Improved back extraction of papain from AOT reverse micelles using alcohols and a counter-ionic surfactant

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1 Biochemical Engineering Journal 25 (2005) Improved back extraction of papain from AOT reverse micelles using alcohols and a counter-ionic surfactant Daliya S. Mathew, Ruey-Shin Juang Department of Chemical Engineering and Materials Science, Yuan Ze University, Chung-Li 320, Taiwan Received 19 October 2004; received in revised form 12 April 2005; accepted 24 May 2005 Abstract The extraction of papain (MW 23 kda, pi 9.6) from water was studied using reverse micelles containing sodium bis(2- ethylhexyl)sulfosuccinate (AOT). Forward extraction of papain was carried out in the ph range of and a maximum extraction of 65 70% was observed at ph 6.3. Papain extracted into the micelles could be back extracted to only 30% into a conventional aqueous phase (high ph and high KCl concentrations). In order to improve the back extraction of papain, 8 10 vol% alcohols were added because it was known that alcohols have an effect on the micelle micelle interactions. However, this way resulted in the loss of papain activity inspite of good back extraction yields (80 90%). The back extraction of papain encapsulated in AOT reverse micelles was then carried out by adding a counter-ionic surfactant tri-n-octylmethyl-ammonium chloride (TOMAC). This technique resulted in faster back extraction compared to conventional method. Around 80 90% of activity recovery was obtained after back extraction. The improved backward transfer was likely caused by electrostatic interaction between the oppositely charged surfactant molecules, which led to the collapse of reverse micelles Elsevier B.V. All rights reserved. Keywords: Reverse micelle; Back extraction; AOT; Papain; Activity recovery; TOMAC 1. Introduction In recent years, many techniques have been developed in biotechnology to achieve a highly efficient and economical separation process. One novel separation technique with the ability to be scaled up easily, to be operated continuously and to be highly selective is liquid liquid extraction using microemulsions [1,2]. Aggregates of surfactant molecules are spontaneously generated in organic solvents as a result of molecular self-assembly [3,4]. These aggregates can solubilize water in their polar cores giving rise to water-in-oil microemulsions, commonly referred to as reverse micelles [5]. Several studies have shown that proteins can be solubilized within these reverse micelles in active form [6 8]. This method is more suitable for separating proteins than regular liquid liquid extraction or other separation methods that were used in the past because the transfer of proteins into Corresponding author. Tel.: x2555; fax: address: rsjuang@ce.yzu.edu.tw (R.-S. Juang). solvents often results in irreversible denaturation and loss of biological activity. In order to employ reverse micelles for protein separations, the micelles should exhibit two characteristic features. Firstly, they should be capable of solubilizing proteins selectively. This protein uptake into the reverse micelles is referred to as forward extraction process. Second, it should be possible to release the protein from the reverse micelles so that a quantitative recovery of purified protein can be achieved. This is referred to as the back extraction process. Extensive studies have been done on reverse micellar extraction of proteins using an anionic surfactant, bis(2- ethylhexyl)sulfosuccinate (AOT) [9 11]. The distribution of proteins between the micellar phase and an aqueous phase is largely determined by the environments of bulk aqueous phase, i.e., ph, ionic strength and type of salt. Parameters related to the organic phase also affect the partition of protein, such as the concentration and type of surfactant, presence of co-surfactant and type of solvent [9]. By controlling these parameters, the extracted fraction can be varied via variations of protein micelle electrostatic, X/$ see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.bej

2 220 D.S. Mathew, R.-S. Juang / Biochemical Engineering Journal 25 (2005) hydrophobic and steric interactions. Among these, the electrostatic interaction was considered as the main driving force especially in forward extraction process [10]. In contrast to a large number of studies examining forward transfer, release and recovery of proteins from the reverse micelles have received little attention. Most of the existing studies tacitly assume that the conditions, which normally prevent protein uptake in forward transfer experiments, would promote their release in back transfer experiments. However, in most reported attempts to release the solubilized proteins from the reverse micelles, low yields were obtained [11 13], which explains that in the reverse micellar extractions, particularly the back extraction steps, the micelle micelle interaction has to be considered one of the important factors. The strategy of recovery improvement could be considered in two aspects. One is dealing with surfactant organic phase by concentration, species of surfactant and type of organic solution. The other is dealing with the stripping aqueous phase by ph, concentration, species of salts and adding various alcohols [14 16]. The use of counterionic surfactants, such as tri-n-octylmethyl-ammonium chloride (TOMAC) and dodecyltrimethylammomium bromide (DTAB) for improving back extraction yield of some proteins has been actually reported [17]. In the present work, we show that papain cannot be completely back extracted from the AOT/isooctane system under normal conditions, i.e., in the presence of mol/dm 3 KCl or at ph above the pi of papain. Back extraction of papain was thus examined in the presence of various alcohols (8 10%) or a counter-ionic surfactant TOMAC (7 8%). The effect of various alcohols on reverse micelle system was studied using the percolation process [18], which can quantify the micelle micelle interaction easily by measuring the electrical conductivity of the reverse micellar system. A sharp increase in the electrical conductivity caused by percolation phenomenon well demonstrates the interaction between the micelles [19]. Although the addition of 8 10% alcohol could bring about complete back extraction of papain, its activity was lost to a considerable extent. It will be shown that the addition of TOMAC to the reverse micellar system during back extraction gave both good back extraction and activity recovery. 2. Materials and methods 2.1. Enzyme and solutions Papain from papaya latex (EC ) (MW 23 kda, pi 9.6) was obtained from Sigma Co. The surfactant sodium bis(2-ethylhexyl)sulfosuccinate was purchased from Sigma Aldrich Co. and was used without further purification. Isooctane, TOMAC, N -benzoyl-l-arginine ethyl ester, cystein HCl and 2-mercaptoethanol were all purchased from Sigma Aldrich Co. For all studies, 0.01 mol/dm 3 phosphate buffer solutions were employed. All alcohols (ethanol, n- propanol, isopropyl alcohol (IPA) and n-hexanol) used in this work were of analytical grade Measurement of the content and activity of papain The content of extracted papain was determined by measuring the absorbance of the papain-loaded aqueous phase at 280 nm on a UV visible spectrophotometer (Jasco V-550, Japan). The extraction yield was calculated according to the standard curve. Activity of papain was assayed by titrimetric determination of the acid produced during the hydrolysis of benzoyl-larginine ethyl ester [20]. One unit of papain will hydrolyze 1 mol of benzoyl-l-arginine ethyl ester per minute at 25 C and ph 6.2 under the specified conditions Measurement of water content The water content of reverse micellar phase was determined by Karl Fischer titration using a volumetric titrator (Mettler Toledo DL-38). The chemical reagents used were CombiTitrant 5 (one-component reagent) and combisolvent (methanol-free solvent with one-component reagent) purchased from Merck Co. (Germany). A 1.0 cm 3 solution was injected using a syringe and the coefficient of variation was ±5% Measurement of electrical conductivity The conductivity of the reverse micellar system was measured as a function of water content with a conductivity meter (Horiba D-24, Japan). The electrode was inserted into a beaker placed in a water bath (25 C). Electrical conductivity measurements were performed with dropwise addition of an aqueous phase containing papain to the AOT/isooctane or AOT/alcohol/isooctane phase until the percolation behavior was reached. The ratio of the volume of water to the overall volume of the mixture was considered as the aqueous volume fraction φ aq. The percolation thresholds with alcohol and protein and without them, which are defined as the starting point of the sharp increase in conductivity, are abbreviated as φ h, φ p and φ 0, respectively. The values of φ h, φ p and φ 0 were determined by extrapolation for finding an intersecting point between constant line and increasing line of the curve Forward extraction of papain The forward extraction experiments were carried out using an aqueous phase containing 1.0 g/dm 3 of papain in a 25 cm 3 stoppered conical flask at room temperature. Equal volumes of the organic phase (AOT/isooctane) and aqueous phase, which consisted of a fixed KCl concentration and ph value, were mixed and stirred for 30 min. Phase separation was aided by centrifugation at 2000 rpm for 10 min. The upper organic phase was gathered for following back extraction.

3 The concentration of papain solubilized into the organic phase was assayed by measuring the absorbance of lower aqueous phase at 280 nm. In order to prevent the interference of other species during UV measurements, the sample analyses were performed against appropriate blank solutions, which were prepared simultaneously with the protein sample. The forward extraction yield was calculated Back extraction of papain The organic phase loaded with papain from the forward extraction was added to an equivalent volume of a new aqueous phase, which contained a fixed KCl concentration and ph value as well as 8 10% (v/v) alcohol or TOMAC (7 8%). The mixture was stirred for 30 min and then centrifuged for 10 min at 2000 rpm. The papain content was measured with the same method as in the forward extraction studies. The activity of papain in the lower aqueous phase was determined. The back extraction and activity recovery of papain were calculated as follows: backward extraction (%) = D.S. Mathew, R.-S. Juang / Biochemical Engineering Journal 25 (2005) backward extracted papain in the aqueous phase 100; (1) forward extracted papain in the reverse micelles activity of recovered papain (units/mg) activity recovery (%) = activity of feed papain (units/mg) It is noted here that each experiment was at least duplicated under identical conditions. The reproducibility of the measurement was within 6% (mostly within 3%). 3. Results and discussion 3.1. Forward extraction The effect of aqueous ph on papain solubilization into the reverse micellar phase is shown in Fig. 1. Around 65% of papain can be extracted into the reverse micelles at ph < pi because under these conditions, papain is positively charged and is attracted by the AOT head-groups. In general, proteins are only transferred to the reverse micelles at ph at which their net charge is opposite to that of surfactant head-groups because solubilization is usually steered by electrostatic interactions between protein molecule and the surfactant headgroup [9]. This is evident from Fig. 1, where at ph 12, the solubilization drastically decreases to 1% because protein becomes negatively charged at this ph and hence results in electrostatic repulsion from the negatively charged surfactant head-groups. Another factor affecting protein solubilization is the ionic strength. Forward extraction was carried out at ph 6.3 using an aqueous phase consisting of different KCl concentrations. The ionic strength effect on papain solubilized into the reverse micelles is shown in Fig. 2. The experiment reveals that the solubilization of papain is more significant when KCl concentration is higher. This is because at higher ionic strength, the interactions between hydrophilic protein and the surfactant polar head-groups are reduced, smaller micelles being formed [14] (2) Fig. 3 shows the effect of AOT concentration on forward extraction of papain. Experiments were performed at different ph values. It is observed that whatever the ph value of aqueous phase, the solubilization of papain reaches maximum at an AOT concentration of 0.01 mol/dm 3 ; a further increase in AOT concentration will lead to a decrease in the solubilization. The maximum possible forward extraction is near 65%. This is because at surfactant concentrations above a certain value, the micellar interactions occur leading to percolation and interfacial deformation with a change in the micellar shape and micellar clustering [21]. Fig. 1. Effect of aqueous ph on the forward extraction of papain. Fig. 2. Effect of salt concentration in the aqueous phase on the forward extraction of papain.

4 222 D.S. Mathew, R.-S. Juang / Biochemical Engineering Journal 25 (2005) Fig. 3. Effect of AOT concentration on the forward extraction of papain. The variation of electrical conductivity of the reverse micellar solution against the volume fraction of water in the organic phase, φ aq, is shown in Fig. 4. It can also be stated that at high surfactant concentration, monodisperse spherical micelles might not be present predominantly in the solution. This figure shows that, for the reverse micellar system with solubilized papain, the percolation threshold (φ p ) is decreased in the lower value of φ aq compared to papainfree system. These results suggest that the micelle micelle interaction is significantly affected by the protein species and its concentration solubilized into the reverse micelles. Papain would interact with AOT surfactant layer and the protein micelle interaction (electrostatic attraction) seems to decrease the stability of reverse micellar system by decreasing electrostatic repulsion between the micelles. The formation of micellar clusters shows a large hydrophobic attraction than electrostatic repulsive force between the micelles. The micellar clustering thus decreases the interfacial area available to host the biomolecules causing a decrease in the solubilization capacity of the reverse micelles. Fig. 4. Effect of AOT concentration on the percolation process in the presence and absence of papain. Fig. 5. Effect of aqueous ph on the back extraction and activity recovery of papain. Moreover, a phase volume ratio (V org /V aq ) of one and a stirring time of min is sufficient to bring about maximum forward extraction of papain by the AOT reverse micelles Factors affecting the back extraction of papain Back extraction of papain was carried out using a fresh aqueous phase with 0.5 mol/dm 3 of KCl at different ph values. Fig. 5 shows that the back extraction reaches maximum at ph above pi, i.e., 9.6. But the maximum possible back extraction is only 30% with an activity recovery of 80 85%. Further improvement in the extraction is not observed with increasing KCl concentration or ph. It can thus be stated that a strong interaction between the solubilized protein and micelles induces the micelle micelle interaction or micellar cluster formation, resulting in a decrease of back-extracted fractions. Hence, the control of micelle micelle interaction may become a very important factor for the success of back extraction of the proteins. Alcohol is considered to be a good modifying agent for the reverse micelles because alcohol molecules have amphiphilic property as a co-surfactant [18]. The effect of various alcohols on the reverse micellar system using the percolation phenomenon has been reported [18]. The percolation processes clearly quantify the micelle micelle interactions by measuring electrical conductivity of the reverse micellar system as a function of the volume fraction of water. Fig. 6 shows the effect of added alcohols on back extraction yield and activity of papain. The short-chain alcohols, such as ethanol and n-propanol, give slightly lower back extraction (70% at 10%, v/v, alcohol) compared to the branched-chain IPA and long-chain hexanol (90% at 10%, v/v, alcohol). The back extraction yield in all cases increases with increasing volume percentage of the alcohols. Thus, the back extraction of papain in the presence of alcohols considerably increases as compared to 30% of back extraction using the conventional method as described above. In order to understand the role of added alcohols in such back extraction process, electrical conductivities of the

5 D.S. Mathew, R.-S. Juang / Biochemical Engineering Journal 25 (2005) Fig. 6. Effect of added alcohols on the back extraction of papain. Fig. 8. Effect of added alcohols on the activity recovery of papain. reverse micellar systems were measured. A sharp increase in the conductivity demonstrates the interaction between micelles. The cluster formation of the micelles increases the conductivity over the percolation threshold, which can indicate the starting point of the reverse micellar droplet clustering. The variation of the electrical conductivity as a function of the volume fraction of aqueous phase φ aq solubilized into the AOT reverse micelles in the presence of alcohols is shown in Fig. 7. For the reverse micellar system, the volume fraction φ 0, corresponding to the threshold of electrical percolation, varies with the types of alcohols. When alcohol is added into the reverse micellar system, the electrical percolation threshold (φ h ) increases in contrast to the alcohol-free reverse micellar system. These results indicate a decrease of attractive interaction between the micelles when alcohol is added into the reverse micellar system and hence prove a decrease of micelle micelle interaction. Under the conditions studied, the highest back extraction yield is obtained using IPA. This is also supported from the percolation behavior as Fig. 7. Variation of the percolation process of reverse micellar systems with and without alcohols. the micelle micelle interaction is the smallest in the presence of IPA compared to other alcohols. Although good back extraction is obtained using alcohols in the reverse micellar system, the activity of papain is lost to a considerable extent (Fig. 8). The activity is maintained only when the concentration of added alcohol is low enough. However, at least 8 10% alcohol is required to achieve a reasonably good back extraction, but in this case the activity is decreased drastically. The activity recovery is 30 40% using IPA or hexanol (10%, v/v) in the back extraction process, as against 5 10% in the case of short-chain alcohols. A probable explanation can be the location of alcohols when they are added into the reverse micellar system. It has been reported that the short-chain alcohols are trapped in the water pool of reverse micelles resulting in direct contact with proteins whereas the branched- and long-chain alcohols are dispersed in the surrounding solvent and probably incorporated into the micellar shell [22]. This behavior for alcohols can explain the improved activity recovery of papain in the presence of branched- and long-chain alcohols. In order to increase the activity recovery of papain after back extraction, another technique using a counter-ionic surfactant TOMAC was tested. This method can yield higher back extraction yields compared to the conventional methods. Such back transfer mechanism is postulated to be caused by electrostatic interaction between oppositely charged surfactant molecules, which lead to the collapse of the reverse micelle [17]. Fig. 9 shows the effect of TOMAC concentration on backward extraction of papain at ph > pi and <pi. The highest yield of 90% is obtained at ph at a TOMAC concentration of 0.01 mol/dm 3.AtpH>pI, the back extraction decreases with increasing TOMAC concentration beyond 0.02 mol/dm 3. This is probably due to the occurrence of some precipitation at the interface. On the other hand, at ph < pi, only 30 40% of back extraction is possible. One reason for enhanced back extraction of papain at ph > pi is the electrostatic repulsion effect due to the negative charges on protein

6 224 D.S. Mathew, R.-S. Juang / Biochemical Engineering Journal 25 (2005) Fig. 9. Effect of TOMAC concentration on the back extraction of papain. at this ph. Since there is only 30 40% of back extraction at ph<pi besides electrostatic repulsion effect, there are other factors which contribute to the back extraction because otherwise there must not be any back extraction at ph < pi as in the conventional methods. One probable factor, which may be contributing to the above phenomenon, is the location of protein with respect to the ph of water pool. When the ph of water pool is changed, the protein previously located in the water pool can migrate at the interface [23]. Further studies need to be done in order to confirm this subject Backward transfer mechanism The mechanism of backward transfer was studied by examining the changes of water content in the organic phase (W 0 ) when various concentrations of TOMAC were added. It is found from Fig. 10 that there is a decrease in water content with increasing TOMAC concentration and it almost becomes constant at higher TOMAC concentrations. Fig. 10. Variation of water content (W 0 ) of the AOT reversed micelles after back extraction of papain. Fig. 11. Effect of TOMAC concentration on the activity recovery of papain. This behavior may be attributed to the constant collision of reverse micelles, which leads to a temporary fusion of two reverse micelles. Consequently, TOMAC may interfere with the dynamics of AOT reverse micelles by preventing them from reforming after collisions, causing the water content to drop. In principle, only a few TOMAC molecules could cause such breakage. At higher TOMAC concentrations, an increased number of non-charged and hydrophobic AOT TOMAC complexes may compete with TOMAC interaction with the reverse micelles and cause a reduction in the water content. The activity recovery of papain was also examined with respect to TOMAC concentration and ph (Fig. 11). With increasing TOMAC concentration, there is an increase in the activity recovery and the activity is always greater than 80% above and below the pi. Hence, the use of TOMAC brings about an activity recovery to 80 90%. Taking into account all the above results, it can be concluded that the electrostatic interaction between TOMAC and AOT is stronger than that between AOT and the oppositely charged groups on protein surface, thus making the release of protein much easier as compared to the conventional back extraction where the protein is tightly bound to AOT. Although back extraction using counter-ionic surfactants like TOMAC gives good yield and high protein recovery, the resulting organic phase contains ion-pairs. Though this phase contains AOT, it cannot be further reused for protein extraction. To make the present method commercially viable, the organic phase must be recycled. One way of doing this is by adsorption of ion-pairs onto suitable adsorbents. It was actually reported that montmorillonite is capable of adsorbing and removing the ion-pairs well from the solvent phase [17]. An important application of this work would be the selective extraction of papain from other components of papaya latex. Though it cannot be stated at this moment about the exact selectivity, we believe that the information available from our current work will definitely play a vital role.

7 D.S. Mathew, R.-S. Juang / Biochemical Engineering Journal 25 (2005) Conclusions AOT reverse micelles were chosen for separation of papain by a two-step extraction process. About 60 65% of papain was forward extracted (1st step) without much difficulty, but the back extraction (2nd step) posed many problems. A strong interaction between the solubilized protein and micelles induced the micelle micelle interaction or micellar cluster formation, resulting in a decrease of back-extracted yield. Various alcohols were thus tried to control such micelle micelle interactions and to improve back extraction. Under the conditions studied, isopropyl alcohol and hexanol gave the best back extraction of 80 90% but an activity recovery of only 30 40%. In addition, the percolation process could quantify and explain the effects of added alcohols on the micelle micelle interactions. The back extraction of papain encapsulated in AOT reverse micelles was further improved by adding a counter-ionic surfactant, TOMAC. This technique gave not only high back extraction of 80 90%, but also high activity recovery of 85 90%. The improved back extraction was supposed to be caused by electrostatic interactions between oppositely charged AOT and TOMAC molecules. These interactions led to a rapid collapse of reverse micelles and a subsequent decrease of water content in the organic phase, therefore, resulting in a rapid partitioning of proteins into the aqueous phase. This technique could definitely become a commercially viable process if papain could be extracted selectively from other components of papaya latex. Acknowledgement Financial support for this work by the National Science Council, Taiwan, ROC, under Grant NSC E is gratefully acknowledged. References [1] M.R. Gasco, in: C. Solans, R. Poms, H. 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