efficiency of extraction and stripping steps (including kinetics and yield or concentration factor at equilibrium), the selectivity of the separation between several proteins, a n d the biochemical constraint of achieving a non-degrading separation. For that purpose, two main areas were investigated: the first, and still the most studied, is the optimization of phase composition - reversed micellar and stripping solutions and to a lesser extent the aqueous feed; the second deals with process development i n view of scale-up. From the observation that the partitioning behavior of each protein was not affected by the presence of the others (38, 49), Göklen and Hatton resolved a mixture of cytochrome-c, ribonuclease-A and lysozyme, three l o w molecular weight proteins comprised i n the range 12.4 - 14.3 k D a . W i t h the same reversed micellar phase, W o l l et aL (49) showed that the selectivity of extraction between ribonuclease-A and concanavalin-A could be modulated by varying the surfactant concentration; a 40% enhancement


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Figure 6. Schematic ternary phase diagram of an oil-water-surfactant microemulsion system. (Reproduced with permission from Ref. 63. Copyright 1988 M . Dekker.)



5. JOLIVALT ET AL.


of the maximal selectivity being obtained by increasing A O T concentration from 50 to 100 m M . For larger proteins, such as concanavalin-A (a dimer of 55 kDa), size exclusion plays a discriminating role i n addition to the electrostatic interactions. This was underlined by Armstrong and L i (66) w h o developed a reversed micellar l i q u i d membrane, u s i n g A O T i n hexane. Effects of p H and surfactant concentration enabled the separation of a mixture of cytochrome-c, lysozyme, myoglobin and bovine serum albumine (BSA). It is noteworthy that the large BS A (66 kDa) could transfer through the membrane for A O T concentration above 0.5 M . Further enhancement of the selectivity was obtained by exploiting biospecific interactions between an additional surfactant and the protein. W o l l et aL (49, 67) showed that small amounts (2 to 10% of total surfactant concentration) of a biological detergent (octyl ß-D-glucopyranoside) increased, b y more than one order of magnitude, the selectivity of extraction of concanavalin-A against ribonuclease-A, i n an AOT/isooctane system. The affinity of the carbohydrate-binding concanavalin-A for the ligand was essentially responsible for its increased extraction, w h i l e ribonuclease-A was not specifically affected. In the previous examples, where the principal surfactant of reversed micellar phases was A O T , apparently no problem of protein denaturation was encountered and i n favorable cases quantitative back extractions were performed. However, these studies were carried out using synthetic feed solutions. Experiments i n more realistic conditions are reported by Rahaman et a_L (68), w h o extracted an extracellular alkaline protease ( M = 33 kDa) from a fermentation broth of bacillus strain, w i t h an AOT/isooctane system. In one extraction-stripping stage, an activity yield of 22% and a relative specific activity of 2.2 (compared to feed phase) were achieved; the use of multi-stage cascaded operations and the variation of the volume ratios of the phases permitted i m p r o v e d results (relative specific activity u p to 6). These experiments underlined the importance of extraction kinetics, since the protease exhibited m a r k e d instability i n presence of the organic phase at high p H and since the back transfer was surprisingly slower than i n simple synthetic protein solutions; m i x i n g times were optimized at 5 minutes for extraction a n d 15 minutes for stripping. The influence of broth impurities on separation features was, however, not really evaluated. Another application of reversed micelles on complete broths was reported b y G i o v e n c o a n d Verheggen (69) for the p u r i f i c a t i o n of intracellular dehydrogenases of Azotobacter vinelandii. The surfactant cetyltrimethyl a m m o n i u m b r o m i d e , dissolved i n an hexanol-octane solution, served also as a disrupting agent of cell membranes. The activity recovered i n the stripping phase was superior to 100% - explained by the supposed removal of inhibitors - with a relative specific activity of 6, for the smaller enzymes ß-hydroxybutyrate and isocitrate dehydrogenases. The largest glucose-6-phosphate dehydrogenase was not extracted i n an active form.


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Most of the examples of extraction by reversed micelles reported above were performed i n a batch mode, with successive steps of extraction and stripping, and little consideration was taken with regard to the process itself. U s i n g reversed micellar phase as a l i q u i d membrane, A r m s t r o n g and L i (66) could perform continuous extraction from and stripping to aqueous solutions i n either side of the membrane. However, improvements of kinetic mass transfer parameters, as influenced by membrane thickness a n d stability i n c o n d i t i o n s of a p p r o p r i a t e hydrodynamics, remain necessary. Dekker et aL (70, 71) developed a 2-stage mixer-settler device to achieve continuous concentration of α-amylase, the T O M A C /octanol/ isooctane reversed micellar phase being recirculated from the stripping settler to the first extracting mixer. O n condition that losses of surfactant be compensated by its addition during the course of the experiment, the efficiency of the separation was maintained over six circulations, w i t h a concentration factor of 8 for the active enzyme. A g a i n , this study made clear the importance of achieving rapid mass transfer, since first order kinetics were found for the inactivation of α-amylase d u r i n g the forward extraction step (the total activity loss was 30% during the procedure). O n the other hand, an overly strong mixing w o u l d increase the duration of settling and possibly cause similar drawbacks. The use of hollow fibers tries to answer the need of increasing the surface/volume ratio of the extracting phase to the feed volume, by means other than higher agitation and the resulting undesired emulsification. Dekker et aL (71) and Dahuron and Cussler (72) reported experiments using microporous p o l y p r o p y l e n e membrane to stabilize the interface between the two streams of aqueous feed and reversed micellar phases. Mass transfer coefficients were determined for the extraction of cytochrome-c and α-chymotrypsin by AOT/octane (72) and α-amylase by T O M A C / R e w o p a l HV5/octanol/isooctane system (71). A n attractive new application of reversed micelles has also been mentioned for the refolding of proteins. M a n y of the proteins produced by recombinant D N A technology have to be extracted from cells using strong solubilizing dénaturants. The step of refolding, by dénaturant removal, generally necessitates operating i n very dilute solutions, i n order to avoid aggregation of the partially refolded intermediates. The method tested by Hagen et aL (73) takes advantage of the isolation of single proteins inside micelles to prevent harmful interactions. They demonstrated that it was possible to extract a denaturated R N A s e by an AOT/isooctane phase, and to remove the dénaturant guanidine hydrochloride by successive aqueous phase contacting. The reoxidation of disulfide bonds by a glutathione mixture permitted the total reactivation of the enzyme. The last step of stripping led to the recovery of a refolded and fully active R N A s e , with an overall yield of 50 percent.


5. JOLIVALT ET AL.



CONCLUSION A significant amount of w o r k has demonstrated the feasibility and the interest of reversed micelles for the separation of proteins and for the enhancement or inhibition of specific reactions. The number of micellar systems presently available and studied i n the presence of proteins is still limited. A n effort should be made to increase the number of surfactants used as well as the set of proteins assayed and to characterize the molecular mechanism of solubilization and the microstructure of the laden organic phases i n various systems, since they determine the efficiency a n d selectivity of the separation a n d are essential to u n d e r s t a n d the phenomena of bio-activity loss or preservation. A s the features of extraction depend on many parameters, particular attention should be paid to controlling a l l of them i n each phase. S i m p l i f i e d thermodynamic models begin to be developed for the representation of partition of simple ions a n d proteins between aqueous a n d micellar phases. Relevant experiments a n d more complete data sets o n d i s t r i b u t i o n of salts, cosurfactants, should promote further developments i n m o d e l l i n g i n relation with current investigations on electrolytes, polymers and proteins. This work could be connected with distribution studies achieved i n related areas as microemulsions for o i l recovery or supercritical extraction (74). In addition, the contribution of physico-chemical experiments s h o u l d be taken into account to evaluate the size and structure of the micelles.

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Kadam, K. L. Enzyme Microb. Technol. 1986, vol 8, ρ 266. Hatton, T. A. In ACS Symposium Series; Hinze, W. L., Armstrong, D. N., Eds.; 1987; Chapter 9. Hatton, T. A. In Surfactants and Separation; Scamehorn, J. F. and Harwell J. H., Eds.; New York, 1987. Abbott, N. L.; Hatton, T. A. Chem. Eng. Prog. August 1988,p31-40. Martinek, K.; Levashov, Α. V.; Klyachko, N. L.; Pantin, V. I.; Berezin, I.V. Biochimica and Biophysica Acta 1981, 657, 277-294. Fletcher, P. D. I.; Freedman, R. B.; Mead, J.; Oldfield, C.; Robinson, Β. H. Colloids and Surfaces 1984, 10, 193-203. Laane, C.; Verheart, R. Isr. J. of Chemistry 1987-1988, 28, 17-22. Shield, J. W.; Fergusson, H. D.; Bommarius, A. S.; Hatton, T. A. Ind. Eng. Chem.Fundam. 1986, 25, 603-612. de Kruijff, B.; Cullis, P. R. Biochim. Biophys. Acta 1980, 602, 477-490. Gierash, L. M.; Thompson, K. F. Surfactants in Solution; Mittal, K. L. and Lindman, B., Eds., New York, 1984,p265. Thompson, K. F.; Gierasch, L. M. J. Am. Chem. Soc. 1984, 106, 3648-52. Nicot, C.; Vacher, M.; Vincent, M.; Gallay, J.; Waks, M. Biochemistry 1985, 24, 70-84. Delahodde, Α.; Vacher, M.; Nicot, C.; Waks, M. FEBS Lett. 1984, 172(2), 343-47. Steinmann, B., Jäckie, H.; Luisi, P. L. Biopolymers 1986, 25, 1133-56. Marco, A. D.; Zetta, L.; Menegatti, E.; Luisi, P. L. J. Biochem. Biophys. Methods 1986, 12, 335-347. Luisi, P. L. Angewandte Chemie 1985, 24(6), 439-528.


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106 17. 18. 19. 20. 21.


22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

DOWNSTREAM PROCESSING AND BIOSEPARATION O'Connor, C. J.; Lomax, T. D.; Ramage, R. E. Adv. Colloid Int. Sci. 1984, 20, 21-97. Waks, M. Proteins 1986, vol. 1,p4-15. Luisi, P. L.; Laane, C. Tibtech 1986,p153-161. Luisi, P. L.; Magid, L. J. CRC Critical Reviews in Biochemistry 1986, vol. 20(4),p409474. Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, Β. H. Biochemica Biophys. Acta 1988, 947, 209-246. Fendler, J. H. Accounts of Chemical Research 1976, 9, 153-161. Martinek, K.; Levashov, A.V.; Klyachko, N.; Khmelnitski, Y. L.; Berezin, I. V. Eur. J. Biochem. 1986, 155, 453-468. Mitchell, D. J.; Ninham, B. W. J. Chem. Soc. Faraday Trans. 2 1981, 77, 601-629. Zulauf, M.; Eicke, H. F. J. Phys. Chem. 1979, 83(4),p480. Jean, Y. C.; Ache, H. J. JACS 1978, 100(3), 984. Maitra, A. J. Phys. Chem. 1984, 88, 3122-25. Wong, M.; Thomas, J. K.; Nowak, T. JACS 1977, 99(14),p4730. Wong, M.; Thomas, J. K.; Grätzel, M. JACS 1976, 98(9),p2391. Kumar, V. V.; Raghvnathan, P. Lipids 1986, 21(12),p764. Keh E.; Valeur, Β. J. Colloid Int. Sci. 1981, 79(2),p465. Howe, Α. M.; McDonald, J. Α.; Robinson, Β. Η. J. Chem. Soc. Faraday Trans. 1 1987, 83, 1007-1027. Kotlarchyk, M.; Huang J. S. J. Phys. Chem. 1985, 89, 4382-4386. Luisi, P. L.; Henninger, F.; Joppich, M. Biochem. Biophys. Res. Com. 1977, 74(4), p 1384. Luisi, P. L.; Bonner, F. J.; Pellegrini, Α.; Wiget, P.; Wolf, R. Helv. Chem. Acta 1979, vol 62(3), n° 77,p740. Jolivalt, C.; Minier, M.; Renon, H., in press, J. Colloid Int Sci., 1989. Thien, M. P.; " Separation and Concentration of Amino Acids Using Liquid Emulsion Membranes", Doc. of Sci. Thesis, M.I.T., 1988. Göklen, Κ. Ε.; Hatton, T. A. Sep. Sci. Techn. 1987, 22(2-3), 831-841. Göklen, Κ. E.; Hatton, T. A. Proc. ISEC 1986,p587-595. Van't Riet, K.; Dekker, M. 3rd Eur. Cong. Biochem. 1984,p540. Paatero, J. Dept. Inst. Ind. Chem. Abo Akademi 1975, n° 106. Göklen, Κ. E.; Hatton, T. A. Biotechnology Prog. 1985, vol 1(1),p69. Leodidis, Ε. Β.; Hatton Τ. Α.; Submitted to Langmuir, July 1988. Dekker, M.; Van't Reit, Κ.; Baltussen, J. W. Α.; Bijsterboch, B.H.; Hilhorst, R.; Laane, C. Proc. 4th Eur.Cong.on Biotechnology 1987,p507. Fletcher, P.; J. Chem. Soc. Faraday Trans. 1 1986, 82, 2651-2664. Göklen, Κ. Ε., Ph.D. Thesis, MIT 1986. Leser, Μ. Ε.; Wei, G.; Luisi, P. L.; Maestro, M. Biochem. Biophy. Res. Com. 1986, 135(2),p629. Göklen, Κ. Ε.; Hatton, T. A. AIChE Annual Mtg. 1984, n° 5a. Woll, J.; Willon, A. S.; Rahaman, R. S.; Hatton, Τ. Α., Protein Purification - Micro to Macro 1987,p117-130. Dekker, M.; Baltussen, J. W. Α.; Van't Riet, K., Bijsterbosch, Β. Η.; Laane, C.; Hilhorst, R. Biocatalysis in Organic Media; Laane, C.; Tromper, J.; Lilly, M.D., Eds.; Elsevier 1987;p285-288. Kirgios,I.;Hansec, R.; Rhein, H. B.; Schugerl, K. Preprint ISEC München 1986, 3, 623 Levashov, Α. V.; Khmelnitsky, Y. L.; Klyachko, N.L.; Chernyak, V.Ya.; Martinek, K. J. Coll. Int. Sci. 1982, 88(2), 444. Bonner, F. J.; Wolf, R.; Luisi, P. L. J., Solid Phase Biochemistry 1980, 5(4), 255. Zampieri, G. G.; Jackie, H.; Luisi, P. L. J. Phys. Chem. 1986, 90, 1849-1853. Sheu, E.; Göklen, K. E.; Hatton, T. Α.; Chen, S.H. Biotechnology Progress 1986, 2(4), p 175. Chatenay, D.; Orbach, W.; Cazabat, A. M.; Vacher, M.; Waks, M. Biophys. J. 1985, 48, 893-898.


5. JOLIVALT ET AL. 57. 58. 59. 60. 61. 62.


63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.


107

Pileni, M. P.; Zemb, T.; Petit, C. Chem. Phys. Lett. 1985, 118,p414. Caselli, M.; Luisi, P. L.; Maestro, M.; Roselli, R. J. Phys. Chem. 1988, 92, 3899-3905. Woll, J. M.; Hatton, Τ. Α.; "A Simple Phenomenological Thermodynamic Model for Protein Partitioning in Reversed Micellar Systems"; Bioproc. Eng., in press. Biais, J.; Bothorel, P.; Clin, B.; Lalanne, P. J. Dispersion Sci. and Tl. 1981, 2, 67. Eicke, H. F. Top. Curr. Chem. 1980, 87:86. Bellocq, A. M.; Biais, J.; Bothorel, P.; Clin, P.; Fourche, G.; Lalanne, P.; Lemaire, B.; Lemanceau, B.; Roux, D. Adv. Colloid Int. Sci. 1984, 20, 167 Leung, R.; Mean Jeng Hou; Do Shah. Surfactants in Chemical Process Engineering, Surfactant Science Series 1988, vol 28, 315-67 Mukhiya, S.; Miller, C. Α., Fort, T. J. Colloid Int. Sci. 1983, 81, 223 Miller, C. Α.; Neogi, P. AIChE J. 1980, 26, 212 Armstrong, D. W.; Li, W. Anal. Chem. 1988, 60, 88-90. Woll, J. M.; Hatton, T. Α.; Yarmush, M.L.; Biotech. Progr., 1989, 5,2, 57-62. Rahaman, R. S.; Chee, J. Y.; Cabral, J. M. S.; Hatton, T.A.; Biotech. Progr. 1988, 4,4, 218-224. Giovenco, S. and Verheggen, F., Enzyme Microb. Technol. 1987, 9, 470-473. Dekker, M.; Van't Riet, K.; Weijers, S. R. Chem. Eng. J. 1986, 33, B27-B33. Dekker, M.; Van't Riet, K.; Wijnans, J. M. G. M.; Baltussen, J. W. Α.; Bijsterbosch, B. H.; Laane, C. Proc. IOCM 1987,p793. Dahuron, L.; Cussler, E. L. AIChE J. 1988, 34, 130-136. Hagen, A. J.; Hatton, T. Α.; Wang, D. I. C. Proc. ACS Mtg. 1988. Gale, R. W.; Fulton, J. L.; Smith, R. D. J. Am. Chem. Soc. 1987, 109, 920-21.

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