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Isolating enzymes by reversed micelles
178,217-226
ANALYTICALBIOCHEMISTRY
(1989)
REVIEW Isolating Enzymes by Reversed Micelles Matthijs Dekker,*p’ Riet Hilhorst,?
and Colja LaaneS
*Food and Bioengineering Group, Department of Food Science, Agricultural University, P.O. Box 8129,670O EV Wageningen, The Netherlands; TDepartment of Bioch.emistry, Agricultural University, P.O. Box 8128,670O ET Wageningen, The Netherlands; and $Unilever Research Lab, P.O. Box 114,313O AC Vluardingen, The Netherlands 1.
INTRODUCTION
Reversed micelles are aggregates of surfactant molecules containing an inner core of water molecules, dispersed in a continuous organic solvent medium. These systems are optically transparent and thermodynamically stable. The considerable biotechnological potential of these systems is derived principally from the ability of the water droplets to dissolve enzymes, without loss of activity, in much the same way as does bulk water. The use of these systems in the biotransformation of apolar compounds, present in the bulk oil phase, has been described in a number of publications (l-5). Recently, it has been found that enzymes can be transported from one bulk aqueous phase to another via an intermediate reversed micellar phase, the transported protein molecules being transiently accommodated within the reversed micelles in this case. This phenomenon is a consequence of the reversibility of the phase transfer process. In addition to transport from a homogeneous solution phase, proteins may also be transported from a solid phase, and even from the cytoplasmic compartment of bacteria. The latter phenomenon is based on the prior disruption of the bacterial cell wall by surfactant partitioning out of the reversed micellar phase. Here, recent advances in separating enzymes by the use of reversed micelles are highlighted. The use of these media to convert apolar compounds enzymatically has been reviewed elsewhere (35). 2. RECOVERY SOLUTION
OF ENZYMES
FROM
AQUEOUS
The isolation of specific extracellular enzymes from a fermentation broth by conventional processes consists of the stepwise removal of undesired compounds from the broth. Because these steps are usually not specific 1 To whom
correspondence
should
oooa-2697/&39 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form
be addressed.
Inc. reserved.
for the desired enzymes, several steps are necessary to obtain an enzyme preparation of the required purity. Consequently, new isolation techniques, more selective for the required enzyme and easier to scale up, are desirable. Liquid-liquid extraction procedures involving the use of reversed micellar systems have some very promising features in this respect. The principle of the method is depicted schematically in Fig. 1. First, the enzyme is extracted from the aqueous phase to the reversed micellar phase under conditions such that the extent of phase transfer of the protein is maximal. Second, the enzyme is recovered from the organic phase, by extraction of the reversed micellar phase with a second aqueous phase, under such conditions that the transfer of the enzyme from the reversed micellar phase to the aqueous phase is maximal. By appropriate manipulation of physical parameters such as solution pH and ionic strength, which determine the distribution behavior of the enzyme, it is possible to obtain purification and concentration of the required enzyme in a straightforward process. The first reports on the transfer of proteins from an aqueous phase to a reversed micellar phase and vice versa were those of Luisi et al. (6,7), who illustrated this principle using a range of proteins. These investigations have been extended in more recent studies by groups at MIT (B-12) and in Wageningen (13-19) for the development of a recovery process for proteins from aqueous solutions. In the first part of this section the factors affecting the distribution of proteins between an aqueous phase and a conjugate reversed micellar phase are discussed. In the second part the development and performance of continuous extraction processes are described. 2.1. The Distribution of Proteins There is a large body of evidence which indicates that electrostatic interactions play a very important role in determining the distribution coefficient of a protein be217
218
DEKKER,
FIG. 1. Schematic representation enzymes between an aqueous phase enzyme).
HILHORST,
of the liquid-liquid extraction and a reversed micellar phase
of (E,
tween a reversed micellar phase and a conjugate aqueous phase. This influence is demonstrated clearly by the effect of aqueous phase pH, ionic strength, and surfactant type (anionic, cationic, or nonionic) on this distribution behavior. The individual effects exerted by each of these parameters are discussed separately. 2.1.1. Aqueous phase PH. The aqueous phase pH determines the ionization state of the surface-charged groups on the protein molecule. Attractive electrostatic interactions between the protein molecule and the surfactant head groups, which form the internal surface of the reversed micelle, will occur if the overall charge of the protein is opposite to the charge of the surfactant head groups. This implies that, for cationic surfactants, solubilization of the protein in reversed micelles is favored at pH values above the isoelectric point (~1) of the protein, while the opposite is true for anionic surfactants. Luisi et al. (7) showed that the aqueous phase pH influences the transfer of cY-chymotrypsin and pepsin to a reversed micellar phase of the cationic surfactant trioctylmethylammonium chloride (TOMAC)2 in cyclohexane. These results, however, could not be interpreted on basis of the pl values of these proteins. The phase transfer of the enzyme cY-amylase into reversed micelles of TOMAC/octanol in isooctane was found to recur only over a narrow pH range, well above the pl of the enzyme (13). These results indicate that not only the sign of the protein charge but also the surface charge density is an important factor in protein solubilization. Using a reversed micellar system consisting of the anionic surfactant sodium di-2-ethylhexyl sulfosuccinate
AND LAANE
(commercial name Aerosol OT or AOT) in isooctane, Goklen and Hatton (10) found almost complete solubilization of the three low-molecular-weight proteins (A& 12,000-14,000), ribonuclease A, cytochrome c, and lysozyme. This solubilization took place in a range of 4 to 6 pH units just below the PI’S of the three proteins (Fig. 2). As the molecular weight of the protein increases, phase transfer can be accomplished only by increasing the value of (pH-~1). Thus, in the case of a-chymotrypsin (AI, 25,000) phase transfer was favored in a pH range approximately 2 to 4 pH units below the p1 of the protein; bovine serum albumin (AI, 68,000) was not transferred at all in the same system. The effect of pH on solubilization for proteins of different molecular weight might be explained by taking into account the fact that for larger proteins the size of the reversed micelle containing a protein molecule must be significantly larger than the size of the empty reversed micelle. This energetically unfavorable transition of the micellar size must be compensated for by more extensive electrostatic interactions in order to make the overall solubilization process feasible. This can be achieved by increasing the charge density of the protein by manipulation of the pH. For small proteins whose size is smaller than the size of the water pool inside a reversed micelle, solubilization occurs as soon as the net protein charge is opposite to that of the reversed micellar interface. This behavior is demonstrated clearly (21) by the linear correlation attained between the A!, of a protein and the difference between the pH of solubilization and the p1 of the protein (Fig. 3). The correlation holds for reversed micelles of both anionic and cationic surfactants. 2.1.2. Ionic strength. The ionic strength of the aqueous phase determines the degree of shielding of the electrostatic potential imposed by a charged surface. This phenomenon causes at least two important effects in reversed micellar extraction. First, it decreases the
103
20
6
8
10
12
14
PH ’ Abbreviations AOT, di-2-ethylhexyl nium bromide.
used: TOMAC, trioctylmethylammonium sulfosuccinate; CTAB, cetyltrimethyl
chloride; ammo-
FIG. 2. The effect of pH on the solubilization of (0) lysozyme, (0) cytochrome c, and (A) ribonuclease A in AOT-isooctane solutions (from Ref. (10)).
ISOLATING
ENZYMES
BY
REVERSED
219
MICELLES
6C
0
2o.m
40.000
6o.m
ao.ooo
M,
FIG. 3. Relation between the molecular weight of a protein and the difference between the protein’s pl and the pH of optimal extraction into a reversed micellar phase of TOMAC in isooctane (from Ref.
(21)). electrostatic interaction between the charged protein molecule and the charged interface in the reversed micelle. Second, it reduces the electrostatic repulsion between the surfactant head groups, resulting in a decrease in the size of the reversed micelles at higher ionic strength. Gijklen and Hatton (9,lO) showed the effect of ionic strength on the phase transfer of ribonuclease A, cytochrome c, and lysozyme in a reversed micellar phase of AOT in isooctane (9,lO). The extent of phase transfer to the reversed micellar phase decreases for all three proteins with increasing potassium chloride concentration, but the concentration required to initiate this decrease was found to be different for each protein (Fig. 4). No rules with respect to the ionization state of the proteins used (expressed as the difference between the aqueous phase pH (6.7-7.1 in this study) and the p1 of the proteins) can be deduced from these data. Meier et al. (20) observed an increase in the amount of trypsin and peroxidase transferred to a reversed micellar
100
t
OL 0
02
0.4
0.6
KCI ccnc
0.6
1
[Ml
FIG. 4. The effect of the potassium chloride concentration on the solubilization of (Cl) lysozyme, (0) cytochrome c, and (A) ribonuclease A in AOT-isooctane solutions (from Ref. (10)).
10.5
11
PH FIG. 5. Effect of pH on the transfer of cY-amylase to a reversed micellar phase of TOMAC in isooctane at different ionic strengths (from Ref. (18)). (0) 0 mM NaCl, (0) 20 mM NaCl, and (A) 50 mM NaCl.
phase of AOT in isooctane when using sodium salts to increase the ionic strength but a decrease when using calcium salts. These effects were observed at both pH 7 and 10. The fact that an increase in the ionic strength results in a shift in the pH profile of the distribution behavior was demonstrated by Dekker et al. (18). The transfer of the enzyme a-amylase to a reversed micellar phase of TOMAC/octanol in isooctane was studied for different concentrations of sodium chloride as a function of the pH (Fig. 5). As the salt concentration increased, a higher pH (resulting in a higher charge density on the protein) was required for maximal phase transfer. Thus once again, electrostatic interactions are implicated as being of primary importance in the phase transfer mechanism. For the results shown, it can be seen that increasing the ionic strength at pH 10.1, for example, first causes an increase in solubilization of cu-amylase in the reversed micellar phase followed by a decrease in solubilization beyond an optimal ionic strength. The observation that not only the ionic strength, but also the type of ion, influences the distribution behavior of proteins implies specific interaction of ions with the protein and/or the surfactant head groups. The differences in the distribution behavior of proteins on the pH and ionic strength of the aqueous phase have been used to advantage in the separation of a protein mixture (10). An aqueous solution of ribonuclease A, cytochrome c, and lysozyme was extracted with a reversed micellar phase of AOT in isooctane. By using the results from Figs. 2 and 4 the three proteins could be separated by a series of three extractions. Woll et al. (11) showed that the aqueous phase pH and ionic strength could be used to selectively extract an alkaline protease from a fermentation broth. By perform-
220
DEKKER,
I
0 9
9.5
10
HILHORST,
I 11
10.5
PH FIG. 6.
Solubilization of a-amylase in a reversed micellar phase of TOMAC in isooctane in relation to the pH in the aqueous phase. Effect of the addition of nonionic surfactant to the reversed micellar phase (from Ref. (16)). (Cl) -Rewopal Hv5, (m) +Rewopal Hv5.
ing two series of extractions 42% of the enzyme was recovered with an 2.2-fold increase in specific activity. 2.1.3 Surfactant type. As shown in the previous two sections, the distribution of proteins is dependent primarily on the difference in charge between the protein and the charged surfactant head groups. Therefore, in the absence of other effects, an opposite pH dependence of the distribution behavior can be expected for reversed micelles stabilized by cationic and anionic surfactants. In addition to the surfactant charge, other surfactantdependent parameters may be cited. Among these are the size of the reversed micelles that are formed, the energy required to enlarge the reversed micelles, and the charge density on the inner surface of the reversed micelle. Dekker et al. (16) showed the effect of the addition of a nonionic surfactant (Rewopal HV5: nonylphenolpentaethoxylate) to a reversed micellar phase of the cationic surfactant TOMAC in isooctane. The partitioning of cyamylase between the reversed micellar and the conjugate aqueous phase was found to be a strong function of the ratio of nonionic to ionic surfactant. An increase in the proportion of nonionic surfactant resulted in increased transfer of the enzyme to the reversed micellar phase. Additionally, phase transfer took place over a wider range of pH (Fig. 6). This change in distribution behavior might be explained by two effects: first, the charge density of the micellar interface will be influenced by the presence of the nonionic surfactant. Second, the size and flexibility of the reversed micelles will be changed by the addition of the one-tail nonionic surfactant. Until now we have discussed only the use of electrostatic interactions between the protein and the surfac-
AND
LAANE
tant head groups to promote transfer to a reversed micellar phase. Another possibility is the use of biospecific interactions between an enzyme and a ligand (substrate analog, product, or an inhibitor). In principle, the ligand can be confined to the reversed micellar phase by conjugation to a suitable hydrophobic tail; in the reversed micelles, such a (polar) ligand would form a site-specific surfactant head group. The effect of such an affinity surfactant, octyl-/3-D-glucopyranoside, has been shown for the solubilization of concanavalin A in AOT reversed micelles (11). The protein was transferred to the reversed micellar phase at pH values at which no transfer was observed without the affinity surfactant. This transfer was inhibited by the addition of free ligand in the aqueous phase, indicating that an affinity interaction was required for the solubilization at these pH values. In this system the solubilization is most likely due to a combination of electrostatic, affinity, and possibly hydrophobic interactions. It would be interesting to explore this phenomenon further by substituting a nonionic surfactant for the ionic surfactants used previously. In this case electrostatic interactions between the protein and the surfactant head groups are eliminated and affinity interactions, perhaps in combination with hydrophobic interactions, become decisive in determining the extent of phase transfer. This should result in a very selective extraction process. 2.1.4. Surfactant concentration. The concentration of surfactant in a reversed micellar phase that is in equilibrium with an aqueous phase has little effect on the size and structure of the reversed micelles. The extent of protein uptake from a conjugate aqueous phase therefore increases in proportion to the surfactant concentration in the reversed micellar phase (12,22). Solubilization of ribonuclease A and concanavalin A as a function of the AOT concentration could be described by a model based on a thermodynamic equilibrium between the concentration of protein-reversed micelle complexes and the concentration of free reversed micelles and free protein in the aqueous phase (12). The distribution of a-chymotrypsin and pepsin in AOT-reversed micelles has been described (22) by expressing the protein partitioning based on the protein concentration in the water pools of the reversed micelles divided by the bulk aqueous phase concentration. This approach resulted in a partition coefficient which was independent of the surfactant concentration. Since the total volume of the water pools varies linearly with the surfactant concentration (w~,~~~,the molar ratio of water to surfactant in the micellar phase is approximately independent of the concentration of surfactant), the two descriptions are essentially the same. In this context, the partitioning behavior of the enzyme lysozyme proved to be exceptional (22); below a certain surfactant concentration the enzyme precipi-
ISOLATING
ENZYMES
BY
REVERSED
MICELLES
221
distribution of charged groups on the surface of proteins on their distribution behavior. Proteins with a more asymmetric charge distribution were found to partition more easily into the reversed micellar phase.
tated at the interface between the two phases. Yet increasing the surfactant concentration above this point resulted in the transfer of all of the protein to the reversed micellar phase. They estimated this concentration to be just sufficient to provide a monolayer coverage for the total enzyme in the system.
2.2. Process Development
2.1.5. Thermodynamic modeling. Several models of the distribution behavior of proteins between a bulk aqueous phase and a reversed micellar phase have been developed (11,23,24). They are all based on electrostatic interactions between the charges on the protein molecule and the charges on the inner surface of the reversed micelle due to the ionic head groups of the surfactants. In addition to free energy change resulting directly from this interaction, an additional contribution arising from the redistribution of surfactant and protein counterions, of other free ions, of water, and of surfactant on phase transfer must be considered. Calculations have been made of the total free energy change on the uptake of a hypothetical small protein in reversed micelles (23); no comparisons were made with experimentally determined distribution data. In a more phenomenological approach, the distribution behavior of cytochrome c between an aqueous phase and a reversed micellar phase of the cationic surfactant TOMAC in isooctane was analyzed with respect to the aqueous phase pH and ionic strength (24). From the experimental data, calculations were made on the copartitioning of protons and other small ions accompanying protein uptake in reversed micelles. Around the optimal pH for protein transfer to the reversed micellar phase, both protons and surfactant counterions (chloride) were found to be redistributed. A third approach (11) described the partitioning of ribonuclease A and concanavalin A as a function of aqueous phase pH and surfactant concentration. Four coefficients which depend on the reversed micellar system and the protein used were employed. All of the above models are useful in analyzing experimental data of protein partitioning and in indicating the most important system parameters affecting the value of the distribution coefficient. At present, however, the models cannot give accurate predictions of the distribution coefficient of a protein; many parameters are unknown or difficult to quantify. These include hydrophobic interactions of the protein with the apolar phase, specific interactions of ions with the protein and surfactant, the free energy changes associated with the change in size of the reversed micelles on protein uptake, and the distribution of charged groups on the protein molecules. This last parameter has been neglected in all three approaches; they all assume an equal distribution of surface charge on a globular protein molecule. Wolbert et al. (21) showed a strong effect of the symmetry of the
For the large-scale recovery of extracellular enzymes, an extraction with an organic solvent containing reversed micelles has interesting advantages over existing processes. A reversed micellar extraction combines the potential for concentration and purification of the enzyme in a single process. The liquid-liquid extraction technique in general is well known and apparatus and scale-up rules have been established for numerous applications, including the use in recovery processes for antibiotics and organic acids from fermentation broths. The success of the type of extraction processes described here is critically dependent on the ability to direct the distribution coefficient of the desired enzyme between the aqueous phase and the reversed micellar phase, as discussed under Section 2.1. 2.2.1. Forward and back extractions. Enzyme recovery from an aqueous phase by liquid-liquid extraction using reversed micelles consists of a first step whereby the desired enzyme is transferred selectively from the aqueous phase to the reversed micellar phase (forward extraction). The enzyme is subsequently recovered from the reversed micellar phase by extraction with a second aqueous phase (back extraction). These two extractions can be performed in a continuous mode, with the reversed micellar phase circulating between the two extraction units. No specialized equipment is required, that developed for conventional liquidliquid extraction processes is, in principle, also suitable for this application. The most important types of equipment are mixer/settler, agitated column, centrifugal, and membrane extractors. For some reversed micellar systems the use of mixer/settlers and agitated columns might be limited by emulsion formation between the aqueous phase and the reversed micellar phase because of the presence of surfactants that stabilize those emulsions. In such cases centrifugal extraction may be applied to reduce the settling time. Additionally, membrane extraction techniques may be adapted for use with these types of systems, the membrane serving to stabilize the reversed micellar/aqueous phase interface. For systems where short residence times are required centrifugal extractors might be very useful. At the present time process development has centered exclusively on the use of mixer/settlers (14-16,18,19) and membrane extractors (17,25). 2.2.2. Mixer/settler extraction. The performance of a continuous forward and back extraction for the recovery of an enzyme was demonstrated (14) with two mixer/settler units, one for each extraction step. The re-
222
DEKKER,
,
reversed
micellar
phase
HILHORST, ,
+ 3,--m
-b--4
mixer1
wl,in
mixer2 FORWARD
BACK
EXTRACTION
EXTRACTION
Wl.out
w2, I”
W2,out
FIG. ‘7. Flow sheet of the combined forward and back extraction for two mixer/settler units, with the reversed micellar phase circulating between the two extraction units (from Ref. (14)).
versed micellar phase was circulated between the two units. The flow sheet for this extraction is given in Fig. 7. This design was used to transfer cr-amylase from an aqueous phase to a reversed micellar phase of TOMAC in isooctane during the forward extraction and to transfer it to a second aqueous phase during the back extraction. The distribution coefficient of the enzyme was manipulated by adjusting the pH and the ionic strength of the aqueous phases. Forward extraction was favored by a high pH and low ionic strength; back extraction was favored by a low pH and high ionic strength. The value of this distribution coefficient (reversed micellar concentration divided by the aqueous phase concentration of the enzyme) was determined to be at least 10 during the forward extraction and at most 0.004 during the back extraction. In this way, an eightfold concentration of the enzyme a-amylase was obtained, without optimization of the process. The enzyme recovered in the second aqueous phase accounted for 45% of the initial enzymatic activity. Approximately 30% inactivation of the enzyme was observed during extraction. Surfactant was found to be slowly desorbed from the reversed micellar phase, resulting in a decreased extraction efficiency after 3.5 circulations of the reversed micellar phase, an effect that could be fully restored by the addition of more surfactant to the reversed micellar phase. 2.2.3. Modeling and optimization. The reversed micellar extraction process can be described in terms of the distribution coefficients, mass transfer rate coefficient, inactivation rate constants, phase ratios, and residence times during the forward and back extractions (18). For all phases, differential equations have been derived for the concentration of active enzyme in that phase as a function of time. When the individual param-
AND
LAANE
eters on distribution, mass transfer rate, and inactivation rate are known, these equations can be solved numerically to yield the concentrations as a function of extraction time (18,19). For the extraction of the enzyme a-amylase, with a reversed micellar phase of TOMAC in isooctane, the various variables which describe the extraction process have been determined (19). Determination of the inactivation rate constants in this system showed that the inactivation took place predominantly by complexation of the enzyme with surfactant in the first aqueous phase (l&19). Although the equilibrium concentration of TOMAC in the aqueous phase is very low, it is continuously extracted from the reversed micellar phase during forward extraction, resulting in the observed surfactant loss from the reversed micellar phase. In order to minimize the extent of enzyme inactivation the steady-state concentration of enzyme in the first aqueous phase should be as low as possible. This can be achieved by a high mass transfer rate constant and a high distribution coefficient of the enzyme between the reversed micellar and the aqueous phases. In Fig. 8 simulations of the forward and back extractions of a-amylase are given using the experimentally determined values. The effect of the mass transfer rate coefficient during forward extraction on the recovery of active enzyme is shown for two values of the distribution coefficient (19). These model predictions were verified experimentally by changing the distribution coefficient (by the addition of a nonionic surfactant to the reversed micellar phase, see Section 2.1.3.) and the mass transfer rate coefficient of the enzyme during the forward extraction (by increas-
FIG. 8. Simulation of the effect of the mass transfer rate coefficient on the cu-amylase activity recovery by a reversed micellar extraction. The activity in the second aqueous phase is relative to the initial activity in the first aqueous phase. 1, mt = 10; 2, m, = 100. The flow ratio of the two aqueous phases is 20 (from Ref. (19)).
ISOLATING
0
100
200
ENZYMES
BY
400 300 TIME [mini
FIG. 9. Activity recovery of cY-amylase in the second aqueous phase of the combined forward and back extraction. The activity in the second aqueous phase is relative to the initial activity in the first aqueous phase (from Ref. (19)). A (0) Data at low distribution coefficient and low mass transfer rate (dip caused by TOMAC loss from reversed micellar phase). B (m) Data at high distribution coefficient and low mass transfer rate. C (A) Data at high distribution coefficient and high mass transfer rate. (- - -) The model prediction by using the independently found values for the distribution, mass transfer, and inactivation coefficients. (-) Line fitted through steady-state activities.
ing the stirrer speed). The experimental results of this optimization of the mixer/settler extraction thus obtained are shown in Fig. 9 together with the model prediction of the extractions. In this way the performance of the reversed micellar extraction of a-amylase was improved to give an 85% yield of active enzyme in the second aqueous phase and a 17-fold concentration of the enzyme. The surfactant losses were reduced to 2.5% per circulation of the reversed micellar phase. The model predicts that further improvement of the efficiency of the extraction should be possible by using modified extraction techniques, e.g., by reducing the residence time during the extractions in combination with a further increase of the mass transfer rate. The use of centrifugal separators or extractors might be valuable in this respect. A membrane can be 2.2.4. Membrane extraction. used to immobilize an interface between two immiscible solvents. In this way a contacting area between two phases can be created without mechanical dispersion of one phase in the other. Such an approach might have advantages for systems which emulsify upon stirring. By performing a membrane extraction, it might be possible to take advantage of the selective permeability characteristics of these membranes in addition to the selective
REVERSED
MICELLES
223
solubilization characteristics of the reversed micellar phase. Use of hollow fiber modules would be ideal in such applications since it is possible to create an interface of up to lo4 m2/m3, which is comparable to the specific surface area in a mechanical mixer. For protein extraction, the pores of the membrane should be large enough to allow the passage of the protein or the protein-filled reverse micelle. Some preliminary data on reversed micellar extraction of enzymes were obtained by using hollow fiber modules with a microporous polypropylene membrane (17,25). As shown schematically in Fig. 10, the mass transfer rate across the membrane will be determined by three parameters: the resistances of the membrane and of the boundary layers on the aqueous and reversed micellar sides of the membrane. Dahuron and Cussler (25) performed a membrane extraction of a-chymotrypsin and cytochrome c from an aqueous phase to a reversed micellar phase of AOT in isooctane. The observed mass transfer rate coefficients could be interpreted in terms of the three resistances mentioned before. Dekker et al. (17) studied the extraction of cu-amylase to a reversed micellar phase of TOMAC/Rewopal HV5 in isooctane. The mass transfer rate constant of the extraction of the enzyme was found to depend on the aqueous phase flow rate and also on the pressure difference over the membrane. The preliminary results indicate that the mass transfer rate is determined by the resistance of the boundary layer in the aqueous phase and/ or of the aqueous phase displacing the reverse micellar phase inside the membrane. An interesting opportunity of the use of membranes is the performance of a supported liquid membrane extraction. In this type of operation the pores of the membrane, only, are filled with the reversed micellar phase;
FIG. 10. Schematic representation of the membrane extraction with the theoretical concentration profiles of the enzyme during extraction (k., k,, and k,: mass transfer coefficients in aqueous, membrane, and reversed micellar phase, respectively) (from Ref. (17)).
224
DEKKER,
HILHORST,
the enzyme is extracted from the aqueous feed phase on one side of the membrane, while the reextraction into a second aqueous phase takes place at the other side. In this way both the forward and the back extractions can be performed in one membrane module. Luisi et al. (720) did perform a kind of liquid membrane extraction; in their experiments the reversed micellar phase was interposed between two aqueous phases, resulting in a “membrane” with a thickness of a few centimeters. Transport between the two aqueous phase was found to be very slow, and the system required several days to reach equilibrium. This is due, no doubt, to the slow diffusion of protein through the reversed micellar phase, The same technique is used by Armstrong and Li (26), who reduced the thickness of the reversed micellar phase barrier to a few millimeters. They observed transfer of some proteins, but only some 5% of protein was transferred, after which transport stopped. 2.2.5. Mass transfer kinetics. For the scaleup of reversed micellar extractions it is important to know which factors determine the mass transfer rate of proteins to or from a reversed micellar phase. The mechanism of protein transport between two immiscible phases can be divided into three steps: the diffusion of a protein in aqueous solution to or from the interface, the formation or breakup of a protein containing micelle at the interface, and the diffusion of a protein containing micelle in the organic phase to or from the interface. The overall mass transfer rate during the extraction will depend on which of these steps is rate limiting. By using both mixer/settler (dispersion) and stirred cell (constant interfacial area) extraction experiments, Dekker et al. have measured the mass transfer rate coefficients for a-amylase to or from a reversed micellar phase of the cationic TOMAC in isooctane (36). From the dependence of the mass transfer rate on the stirrer speed it was concluded that the forward transfer of the enzyme was controlled by diffusion in the aqueous phase. In the back transfer process, however, the mass transfer rate coefficient was found to be independent of the stirrer speed in the stirred cell, indicating that the rate-limiting step is not the diffusion in either phase. For this transfer the interfacial process of releasing the enzyme from the reversed micelle was found to be rate limiting. This conclusion is supported by the strong effect of the aqueous phase pH on the back transfer rate (Fig. 11). Since the pH is not expected to have a large effect on the diffusion rate of an enzyme or an enzyme-containing reversed micelle it seems most likely that the primary effect of pH is associated with the release of the enzyme from the reversed micelle at the interface. The pH will determine the interaction of charged groups on the protein with the surfactant layer, which may affect the coalescence frequency of the reversed micellar droplets with the interface.
AND
LAANE
4
4.5
5
5.5
6
pHw2 FIG. 11. amylase function
Mass transfer rate coefficients of the hack transfer of QIfrom a reversed micellar phase of TOMAC in isooctane as a of aqueous phase pH as measured in a stirred cell (from Ref.
(36)).
By decreasing the pH during the back transfer, the enzyme will have less interaction with the surfactant layer, resulting in the observed increase in the mass transfer rate. 3. EXTRACTION SOLID STATE
OF PROTEINS
FROM
THE
Proteins can be incorporated in reversed micelles by injecting a protein solution into a reversed micellar solution, by liquid-liquid extraction, and by extraction of proteins from the solid state. In the first and third cases the reversed micellar medium is present, as such, without being in equilibrium with an aqueous phase. Thus, it is possible to adjust the water content of the system at will, thereby enabling the use of w. as selection criterion for protein solubilization. Waks et al. (27,28) reported that the solubilization of two membrane proteins, the Foch-Pi protein and myelin basic protein in AOT-reversed micelles is maximal at w. = 5.6. At this water content hardly any free water is present. The amount of protein solubilized decreases to a steady-state value at higher w. values and depends on the concentration of surfactant present. A few hundred molecules of AOT are necessary to solubilize one molecule of myelin basic protein (28). Leser et al. (29) reported that the solubilization of lysozyme was maximal at w. = 10, decreased steeply with increasing wo, and increased again at w. = 50. a-Chymotrypsin and pepsin showed a different behavior: the amount solubilized in AOT-reversed micelles increased with the water content. To explain the results they suggested that two different mechanisms play a role: at low water content interactions between protein and surfac-
ISOLATING
ENZYMES
BY TABLE
Isolation of Intracellular
Enzyme
Conditions
Isocitrate dehydrogenase
CFE wg= 5 wg= 15 w. = 25
225 49 41 21
262 172 296 83
P-Hydroxybutyrate dehydrogenase
CFE w()= 5 wg = 15 w,, = 25
225 49 41 21
Glucose-6phosphate dehydrogenase
CFE wg= 5 wg= 15 w. = 25
225 49 41 21
CFE
is cell free extract.
Taken
225
MICELLES
1
Enzymes from Intact Cells of Azotobacter uinelnndii Using Reversed Micelles Total protein bd
Note.
REVERSED
from
Total activity (m units)
Specific activity W mg-‘)
Recovery (protein) (%I
Recovery (activity) (%I
Purification factor (Xl
1.2 3.5 7.2 4.0
100 23 18 9
100 65 113 31
1 2.8 6.2 3.4
30 25 33 21
0.13 0.15 0.80 1.0
100 23 18 9
100 85 110 69
1 3.7 6.1 7.6
36 0 0 0
0.16 0 0 0
100 23 18 9
100 0 0 0
1 0 0 0
Ref. (31).
tant determine solubilization and at high water content the enzyme is solubilized in the interior of the water pool. The study was extended to protein solubilization from a crude extract of Escherichia coli and from vegetable meal (30,37). Preliminary results indicated that w. can be used as a selection criterion for protein uptake. At w. = 8.5 only proteins with a molecular weight less than 20,000 were solubilized, whereas at w. = 30 proteins with molecular weights up to 60,000 were taken up. It appears from data available at present that the factors that determine protein extraction from the solid state into reversed micelles are the same as those for liquid-liquid extraction. For extraction into reversed micelles, the ionization state of the protein, i.e., the ionization state in the solution from which the protein was precipitated or lyophilized from, seems to be important (M. E. Leser, personal communication), but too few data are available to evaluate this effect.
pH 8.0 (32). The results of some extractions are shown in Table 1. It can be seen that the amount of protein extracted into the reversed micelles decreases with increasing wo, whereas the recovery of isocitrate dehydrogenase and fi-hydroxybutyrate dehydrogenase activity was optimal at w. = 15. For those enzymes, over 100% of activity (compared to the cell-free extract) could be extracted into the aqueous phase, with a concomitant sixfold purification. For glucose-g-phosphate dehydrogenase, a protein of M, 200,000, activity could be detected neither in the reversed micellar medium nor in the aqueous phase after extraction. This seemsto be due to a size exclusion effect, since analysis of the aqueous phase after extraction by SDS electrophoresis revealed that no proteins of a molecular weight above 80,000 were present. This observation agrees well with the data of Leser et al. (30,37). These results show that reversed micelles can be used for the extraction of intracellular proteins. This seems contradictory to reports by Luisi et al. (33,34) that viable bacterial cells can be solubilized in reversed micelles. 4. EXTRACTION OF INTRACELLULAR ENZYMES However, in these experiments, the nonionic surfactant For protein purification from bacterial cells, disrupTween was used. This surfactant is less likely to disrupt tion of the cells generally is the first step. Recently a bacterial cell membranes than the charged surfactants procedure in which cells are lysed by injection into a soCTAB and AOT (35). Another possible explanation lution containing reversed micelles of cetyltrimethylammight be that the bacterial species tested differ in their monium bromide (CTAB) (31,32) was reported. Upon sensitivity to lysis. injection, a turbid solution which clarified within a minute was obtained. Enzymes liberated by cell lysis were solubilized in the waterpools of reversed micelles, where 5. CONCLUSIONS their activity could be measured. By addition of an exThe recent developments in reversed micellar methcess aqueous solution, back transfer into an aqueous odology clearly demonstrate that reversed micellar mephase could be achieved. The composition of the extracdia have potential in biotechnology as a separation tool tion buffer appears to be very important. The reported for enzymes. Both intra- and extracellular proteins can optimal composition is 0.75 M KBr in 50 mM Tris/HCl, be extracted from various sources and at the same time
226
DEKKER,
HILHORST,
purified and concentrated to some extent by relatively simple means, using processes which are easy to scaleup. Since it has been recognized that protein solubilization by reversed micelles and their transport from one phase to another is governed by the same principles and insight is being gained into the properties both of the system and of the proteins that determine the extent to which a protein can be solubilized in the micellar water pool, improvements have been made to optimize the performance of the various extraction systems with respect to selectivity and efficiency. Further work is necessary to learn whether reversed micellar methodology can cornpete with current downstream processes in the area of protein purification. ACKNOWLEDGMENT The
authors
thank
Dr. C. Oldfield
for editorial
assistance.
REFERENCES 1. Luisi, P. L. (1985) Angew. 2. Martinek, K., and Semenov, 126.
Chem. Znt. Ed. Engl. 24,439-460. A. N. (1981) J. Appl. Biochem. 3,93-
3. Hilhorst, R., Laane, C., and Veeger, C. (1983) FEBS 225-228. 4. Laane, C., Hilhorst, R., and Veeger, C. (1987) in Methods mology (Mosbach, K., Ed.), Vol. 136, pp. 216-229, Press, San Diego, CA. 5. Fletcher,
P. D. I., and Robinson,
B. (1985)
J.
Lett. 169, in EnzyAcademic
Chem. Sot. Faraday
Trans.1 8 1,2667-2679. 6. Luisi, natti,
P. L., Henninger, F., Joppich, M., Dossena, A., and CasG. (1977) Biochem. Biophys. Res. Commun. 74,1384-1389.
7. Luisi, P. L., Bonner, (1979) Helv. Chim.
F. J., Pellegrini,
A., Wiget,
P., and Wolf,
R.
Acta 62,740-753.
8. Goklen, 74.
K. E., and Hatton,
T. A. (1985)
9. Goklen, Munich,
K. E., and Hatton, T. A. (1986) Vol. III, pp. 587-595.
12. Woll,
J. M.,
and Hatton,
T. A. (1988)
14. Dekker, Laane, B33.
LAANE M. (1986) in Surfactants Symp. on Surfactants
15.
Laane, C., and Dekker, ceedings, VIIIth Int. Delhi, Aug. 22-28,1986.
16’
Dekker, M., Baltussen, J. W. A., Van ‘t Riet, K., Bijsterbosch, B. H., Laane, C., and Hilhorst, R. (1987) in Biocatalysis in Organic Media (Laane, C., Tramper, J., and Lilly, M. D., Eds.), pp. 285288, Elsevier, Amsterdam.
17. Dekker, M., Van ‘t Riet, K., Wijnans, J. W. A., Bijsterbosch, B. H., and Laane, ICOM, Tokyo, pp. 793-794.
18. 19 ’
Dekker, M., Van ‘t Riet, and Hilhorst, R. (1989)
K., Bijsterbosch,
B. H., Wolbert,
R. B. G.,
AZChE J., in press.
20. Meier, P., Imre, E., Fleschar, M., and Luisi, P. L. (1984) in Surfactants in Solution (Mittal, K. L., and Lindman, B., Eds.), vol. 2, pp. 999-1012, Plenum, New York. 21. Wolbert, R. B. G., Hilhorst, R., Voskuilen, G., Nachtegaal, H., Dekker, M., Van ‘t Riet, K., and Bijsterbosch, B. H. (1989) submitted for publication. 22. Fletcher, P. D. I. and Parrott, D. (1988) J. Chem. Sot. Faraday Trans. 84(4), 1131-1144. 23. Maestro, M., and Luisi, Proceedings VIIIth Int. Delhi, Aug. 22-28, 1986.
P. L. (1986) in Surfactants in Solution, Symp. on Surfactants in Solution, New
24. Fraaije,
J. G. E. M. (1987) Ph.D. Wageningen, The Netherlands.
25. Dahuron, 26. Armstrong, 27. Delahodde,
L. and Cussler,
thesis,
E. L. (1988)
Agricultural
University
AZChE J. 34,130-136.
D. W. and Li, W. (1988) Anal. Chem. 60,86-88. A., Vacher, M., Nicot, C. and Waks, M. (1984)
FEBS
zktt. 172.343-347. 28. Nicot, (1985) 29. Leser,
C., Vacher,
M.,
Vincent,
M.,
Gallay,
J., and
Waks,
M.
Biochemistry 24,7024-7032. M. E., Wei,
G., Luisi,
P. L., and Maestro,
Bio-
M. (1986)
&em. Biophys. Res. Commun. 135,629-635.
in Proceedings,
31. Giovenco,
Submitted
ProNew
Dekker, M., Van ‘t Riet, K., Baltussen, J. W. A., Bijsterbosch, B. H., Hilhorst, R., and Laane, C. (1987) in Proceedings 4th Eur. Congr. Biotechnol. Amsterdam, Vol. II, pp. 507-510.
30. Leser, M. E., Wei, G., Liithi, P., Haering, Blochliger, E., and Luisi, P. L. (1987) J. 1118.
ISEC86,
in Solution, in Solution,
J. M. G. M., Baltussen, C. (1987) in Proceedings
Biotechnol. Progr. 1,69-
S., Verheggen,
F., and Laane,
G., Hochkoeppler,
A.,
Chim. Phys. 84, 1113-
C. (1987)
Enzyme Microb.
Technol. 9,470-473.
10. Goklen, K. E., and Hatton, T. A. (1987) Sep. Sci. Technol. 22, 831-841. 11. Woll, J. M., Dillon, A. S., Rahaman, R. S., and Hatton, T. A. (1987) in Protein Purification: Micro to Macro (Burgess, R., Ed.), pp. 117-130, A. R. Liss, New York. 13. Van ‘t Riet, K., and Dekker, Congr. Biotechnol, Munich,
AND
for publication.
M. (1984) in Proceedings Vol. III, pp. 541-544.
M., Van ‘t Riet, K., Weijers, S. R., Baltussen, C., and Bijsterbosch, B. H. (1986) Chum. Eng.
3rd Eur. J. W. A.,
J. 33, B27-
32. Giovenco,
S., Laane, C., and Hilhorst, R. (1987) in Proceedings 4th Eur. Congr. Biotechnol., Vol. 2, pp. 503-506, Elsevier, Amsterdam.
33. Haering,
G., Luisi,
F. (1985)
Biochem.
F., Hochkoeppler,
S., and
P. L., and Meussdoerffer,
Biophys. Res. Commun. 127,911-915. 34. Haering, G., Pessina, A., Meussdoerffer, Luisi, P. L. (1987) Ann. N. Y. Acad.
Sci. 506,334-337. Trends Biotechnol. 3,153-161.
35. Luisi, P. L., and Laane, C. (1986) M., et al. (1989) Manuscript in preparation. 36. Dekker, 37. Leser, M. E., Luisi, P. L., and Palmieri, S. (1988) publication.
submitted
for
ANALYTICALBIOCHEMISTRY
(1989)
REVIEW Isolating Enzymes by Reversed Micelles Matthijs Dekker,*p’ Riet Hilhorst,?
and Colja LaaneS
*Food and Bioengineering Group, Department of Food Science, Agricultural University, P.O. Box 8129,670O EV Wageningen, The Netherlands; TDepartment of Bioch.emistry, Agricultural University, P.O. Box 8128,670O ET Wageningen, The Netherlands; and $Unilever Research Lab, P.O. Box 114,313O AC Vluardingen, The Netherlands 1.
INTRODUCTION
Reversed micelles are aggregates of surfactant molecules containing an inner core of water molecules, dispersed in a continuous organic solvent medium. These systems are optically transparent and thermodynamically stable. The considerable biotechnological potential of these systems is derived principally from the ability of the water droplets to dissolve enzymes, without loss of activity, in much the same way as does bulk water. The use of these systems in the biotransformation of apolar compounds, present in the bulk oil phase, has been described in a number of publications (l-5). Recently, it has been found that enzymes can be transported from one bulk aqueous phase to another via an intermediate reversed micellar phase, the transported protein molecules being transiently accommodated within the reversed micelles in this case. This phenomenon is a consequence of the reversibility of the phase transfer process. In addition to transport from a homogeneous solution phase, proteins may also be transported from a solid phase, and even from the cytoplasmic compartment of bacteria. The latter phenomenon is based on the prior disruption of the bacterial cell wall by surfactant partitioning out of the reversed micellar phase. Here, recent advances in separating enzymes by the use of reversed micelles are highlighted. The use of these media to convert apolar compounds enzymatically has been reviewed elsewhere (35). 2. RECOVERY SOLUTION
OF ENZYMES
FROM
AQUEOUS
The isolation of specific extracellular enzymes from a fermentation broth by conventional processes consists of the stepwise removal of undesired compounds from the broth. Because these steps are usually not specific 1 To whom
correspondence
should
oooa-2697/&39 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form
be addressed.
Inc. reserved.
for the desired enzymes, several steps are necessary to obtain an enzyme preparation of the required purity. Consequently, new isolation techniques, more selective for the required enzyme and easier to scale up, are desirable. Liquid-liquid extraction procedures involving the use of reversed micellar systems have some very promising features in this respect. The principle of the method is depicted schematically in Fig. 1. First, the enzyme is extracted from the aqueous phase to the reversed micellar phase under conditions such that the extent of phase transfer of the protein is maximal. Second, the enzyme is recovered from the organic phase, by extraction of the reversed micellar phase with a second aqueous phase, under such conditions that the transfer of the enzyme from the reversed micellar phase to the aqueous phase is maximal. By appropriate manipulation of physical parameters such as solution pH and ionic strength, which determine the distribution behavior of the enzyme, it is possible to obtain purification and concentration of the required enzyme in a straightforward process. The first reports on the transfer of proteins from an aqueous phase to a reversed micellar phase and vice versa were those of Luisi et al. (6,7), who illustrated this principle using a range of proteins. These investigations have been extended in more recent studies by groups at MIT (B-12) and in Wageningen (13-19) for the development of a recovery process for proteins from aqueous solutions. In the first part of this section the factors affecting the distribution of proteins between an aqueous phase and a conjugate reversed micellar phase are discussed. In the second part the development and performance of continuous extraction processes are described. 2.1. The Distribution of Proteins There is a large body of evidence which indicates that electrostatic interactions play a very important role in determining the distribution coefficient of a protein be217
218
DEKKER,
FIG. 1. Schematic representation enzymes between an aqueous phase enzyme).
HILHORST,
of the liquid-liquid extraction and a reversed micellar phase
of (E,
tween a reversed micellar phase and a conjugate aqueous phase. This influence is demonstrated clearly by the effect of aqueous phase pH, ionic strength, and surfactant type (anionic, cationic, or nonionic) on this distribution behavior. The individual effects exerted by each of these parameters are discussed separately. 2.1.1. Aqueous phase PH. The aqueous phase pH determines the ionization state of the surface-charged groups on the protein molecule. Attractive electrostatic interactions between the protein molecule and the surfactant head groups, which form the internal surface of the reversed micelle, will occur if the overall charge of the protein is opposite to the charge of the surfactant head groups. This implies that, for cationic surfactants, solubilization of the protein in reversed micelles is favored at pH values above the isoelectric point (~1) of the protein, while the opposite is true for anionic surfactants. Luisi et al. (7) showed that the aqueous phase pH influences the transfer of cY-chymotrypsin and pepsin to a reversed micellar phase of the cationic surfactant trioctylmethylammonium chloride (TOMAC)2 in cyclohexane. These results, however, could not be interpreted on basis of the pl values of these proteins. The phase transfer of the enzyme cY-amylase into reversed micelles of TOMAC/octanol in isooctane was found to recur only over a narrow pH range, well above the pl of the enzyme (13). These results indicate that not only the sign of the protein charge but also the surface charge density is an important factor in protein solubilization. Using a reversed micellar system consisting of the anionic surfactant sodium di-2-ethylhexyl sulfosuccinate
AND LAANE
(commercial name Aerosol OT or AOT) in isooctane, Goklen and Hatton (10) found almost complete solubilization of the three low-molecular-weight proteins (A& 12,000-14,000), ribonuclease A, cytochrome c, and lysozyme. This solubilization took place in a range of 4 to 6 pH units just below the PI’S of the three proteins (Fig. 2). As the molecular weight of the protein increases, phase transfer can be accomplished only by increasing the value of (pH-~1). Thus, in the case of a-chymotrypsin (AI, 25,000) phase transfer was favored in a pH range approximately 2 to 4 pH units below the p1 of the protein; bovine serum albumin (AI, 68,000) was not transferred at all in the same system. The effect of pH on solubilization for proteins of different molecular weight might be explained by taking into account the fact that for larger proteins the size of the reversed micelle containing a protein molecule must be significantly larger than the size of the empty reversed micelle. This energetically unfavorable transition of the micellar size must be compensated for by more extensive electrostatic interactions in order to make the overall solubilization process feasible. This can be achieved by increasing the charge density of the protein by manipulation of the pH. For small proteins whose size is smaller than the size of the water pool inside a reversed micelle, solubilization occurs as soon as the net protein charge is opposite to that of the reversed micellar interface. This behavior is demonstrated clearly (21) by the linear correlation attained between the A!, of a protein and the difference between the pH of solubilization and the p1 of the protein (Fig. 3). The correlation holds for reversed micelles of both anionic and cationic surfactants. 2.1.2. Ionic strength. The ionic strength of the aqueous phase determines the degree of shielding of the electrostatic potential imposed by a charged surface. This phenomenon causes at least two important effects in reversed micellar extraction. First, it decreases the
103
20
6
8
10
12
14
PH ’ Abbreviations AOT, di-2-ethylhexyl nium bromide.
used: TOMAC, trioctylmethylammonium sulfosuccinate; CTAB, cetyltrimethyl
chloride; ammo-
FIG. 2. The effect of pH on the solubilization of (0) lysozyme, (0) cytochrome c, and (A) ribonuclease A in AOT-isooctane solutions (from Ref. (10)).
ISOLATING
ENZYMES
BY
REVERSED
219
MICELLES
6C
0
2o.m
40.000
6o.m
ao.ooo
M,
FIG. 3. Relation between the molecular weight of a protein and the difference between the protein’s pl and the pH of optimal extraction into a reversed micellar phase of TOMAC in isooctane (from Ref.
(21)). electrostatic interaction between the charged protein molecule and the charged interface in the reversed micelle. Second, it reduces the electrostatic repulsion between the surfactant head groups, resulting in a decrease in the size of the reversed micelles at higher ionic strength. Gijklen and Hatton (9,lO) showed the effect of ionic strength on the phase transfer of ribonuclease A, cytochrome c, and lysozyme in a reversed micellar phase of AOT in isooctane (9,lO). The extent of phase transfer to the reversed micellar phase decreases for all three proteins with increasing potassium chloride concentration, but the concentration required to initiate this decrease was found to be different for each protein (Fig. 4). No rules with respect to the ionization state of the proteins used (expressed as the difference between the aqueous phase pH (6.7-7.1 in this study) and the p1 of the proteins) can be deduced from these data. Meier et al. (20) observed an increase in the amount of trypsin and peroxidase transferred to a reversed micellar
100
t
OL 0
02
0.4
0.6
KCI ccnc
0.6
1
[Ml
FIG. 4. The effect of the potassium chloride concentration on the solubilization of (Cl) lysozyme, (0) cytochrome c, and (A) ribonuclease A in AOT-isooctane solutions (from Ref. (10)).
10.5
11
PH FIG. 5. Effect of pH on the transfer of cY-amylase to a reversed micellar phase of TOMAC in isooctane at different ionic strengths (from Ref. (18)). (0) 0 mM NaCl, (0) 20 mM NaCl, and (A) 50 mM NaCl.
phase of AOT in isooctane when using sodium salts to increase the ionic strength but a decrease when using calcium salts. These effects were observed at both pH 7 and 10. The fact that an increase in the ionic strength results in a shift in the pH profile of the distribution behavior was demonstrated by Dekker et al. (18). The transfer of the enzyme a-amylase to a reversed micellar phase of TOMAC/octanol in isooctane was studied for different concentrations of sodium chloride as a function of the pH (Fig. 5). As the salt concentration increased, a higher pH (resulting in a higher charge density on the protein) was required for maximal phase transfer. Thus once again, electrostatic interactions are implicated as being of primary importance in the phase transfer mechanism. For the results shown, it can be seen that increasing the ionic strength at pH 10.1, for example, first causes an increase in solubilization of cu-amylase in the reversed micellar phase followed by a decrease in solubilization beyond an optimal ionic strength. The observation that not only the ionic strength, but also the type of ion, influences the distribution behavior of proteins implies specific interaction of ions with the protein and/or the surfactant head groups. The differences in the distribution behavior of proteins on the pH and ionic strength of the aqueous phase have been used to advantage in the separation of a protein mixture (10). An aqueous solution of ribonuclease A, cytochrome c, and lysozyme was extracted with a reversed micellar phase of AOT in isooctane. By using the results from Figs. 2 and 4 the three proteins could be separated by a series of three extractions. Woll et al. (11) showed that the aqueous phase pH and ionic strength could be used to selectively extract an alkaline protease from a fermentation broth. By perform-
220
DEKKER,
I
0 9
9.5
10
HILHORST,
I 11
10.5
PH FIG. 6.
Solubilization of a-amylase in a reversed micellar phase of TOMAC in isooctane in relation to the pH in the aqueous phase. Effect of the addition of nonionic surfactant to the reversed micellar phase (from Ref. (16)). (Cl) -Rewopal Hv5, (m) +Rewopal Hv5.
ing two series of extractions 42% of the enzyme was recovered with an 2.2-fold increase in specific activity. 2.1.3 Surfactant type. As shown in the previous two sections, the distribution of proteins is dependent primarily on the difference in charge between the protein and the charged surfactant head groups. Therefore, in the absence of other effects, an opposite pH dependence of the distribution behavior can be expected for reversed micelles stabilized by cationic and anionic surfactants. In addition to the surfactant charge, other surfactantdependent parameters may be cited. Among these are the size of the reversed micelles that are formed, the energy required to enlarge the reversed micelles, and the charge density on the inner surface of the reversed micelle. Dekker et al. (16) showed the effect of the addition of a nonionic surfactant (Rewopal HV5: nonylphenolpentaethoxylate) to a reversed micellar phase of the cationic surfactant TOMAC in isooctane. The partitioning of cyamylase between the reversed micellar and the conjugate aqueous phase was found to be a strong function of the ratio of nonionic to ionic surfactant. An increase in the proportion of nonionic surfactant resulted in increased transfer of the enzyme to the reversed micellar phase. Additionally, phase transfer took place over a wider range of pH (Fig. 6). This change in distribution behavior might be explained by two effects: first, the charge density of the micellar interface will be influenced by the presence of the nonionic surfactant. Second, the size and flexibility of the reversed micelles will be changed by the addition of the one-tail nonionic surfactant. Until now we have discussed only the use of electrostatic interactions between the protein and the surfac-
AND
LAANE
tant head groups to promote transfer to a reversed micellar phase. Another possibility is the use of biospecific interactions between an enzyme and a ligand (substrate analog, product, or an inhibitor). In principle, the ligand can be confined to the reversed micellar phase by conjugation to a suitable hydrophobic tail; in the reversed micelles, such a (polar) ligand would form a site-specific surfactant head group. The effect of such an affinity surfactant, octyl-/3-D-glucopyranoside, has been shown for the solubilization of concanavalin A in AOT reversed micelles (11). The protein was transferred to the reversed micellar phase at pH values at which no transfer was observed without the affinity surfactant. This transfer was inhibited by the addition of free ligand in the aqueous phase, indicating that an affinity interaction was required for the solubilization at these pH values. In this system the solubilization is most likely due to a combination of electrostatic, affinity, and possibly hydrophobic interactions. It would be interesting to explore this phenomenon further by substituting a nonionic surfactant for the ionic surfactants used previously. In this case electrostatic interactions between the protein and the surfactant head groups are eliminated and affinity interactions, perhaps in combination with hydrophobic interactions, become decisive in determining the extent of phase transfer. This should result in a very selective extraction process. 2.1.4. Surfactant concentration. The concentration of surfactant in a reversed micellar phase that is in equilibrium with an aqueous phase has little effect on the size and structure of the reversed micelles. The extent of protein uptake from a conjugate aqueous phase therefore increases in proportion to the surfactant concentration in the reversed micellar phase (12,22). Solubilization of ribonuclease A and concanavalin A as a function of the AOT concentration could be described by a model based on a thermodynamic equilibrium between the concentration of protein-reversed micelle complexes and the concentration of free reversed micelles and free protein in the aqueous phase (12). The distribution of a-chymotrypsin and pepsin in AOT-reversed micelles has been described (22) by expressing the protein partitioning based on the protein concentration in the water pools of the reversed micelles divided by the bulk aqueous phase concentration. This approach resulted in a partition coefficient which was independent of the surfactant concentration. Since the total volume of the water pools varies linearly with the surfactant concentration (w~,~~~,the molar ratio of water to surfactant in the micellar phase is approximately independent of the concentration of surfactant), the two descriptions are essentially the same. In this context, the partitioning behavior of the enzyme lysozyme proved to be exceptional (22); below a certain surfactant concentration the enzyme precipi-
ISOLATING
ENZYMES
BY
REVERSED
MICELLES
221
distribution of charged groups on the surface of proteins on their distribution behavior. Proteins with a more asymmetric charge distribution were found to partition more easily into the reversed micellar phase.
tated at the interface between the two phases. Yet increasing the surfactant concentration above this point resulted in the transfer of all of the protein to the reversed micellar phase. They estimated this concentration to be just sufficient to provide a monolayer coverage for the total enzyme in the system.
2.2. Process Development
2.1.5. Thermodynamic modeling. Several models of the distribution behavior of proteins between a bulk aqueous phase and a reversed micellar phase have been developed (11,23,24). They are all based on electrostatic interactions between the charges on the protein molecule and the charges on the inner surface of the reversed micelle due to the ionic head groups of the surfactants. In addition to free energy change resulting directly from this interaction, an additional contribution arising from the redistribution of surfactant and protein counterions, of other free ions, of water, and of surfactant on phase transfer must be considered. Calculations have been made of the total free energy change on the uptake of a hypothetical small protein in reversed micelles (23); no comparisons were made with experimentally determined distribution data. In a more phenomenological approach, the distribution behavior of cytochrome c between an aqueous phase and a reversed micellar phase of the cationic surfactant TOMAC in isooctane was analyzed with respect to the aqueous phase pH and ionic strength (24). From the experimental data, calculations were made on the copartitioning of protons and other small ions accompanying protein uptake in reversed micelles. Around the optimal pH for protein transfer to the reversed micellar phase, both protons and surfactant counterions (chloride) were found to be redistributed. A third approach (11) described the partitioning of ribonuclease A and concanavalin A as a function of aqueous phase pH and surfactant concentration. Four coefficients which depend on the reversed micellar system and the protein used were employed. All of the above models are useful in analyzing experimental data of protein partitioning and in indicating the most important system parameters affecting the value of the distribution coefficient. At present, however, the models cannot give accurate predictions of the distribution coefficient of a protein; many parameters are unknown or difficult to quantify. These include hydrophobic interactions of the protein with the apolar phase, specific interactions of ions with the protein and surfactant, the free energy changes associated with the change in size of the reversed micelles on protein uptake, and the distribution of charged groups on the protein molecules. This last parameter has been neglected in all three approaches; they all assume an equal distribution of surface charge on a globular protein molecule. Wolbert et al. (21) showed a strong effect of the symmetry of the
For the large-scale recovery of extracellular enzymes, an extraction with an organic solvent containing reversed micelles has interesting advantages over existing processes. A reversed micellar extraction combines the potential for concentration and purification of the enzyme in a single process. The liquid-liquid extraction technique in general is well known and apparatus and scale-up rules have been established for numerous applications, including the use in recovery processes for antibiotics and organic acids from fermentation broths. The success of the type of extraction processes described here is critically dependent on the ability to direct the distribution coefficient of the desired enzyme between the aqueous phase and the reversed micellar phase, as discussed under Section 2.1. 2.2.1. Forward and back extractions. Enzyme recovery from an aqueous phase by liquid-liquid extraction using reversed micelles consists of a first step whereby the desired enzyme is transferred selectively from the aqueous phase to the reversed micellar phase (forward extraction). The enzyme is subsequently recovered from the reversed micellar phase by extraction with a second aqueous phase (back extraction). These two extractions can be performed in a continuous mode, with the reversed micellar phase circulating between the two extraction units. No specialized equipment is required, that developed for conventional liquidliquid extraction processes is, in principle, also suitable for this application. The most important types of equipment are mixer/settler, agitated column, centrifugal, and membrane extractors. For some reversed micellar systems the use of mixer/settlers and agitated columns might be limited by emulsion formation between the aqueous phase and the reversed micellar phase because of the presence of surfactants that stabilize those emulsions. In such cases centrifugal extraction may be applied to reduce the settling time. Additionally, membrane extraction techniques may be adapted for use with these types of systems, the membrane serving to stabilize the reversed micellar/aqueous phase interface. For systems where short residence times are required centrifugal extractors might be very useful. At the present time process development has centered exclusively on the use of mixer/settlers (14-16,18,19) and membrane extractors (17,25). 2.2.2. Mixer/settler extraction. The performance of a continuous forward and back extraction for the recovery of an enzyme was demonstrated (14) with two mixer/settler units, one for each extraction step. The re-
222
DEKKER,
,
reversed
micellar
phase
HILHORST, ,
+ 3,--m
-b--4
mixer1
wl,in
mixer2 FORWARD
BACK
EXTRACTION
EXTRACTION
Wl.out
w2, I”
W2,out
FIG. ‘7. Flow sheet of the combined forward and back extraction for two mixer/settler units, with the reversed micellar phase circulating between the two extraction units (from Ref. (14)).
versed micellar phase was circulated between the two units. The flow sheet for this extraction is given in Fig. 7. This design was used to transfer cr-amylase from an aqueous phase to a reversed micellar phase of TOMAC in isooctane during the forward extraction and to transfer it to a second aqueous phase during the back extraction. The distribution coefficient of the enzyme was manipulated by adjusting the pH and the ionic strength of the aqueous phases. Forward extraction was favored by a high pH and low ionic strength; back extraction was favored by a low pH and high ionic strength. The value of this distribution coefficient (reversed micellar concentration divided by the aqueous phase concentration of the enzyme) was determined to be at least 10 during the forward extraction and at most 0.004 during the back extraction. In this way, an eightfold concentration of the enzyme a-amylase was obtained, without optimization of the process. The enzyme recovered in the second aqueous phase accounted for 45% of the initial enzymatic activity. Approximately 30% inactivation of the enzyme was observed during extraction. Surfactant was found to be slowly desorbed from the reversed micellar phase, resulting in a decreased extraction efficiency after 3.5 circulations of the reversed micellar phase, an effect that could be fully restored by the addition of more surfactant to the reversed micellar phase. 2.2.3. Modeling and optimization. The reversed micellar extraction process can be described in terms of the distribution coefficients, mass transfer rate coefficient, inactivation rate constants, phase ratios, and residence times during the forward and back extractions (18). For all phases, differential equations have been derived for the concentration of active enzyme in that phase as a function of time. When the individual param-
AND
LAANE
eters on distribution, mass transfer rate, and inactivation rate are known, these equations can be solved numerically to yield the concentrations as a function of extraction time (18,19). For the extraction of the enzyme a-amylase, with a reversed micellar phase of TOMAC in isooctane, the various variables which describe the extraction process have been determined (19). Determination of the inactivation rate constants in this system showed that the inactivation took place predominantly by complexation of the enzyme with surfactant in the first aqueous phase (l&19). Although the equilibrium concentration of TOMAC in the aqueous phase is very low, it is continuously extracted from the reversed micellar phase during forward extraction, resulting in the observed surfactant loss from the reversed micellar phase. In order to minimize the extent of enzyme inactivation the steady-state concentration of enzyme in the first aqueous phase should be as low as possible. This can be achieved by a high mass transfer rate constant and a high distribution coefficient of the enzyme between the reversed micellar and the aqueous phases. In Fig. 8 simulations of the forward and back extractions of a-amylase are given using the experimentally determined values. The effect of the mass transfer rate coefficient during forward extraction on the recovery of active enzyme is shown for two values of the distribution coefficient (19). These model predictions were verified experimentally by changing the distribution coefficient (by the addition of a nonionic surfactant to the reversed micellar phase, see Section 2.1.3.) and the mass transfer rate coefficient of the enzyme during the forward extraction (by increas-
FIG. 8. Simulation of the effect of the mass transfer rate coefficient on the cu-amylase activity recovery by a reversed micellar extraction. The activity in the second aqueous phase is relative to the initial activity in the first aqueous phase. 1, mt = 10; 2, m, = 100. The flow ratio of the two aqueous phases is 20 (from Ref. (19)).
ISOLATING
0
100
200
ENZYMES
BY
400 300 TIME [mini
FIG. 9. Activity recovery of cY-amylase in the second aqueous phase of the combined forward and back extraction. The activity in the second aqueous phase is relative to the initial activity in the first aqueous phase (from Ref. (19)). A (0) Data at low distribution coefficient and low mass transfer rate (dip caused by TOMAC loss from reversed micellar phase). B (m) Data at high distribution coefficient and low mass transfer rate. C (A) Data at high distribution coefficient and high mass transfer rate. (- - -) The model prediction by using the independently found values for the distribution, mass transfer, and inactivation coefficients. (-) Line fitted through steady-state activities.
ing the stirrer speed). The experimental results of this optimization of the mixer/settler extraction thus obtained are shown in Fig. 9 together with the model prediction of the extractions. In this way the performance of the reversed micellar extraction of a-amylase was improved to give an 85% yield of active enzyme in the second aqueous phase and a 17-fold concentration of the enzyme. The surfactant losses were reduced to 2.5% per circulation of the reversed micellar phase. The model predicts that further improvement of the efficiency of the extraction should be possible by using modified extraction techniques, e.g., by reducing the residence time during the extractions in combination with a further increase of the mass transfer rate. The use of centrifugal separators or extractors might be valuable in this respect. A membrane can be 2.2.4. Membrane extraction. used to immobilize an interface between two immiscible solvents. In this way a contacting area between two phases can be created without mechanical dispersion of one phase in the other. Such an approach might have advantages for systems which emulsify upon stirring. By performing a membrane extraction, it might be possible to take advantage of the selective permeability characteristics of these membranes in addition to the selective
REVERSED
MICELLES
223
solubilization characteristics of the reversed micellar phase. Use of hollow fiber modules would be ideal in such applications since it is possible to create an interface of up to lo4 m2/m3, which is comparable to the specific surface area in a mechanical mixer. For protein extraction, the pores of the membrane should be large enough to allow the passage of the protein or the protein-filled reverse micelle. Some preliminary data on reversed micellar extraction of enzymes were obtained by using hollow fiber modules with a microporous polypropylene membrane (17,25). As shown schematically in Fig. 10, the mass transfer rate across the membrane will be determined by three parameters: the resistances of the membrane and of the boundary layers on the aqueous and reversed micellar sides of the membrane. Dahuron and Cussler (25) performed a membrane extraction of a-chymotrypsin and cytochrome c from an aqueous phase to a reversed micellar phase of AOT in isooctane. The observed mass transfer rate coefficients could be interpreted in terms of the three resistances mentioned before. Dekker et al. (17) studied the extraction of cu-amylase to a reversed micellar phase of TOMAC/Rewopal HV5 in isooctane. The mass transfer rate constant of the extraction of the enzyme was found to depend on the aqueous phase flow rate and also on the pressure difference over the membrane. The preliminary results indicate that the mass transfer rate is determined by the resistance of the boundary layer in the aqueous phase and/ or of the aqueous phase displacing the reverse micellar phase inside the membrane. An interesting opportunity of the use of membranes is the performance of a supported liquid membrane extraction. In this type of operation the pores of the membrane, only, are filled with the reversed micellar phase;
FIG. 10. Schematic representation of the membrane extraction with the theoretical concentration profiles of the enzyme during extraction (k., k,, and k,: mass transfer coefficients in aqueous, membrane, and reversed micellar phase, respectively) (from Ref. (17)).
224
DEKKER,
HILHORST,
the enzyme is extracted from the aqueous feed phase on one side of the membrane, while the reextraction into a second aqueous phase takes place at the other side. In this way both the forward and the back extractions can be performed in one membrane module. Luisi et al. (720) did perform a kind of liquid membrane extraction; in their experiments the reversed micellar phase was interposed between two aqueous phases, resulting in a “membrane” with a thickness of a few centimeters. Transport between the two aqueous phase was found to be very slow, and the system required several days to reach equilibrium. This is due, no doubt, to the slow diffusion of protein through the reversed micellar phase, The same technique is used by Armstrong and Li (26), who reduced the thickness of the reversed micellar phase barrier to a few millimeters. They observed transfer of some proteins, but only some 5% of protein was transferred, after which transport stopped. 2.2.5. Mass transfer kinetics. For the scaleup of reversed micellar extractions it is important to know which factors determine the mass transfer rate of proteins to or from a reversed micellar phase. The mechanism of protein transport between two immiscible phases can be divided into three steps: the diffusion of a protein in aqueous solution to or from the interface, the formation or breakup of a protein containing micelle at the interface, and the diffusion of a protein containing micelle in the organic phase to or from the interface. The overall mass transfer rate during the extraction will depend on which of these steps is rate limiting. By using both mixer/settler (dispersion) and stirred cell (constant interfacial area) extraction experiments, Dekker et al. have measured the mass transfer rate coefficients for a-amylase to or from a reversed micellar phase of the cationic TOMAC in isooctane (36). From the dependence of the mass transfer rate on the stirrer speed it was concluded that the forward transfer of the enzyme was controlled by diffusion in the aqueous phase. In the back transfer process, however, the mass transfer rate coefficient was found to be independent of the stirrer speed in the stirred cell, indicating that the rate-limiting step is not the diffusion in either phase. For this transfer the interfacial process of releasing the enzyme from the reversed micelle was found to be rate limiting. This conclusion is supported by the strong effect of the aqueous phase pH on the back transfer rate (Fig. 11). Since the pH is not expected to have a large effect on the diffusion rate of an enzyme or an enzyme-containing reversed micelle it seems most likely that the primary effect of pH is associated with the release of the enzyme from the reversed micelle at the interface. The pH will determine the interaction of charged groups on the protein with the surfactant layer, which may affect the coalescence frequency of the reversed micellar droplets with the interface.
AND
LAANE
4
4.5
5
5.5
6
pHw2 FIG. 11. amylase function
Mass transfer rate coefficients of the hack transfer of QIfrom a reversed micellar phase of TOMAC in isooctane as a of aqueous phase pH as measured in a stirred cell (from Ref.
(36)).
By decreasing the pH during the back transfer, the enzyme will have less interaction with the surfactant layer, resulting in the observed increase in the mass transfer rate. 3. EXTRACTION SOLID STATE
OF PROTEINS
FROM
THE
Proteins can be incorporated in reversed micelles by injecting a protein solution into a reversed micellar solution, by liquid-liquid extraction, and by extraction of proteins from the solid state. In the first and third cases the reversed micellar medium is present, as such, without being in equilibrium with an aqueous phase. Thus, it is possible to adjust the water content of the system at will, thereby enabling the use of w. as selection criterion for protein solubilization. Waks et al. (27,28) reported that the solubilization of two membrane proteins, the Foch-Pi protein and myelin basic protein in AOT-reversed micelles is maximal at w. = 5.6. At this water content hardly any free water is present. The amount of protein solubilized decreases to a steady-state value at higher w. values and depends on the concentration of surfactant present. A few hundred molecules of AOT are necessary to solubilize one molecule of myelin basic protein (28). Leser et al. (29) reported that the solubilization of lysozyme was maximal at w. = 10, decreased steeply with increasing wo, and increased again at w. = 50. a-Chymotrypsin and pepsin showed a different behavior: the amount solubilized in AOT-reversed micelles increased with the water content. To explain the results they suggested that two different mechanisms play a role: at low water content interactions between protein and surfac-
ISOLATING
ENZYMES
BY TABLE
Isolation of Intracellular
Enzyme
Conditions
Isocitrate dehydrogenase
CFE wg= 5 wg= 15 w. = 25
225 49 41 21
262 172 296 83
P-Hydroxybutyrate dehydrogenase
CFE w()= 5 wg = 15 w,, = 25
225 49 41 21
Glucose-6phosphate dehydrogenase
CFE wg= 5 wg= 15 w. = 25
225 49 41 21
CFE
is cell free extract.
Taken
225
MICELLES
1
Enzymes from Intact Cells of Azotobacter uinelnndii Using Reversed Micelles Total protein bd
Note.
REVERSED
from
Total activity (m units)
Specific activity W mg-‘)
Recovery (protein) (%I
Recovery (activity) (%I
Purification factor (Xl
1.2 3.5 7.2 4.0
100 23 18 9
100 65 113 31
1 2.8 6.2 3.4
30 25 33 21
0.13 0.15 0.80 1.0
100 23 18 9
100 85 110 69
1 3.7 6.1 7.6
36 0 0 0
0.16 0 0 0
100 23 18 9
100 0 0 0
1 0 0 0
Ref. (31).
tant determine solubilization and at high water content the enzyme is solubilized in the interior of the water pool. The study was extended to protein solubilization from a crude extract of Escherichia coli and from vegetable meal (30,37). Preliminary results indicated that w. can be used as a selection criterion for protein uptake. At w. = 8.5 only proteins with a molecular weight less than 20,000 were solubilized, whereas at w. = 30 proteins with molecular weights up to 60,000 were taken up. It appears from data available at present that the factors that determine protein extraction from the solid state into reversed micelles are the same as those for liquid-liquid extraction. For extraction into reversed micelles, the ionization state of the protein, i.e., the ionization state in the solution from which the protein was precipitated or lyophilized from, seems to be important (M. E. Leser, personal communication), but too few data are available to evaluate this effect.
pH 8.0 (32). The results of some extractions are shown in Table 1. It can be seen that the amount of protein extracted into the reversed micelles decreases with increasing wo, whereas the recovery of isocitrate dehydrogenase and fi-hydroxybutyrate dehydrogenase activity was optimal at w. = 15. For those enzymes, over 100% of activity (compared to the cell-free extract) could be extracted into the aqueous phase, with a concomitant sixfold purification. For glucose-g-phosphate dehydrogenase, a protein of M, 200,000, activity could be detected neither in the reversed micellar medium nor in the aqueous phase after extraction. This seemsto be due to a size exclusion effect, since analysis of the aqueous phase after extraction by SDS electrophoresis revealed that no proteins of a molecular weight above 80,000 were present. This observation agrees well with the data of Leser et al. (30,37). These results show that reversed micelles can be used for the extraction of intracellular proteins. This seems contradictory to reports by Luisi et al. (33,34) that viable bacterial cells can be solubilized in reversed micelles. 4. EXTRACTION OF INTRACELLULAR ENZYMES However, in these experiments, the nonionic surfactant For protein purification from bacterial cells, disrupTween was used. This surfactant is less likely to disrupt tion of the cells generally is the first step. Recently a bacterial cell membranes than the charged surfactants procedure in which cells are lysed by injection into a soCTAB and AOT (35). Another possible explanation lution containing reversed micelles of cetyltrimethylammight be that the bacterial species tested differ in their monium bromide (CTAB) (31,32) was reported. Upon sensitivity to lysis. injection, a turbid solution which clarified within a minute was obtained. Enzymes liberated by cell lysis were solubilized in the waterpools of reversed micelles, where 5. CONCLUSIONS their activity could be measured. By addition of an exThe recent developments in reversed micellar methcess aqueous solution, back transfer into an aqueous odology clearly demonstrate that reversed micellar mephase could be achieved. The composition of the extracdia have potential in biotechnology as a separation tool tion buffer appears to be very important. The reported for enzymes. Both intra- and extracellular proteins can optimal composition is 0.75 M KBr in 50 mM Tris/HCl, be extracted from various sources and at the same time
226
DEKKER,
HILHORST,
purified and concentrated to some extent by relatively simple means, using processes which are easy to scaleup. Since it has been recognized that protein solubilization by reversed micelles and their transport from one phase to another is governed by the same principles and insight is being gained into the properties both of the system and of the proteins that determine the extent to which a protein can be solubilized in the micellar water pool, improvements have been made to optimize the performance of the various extraction systems with respect to selectivity and efficiency. Further work is necessary to learn whether reversed micellar methodology can cornpete with current downstream processes in the area of protein purification. ACKNOWLEDGMENT The
authors
thank
Dr. C. Oldfield
for editorial
assistance.
REFERENCES 1. Luisi, P. L. (1985) Angew. 2. Martinek, K., and Semenov, 126.
Chem. Znt. Ed. Engl. 24,439-460. A. N. (1981) J. Appl. Biochem. 3,93-
3. Hilhorst, R., Laane, C., and Veeger, C. (1983) FEBS 225-228. 4. Laane, C., Hilhorst, R., and Veeger, C. (1987) in Methods mology (Mosbach, K., Ed.), Vol. 136, pp. 216-229, Press, San Diego, CA. 5. Fletcher,
P. D. I., and Robinson,
B. (1985)
J.
Lett. 169, in EnzyAcademic
Chem. Sot. Faraday
Trans.1 8 1,2667-2679. 6. Luisi, natti,
P. L., Henninger, F., Joppich, M., Dossena, A., and CasG. (1977) Biochem. Biophys. Res. Commun. 74,1384-1389.
7. Luisi, P. L., Bonner, (1979) Helv. Chim.
F. J., Pellegrini,
A., Wiget,
P., and Wolf,
R.
Acta 62,740-753.
8. Goklen, 74.
K. E., and Hatton,
T. A. (1985)
9. Goklen, Munich,
K. E., and Hatton, T. A. (1986) Vol. III, pp. 587-595.
12. Woll,
J. M.,
and Hatton,
T. A. (1988)
14. Dekker, Laane, B33.
LAANE M. (1986) in Surfactants Symp. on Surfactants
15.
Laane, C., and Dekker, ceedings, VIIIth Int. Delhi, Aug. 22-28,1986.
16’
Dekker, M., Baltussen, J. W. A., Van ‘t Riet, K., Bijsterbosch, B. H., Laane, C., and Hilhorst, R. (1987) in Biocatalysis in Organic Media (Laane, C., Tramper, J., and Lilly, M. D., Eds.), pp. 285288, Elsevier, Amsterdam.
17. Dekker, M., Van ‘t Riet, K., Wijnans, J. W. A., Bijsterbosch, B. H., and Laane, ICOM, Tokyo, pp. 793-794.
18. 19 ’
Dekker, M., Van ‘t Riet, and Hilhorst, R. (1989)
K., Bijsterbosch,
B. H., Wolbert,
R. B. G.,
AZChE J., in press.
20. Meier, P., Imre, E., Fleschar, M., and Luisi, P. L. (1984) in Surfactants in Solution (Mittal, K. L., and Lindman, B., Eds.), vol. 2, pp. 999-1012, Plenum, New York. 21. Wolbert, R. B. G., Hilhorst, R., Voskuilen, G., Nachtegaal, H., Dekker, M., Van ‘t Riet, K., and Bijsterbosch, B. H. (1989) submitted for publication. 22. Fletcher, P. D. I. and Parrott, D. (1988) J. Chem. Sot. Faraday Trans. 84(4), 1131-1144. 23. Maestro, M., and Luisi, Proceedings VIIIth Int. Delhi, Aug. 22-28, 1986.
P. L. (1986) in Surfactants in Solution, Symp. on Surfactants in Solution, New
24. Fraaije,
J. G. E. M. (1987) Ph.D. Wageningen, The Netherlands.
25. Dahuron, 26. Armstrong, 27. Delahodde,
L. and Cussler,
thesis,
E. L. (1988)
Agricultural
University
AZChE J. 34,130-136.
D. W. and Li, W. (1988) Anal. Chem. 60,86-88. A., Vacher, M., Nicot, C. and Waks, M. (1984)
FEBS
zktt. 172.343-347. 28. Nicot, (1985) 29. Leser,
C., Vacher,
M.,
Vincent,
M.,
Gallay,
J., and
Waks,
M.
Biochemistry 24,7024-7032. M. E., Wei,
G., Luisi,
P. L., and Maestro,
Bio-
M. (1986)
&em. Biophys. Res. Commun. 135,629-635.
in Proceedings,
31. Giovenco,
Submitted
ProNew
Dekker, M., Van ‘t Riet, K., Baltussen, J. W. A., Bijsterbosch, B. H., Hilhorst, R., and Laane, C. (1987) in Proceedings 4th Eur. Congr. Biotechnol. Amsterdam, Vol. II, pp. 507-510.
30. Leser, M. E., Wei, G., Liithi, P., Haering, Blochliger, E., and Luisi, P. L. (1987) J. 1118.
ISEC86,
in Solution, in Solution,
J. M. G. M., Baltussen, C. (1987) in Proceedings
Biotechnol. Progr. 1,69-
S., Verheggen,
F., and Laane,
G., Hochkoeppler,
A.,
Chim. Phys. 84, 1113-
C. (1987)
Enzyme Microb.
Technol. 9,470-473.
10. Goklen, K. E., and Hatton, T. A. (1987) Sep. Sci. Technol. 22, 831-841. 11. Woll, J. M., Dillon, A. S., Rahaman, R. S., and Hatton, T. A. (1987) in Protein Purification: Micro to Macro (Burgess, R., Ed.), pp. 117-130, A. R. Liss, New York. 13. Van ‘t Riet, K., and Dekker, Congr. Biotechnol, Munich,
AND
for publication.
M. (1984) in Proceedings Vol. III, pp. 541-544.
M., Van ‘t Riet, K., Weijers, S. R., Baltussen, C., and Bijsterbosch, B. H. (1986) Chum. Eng.
3rd Eur. J. W. A.,
J. 33, B27-
32. Giovenco,
S., Laane, C., and Hilhorst, R. (1987) in Proceedings 4th Eur. Congr. Biotechnol., Vol. 2, pp. 503-506, Elsevier, Amsterdam.
33. Haering,
G., Luisi,
F. (1985)
Biochem.
F., Hochkoeppler,
S., and
P. L., and Meussdoerffer,
Biophys. Res. Commun. 127,911-915. 34. Haering, G., Pessina, A., Meussdoerffer, Luisi, P. L. (1987) Ann. N. Y. Acad.
Sci. 506,334-337. Trends Biotechnol. 3,153-161.
35. Luisi, P. L., and Laane, C. (1986) M., et al. (1989) Manuscript in preparation. 36. Dekker, 37. Leser, M. E., Luisi, P. L., and Palmieri, S. (1988) publication.
submitted
for