- Email: [email protected]
Forward and backward extraction of BSA using mixed reverse micellar system of CTAB and alkyl halides
Biochemical Engineering Journal 12 (2002) 1–5
Forward and backward extraction of BSA using mixed reverse micellar system of CTAB and alkyl halides Wei Zhang, Huizhou Liu∗ , Jiayong Chen Young Scientist Laboratory of Separation Science and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China Received 29 August 2000; accepted after revision 21 December 2001
Abstract The forward and backward extraction of bovine serum albumin (BSA) has been studied using mixed reverse micellar systems of cetyl trimethyl ammonium bromide (CTAB) and alkyl halides (including 1-chlorobutane (R4 Cl), 1-bromooctane (R8 Br) and 1-iodobutane (R4 I)). The addition of alkyl halide R8 Br to 50 mmol/l CTAB/20% (v/v) hexanol/petroleum ether reverse micellar system could improve the BSA transfer from the aqueous phase to reverse micellar phase, while the addition of R4 Cl or R4 I had almost no effect on the BSA transfer. The mixed reverse micelles formed with CTAB and alkyl halides exhibit excellent backward extraction behavior for BSA. The mixed reverse micelles formed with CTAB and R4 I can realize the recovery of BSA effectively in a wide range of pH up to or higher than the isoelectric point of BSA. The mixed reverse micelles formed with CTAB and R4 Cl, R8 Br or R4 I can obviously enhance the BSA backward transfer at low ionic strength with addition of KBr or KCl as electrolyte. The mixed reverse micellar system indicates that it requires less time to reach the mass transfer equilibrium in comparison with the reverse micellar system with CTAB only. The mechanism of backward extraction proposed that with the addition of alkyl halides to CTAB reverse micelles, the hydrophobic interaction between the reverse micelles decreased. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Reverse micelles; Backward extraction; Alkyl halide; BSA; CTAB
1. Introduction The separation of proteins using reverse micelles is rather easy to scale up and can be operated continuously [1]. Recently, synergism has been applied to the protein extraction by adding a secondary surfactant. Kinugasa et al. [2] have reported that the mixed reverse micelles formed by AOT (sodium bis-(2-ethylhexyl) sulfosuccinate) and DEHPA (di-2-ethylhexyl phosphoric acid) could be used to extract hemoglobin and the transfer can reach up to 80%. Goto et al. [3] reported that the mixed reverse micelles formed with AOT and DOLPA (dioleyl phosphoric acid) showed a high selectivity between the active ␣-chymotrypsin and denatured proteins. Zhang et al. [4] reported that the mixed reverse micelles formed with cetyl trimethyl ammonium bromide (CTAB) and TBP (tributyl phosphate) or TRPO (trialkyl phosphine oxide) show obvious advantage for the extraction of proteins over the reverse micelles formed with CTAB only. Spirovska and Chaudhuri [5] reported that the ∗ Corresponding author. Tel.: +86-10-6255-5005; fax: +86-10-6255-4264. E-mail address: [email protected] (H. Liu).
addition of sucrose in forward aqueous phase increased both the protein recovery and the activity of protein. Shiomori et al. [6] investigated the extraction of proteins using mixed reverse micellar systems of AOT and long chain alkyl amines, suggesting that the mixed micellar systems was a novel and functional system. It is clear that mixed reverse micelles formed by a surfactant with another surfactant or reagent exhibited some advantages over reverse micelles formed with a surfactant only for the extraction of proteins. It is well known that the backward transfer of protein from reverse micelles to the aqueous solution is relatively slow due to high interfacial resistance in mass transfer [7–11]. Conditions of high ionic strength and pH within a certain range, which would not allow the uptake of protein in forward transfer by reverse micelles, could not be used for the complete recovery of proteins solubilized in the forward extraction. A variety of alternative backward transfer methods has been studied. Ermin and Metelitsa [12] and Woll et al. [13] tested by addition of a second solvent to destabilize the reverse micelles to release the solubilized protein. Dekker et al. [14] studied by increasing the operating temperature of backward extraction to separate an excess aqueous phase
1369-703X/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 6 9 - 7 0 3 X ( 0 2 ) 0 0 0 0 6 - 2
2
W. Zhang et al. / Biochemical Engineering Journal 12 (2002) 1–5
that contained the protein in the aqueous phase of the reverse micelles during forward extraction. Phillips et al. [15] pressurized the reverse micelles with ethylene to convert the water pool to clathrate hydrates. Carlson and Nagarajan [16] added 10–50% isopropyl alcohol to facilitate the kinetics of protein release, while Leser and Luisi [17] added silica gel to adsorb the aqueous phase of reverse micelles. Recently, Gupta et al. [18] dehydrated the reverse micelles with molecular sieves. Hayes and Marchio [19] expelled proteins by adding a small portion (0.1 v/v or less) of cosurfactant (e.g., 1-alkanol) to w/o microemulsion solution, sometimes as solids, while most or all of the surfactant (AOT) remained in solution. Jarudilokkul et al. [20] obtained higher backward extraction yields by adding a counterionic surfactant at nearly neutral pH and low salt concentration. The drawback in adding alcohol or counterionic surfactant to the reverse micellar phase is recycling of the solvent for reuse. Ideally, in order to enhance the protein activity recovery during backward extraction, mild conditions such as neutral pH and low salt concentrations in the fresh aqueous phase would be preferred. Apparently, in most of the backward recovery methods studied, this is not feasible. Nevertheless, these may be possible if new mixed reverse micelles were used for extraction. In the present study, the mixed solution of CTAB and alkyl halides was used as a novel reverse micellar system for protein extraction. The forward and backward extraction of the bovine serum albumin (BSA) in the mixed reverse micelles was investigated.
2. Materials and methods BSA (molecular weight 68,000, isoelectric point 4.8) was purchased from Beijing Baitai Biochemical Reagents. CTAB (>99%, analytical grade), hexanol (>98%, chemical grade), petroleum ether (b.p.: 90–120 ◦ C, >99%, analytical grade), 1-chlorobutane (chemical grade), 1-bromooctane (>98%, chemical grade) and 1-iodobutane (chemical grade) were purchased from Beijing Chemical Reagent. Other reagents used in this work were all commercially available reagents of analytical grade and were used as received. Forward extraction procedures for BSA were conducted by mixing the petroleum ether solution containing 0.05 mol/l CTAB and 20% (v/v) hexanol with an aqueous solution in the desired pH containing 1.0 mg/ml BSA with desired amount of salt. Equal volumes (usually 5 ml each) of aqueous and organic solutions were poured gently into a 50 ml flask, which was stoppered to prevent the evaporation of the solvent. The flasks were shaken with the rate of agitation of 3.5 s−1 at room temperature (30 ± 0.5 ◦ C) for 20 min. After centrifugation (4000 rpm, 5 min) for phase separation, the protein concentration in the reverse micellar phase was determined by spectroscopy at 280 nm (UV-7542 spectrophotometer). The water concentration in the organic phase was determined by Karl–Fischer titration (WA-1A).
Backward extraction of BSA was conducted by mixing the reverse micellar solution that had extracted BSA in advance with a fresh aqueous phase of desired pH and ionic strength at room temperature (30 ± 0.5 ◦ C). Equal volumes (usually 5 ml) of aqueous and organic solutions were poured gently into a 50 ml stoppered flask. The flasks were shaken with the rate of agitation of 4.0 s−1 at room temperature (30±0.5 ◦ C) for 60 min. The mixture was then centrifuged (4000 rpm, 5 min), and the protein concentration of the stripped aqueous phase was determined by both Bradford method [21] and 280 nm spectroscopic method. The yield of the backward transfer of BSA was based on the percentage of BSA recovered in the aqueous phase relative to the amount of BSA extracted into the reverse micellar phase. The protein mass balances for a set of forward and backward extractions were generally within the experimental error of ±5%. 3. Results and discussion 3.1. Forward extraction The solubilization of water in the reverse micelles formed with CTAB and alkyl halides was examined with no protein extraction. The effect of the amount of alkyl halide addition on water content was shown in Fig. 1. The results indicated that the amount of water inside the reverse micelles did not change very much with the addition of alkyl halides, implying that there was not much of a change in the size of the reverse micelles. The effects of pH, ionic strength and the amount of alkyl halides added on BSA transfer were investigated. The addition of alkyl halide R8 Br to CTAB reverse micellar system could improve the BSA transfer from the aqueous phase to reverse micellar phase, while the addition of R4 Cl or R4 I had almost no effect on the BSA transfer. The results indicated that the formation of the mixed reverse micelles of CTAB and R4 Cl or R4 I had no obvious effect on protein
Fig. 1. Effect of concentration of alkyl halides added on the water content of CTAB reversed micelles. Aqueous phase: pH = 7.0, 0.1 mol/l KCl. Organic phase: 50 mmol/l CTAB/5% (v/v) alkyl halide/20% (v/v) hexanol/petroleum ether.
W. Zhang et al. / Biochemical Engineering Journal 12 (2002) 1–5
3
Fig. 2. Effect of the amount of alkyl halide added on BSA transfer. Aqueous phase: 1.0 mg/ml BSA, 0.1 mol/l KCl, pH = 7.0. Organic phase: 50 mmol/l CTAB with different amount of alkyl halide added/20% (v/v) hexanol/petroleum ether.
extraction, which might result from not much of change in the size of the reverse micelles. The effect of the amount of alkyl halides added to CTAB reverse micelles on the BSA transfer was shown in Fig. 2 at pH 7.0. The BSA transfer to reverse micelles formed by CTAB and R8 Br increased with the increase of the amount of R8 Br added. The BSA transfer was increased by 12% when 5% (v/v) R8 Br was added into CTAB reverse micelles. The BSA transfer increased by 4% to reverse micelles when 1.0% (v/v) R4 Cl was added into CTAB reverse micelles, but it did not increase with the further increase of the amount of R4 Cl. The BSA transfer of reverse micelles formed by CTAB and R4 I increased slightly by 2% when compared with the reversed micelles formed by CTAB only. The alkyl halides are weak Lewis alkali, while the CTAB head groups in the reverse micelles carry positive charge. Halogen atoms present in reverse micellar inner core could separate surfactant molecules such as CTAB. The addition of alkyl halides could decrease the electrostatic repulsion between the surfactant molecules and the positive region of proteins while the hydrophobic interaction between the surfactant head groups and the hydrophobic region of proteins could lead to the increase of BSA transfer. Although the electronegativity of the chlorine atom is highest, its size is very small, thus the improvement of R4 Cl addition for the BSA transfer is limited. Though the size of the iodine atom is highest, its electronegativity is the lowest, thus the improvement of R4 I addition for the BSA transfer is also very limited. The bromine atom has relatively strong electronegativity, and with large size, the improvement of R8 Br addition on the BSA forward transfer is obvious. Additionally, the alkyl chain of R8 Br is longer than that of R4 Cl or R4 I, which might influence the hydrophobicity of alkyl halides. 3.2. The backward transfer Effect of solution pH. The protein extracted into the reverse micelles can be recovered by the backward transfer
Fig. 3. Effect of backward transfer aqueous phase pH on BSA backward transfer. Forward transfer aqueous phase: 1.0 mg/ml BSA, pH = 9.10, 0.1 mol/l KCl. Organic phase: 50 mmol/l CTAB/4% (v/v) alkyl halide/20% (v/v) hexanol/petroleum ether. Backward transfer aqueous phase: 1.0 mol/l (a) KBr, (b) KCl. Backward transfer time is 60 min.
through the result of electrostatic repulsion by changing the solution pH, or through the size exclusion effect by increasing the salt concentration in the aqueous phase used for backward transfer [22]. The effect of pH of aqueous phase used for the backward transfer of the BSA was shown in Fig. 3. BSA could be recovered only under conditions with pH below pI using 1.0 mol/l KBr or KCl solution when using CTAB, CTAB–R4 Cl or CTAB–R8 Br to form reverse micelles. As shown in Fig. 3, the addition of R4 Cl or R8 Br to CTAB to form reversed micelles will lead to the recovery of BSA in a similar pH range as no alkyl halide was added. Surprisingly, BSA could be recovered with high back-transfer in a much wider pH range when using mixed reverse micelles formed by CTAB and R4 I. The result may be explained by the fact that the addition of alkyl halides to CTAB will decrease the hydrophobic interaction between the hydrophobic regions of protein and surfactant polar heads. The degree of decrease of hydrophobic interaction caused by the presence of R4 I is much higher in comparison with those caused by R4 Cl and R8 Br. BSA shows higher hydrophobicity when aqueous phase pH is closer to its isoelectric point (pI = 4.8), so the backward transfer of BSA is lower. The difference in the results obtained between using KBr and KCl (Fig. 3a and b) might be caused by the effect of anions on the protein
4
W. Zhang et al. / Biochemical Engineering Journal 12 (2002) 1–5
Fig. 4. Effect of ionic strength of aqueous phase on BSA backward transfer. Backward transfer aqueous phase: pH = 4.3, (a) KBr, (b) KCl. Other conditions are the same as given in Fig. 3.
Fig. 5. Effect of time of backward transfer on BSA backward transfer. Backward transfer aqueous phase: pH = 4.3, 1.0 mol/l (a) KBr, (b) KCl. Other conditions are the same as given in Fig. 3.
transfer, which could be explained by the competition between the anions acting as counterions of the surfactant [23]. Effect of solution ionic strength. The salt concentration in the aqueous phase can affect the efficiency of protein transfer through the size exclusion effect. The effect of ionic strength of aqueous phase in the backward extraction on BSA back-transfer was shown in Fig. 4. The mixed reverse micelles exhibit much better backward transfer behavior for BSA. Fig. 4a shows that the mixed reverse micelles formed with CTAB and by R4 Cl, R8 Br or R4 I obviously improved the BSA backward transfer at low ionic strength (0.2–1.0 mol/l KBr). For example, the BSA back-transfer of mixed reverse micelles formed with CTAB and by the addition of R4 Cl, R8 Br and R4 I at 0.4 mol/l KBr was 35, 68 and 70%, respectively. The BSA back-transfer from reverse micelles formed with CTAB only was 21% under the same conditions. Fig. 4b shows that the mixed reverse micelles formed by CTAB and R8 Br or R4 I clearly improved the BSA back-transfer at low ionic strength when KCl was used as salt, while the mixed reverse micelles formed with CTAB and R4 Cl give almost no effect on the BSA back-transfer. The mixed reverse micelles show very good protein back-transfer at low ionic strength that is very interesting, because low ionic strength of aqueous phase for backward transfer will be useful to protect protein and for recovery of enzyme activity.
Time required for backward extraction. It is well known that the rate of backward transfer of protein from reversed micelles to an aqueous phase is relatively slow. We examined the effect of the time of back-transfer on the BSA recovery as shown in Fig. 5. The mixed reverse micelles exhibited excellent backward extraction behavior for BSA. As shown in Fig. 5a, the mixed reverse micelles require less time to reach the maximum amount of protein recovery when KBr was used as salt. The improvement of CTAB–R4 I reverse micelles for BSA recovery was remarkable in comparison with those using CTAB–R4 Cl and CTAB–R8 Br reverse micelles. As shown in Fig. 5b, the mixed reverse micelles require less time to approach the mass transfer equilibrium when KCl was used as salt. The BSA back-transfer was remarkably enhanced when mixed reverse micelles was used for the same period of contact time. For example, the BSA back-transfer recovery from CTAB, CTAB–R4 Cl, CTAB–R8 Br and CTAB–R4 I reverse micelles in 10 min were 17, 46, 44 and 84%, respectively. The results indicated that the addition of alkyl halides to CTAB reverse micelles might decrease the hydrophobic interaction between the protein and the surfactant polar head groups. Mechanism of backward extraction of proteins with addition of alkyl halides. It is reasonable and widely acceptable that the backward transfer of protein from the reverse
W. Zhang et al. / Biochemical Engineering Journal 12 (2002) 1–5
micelles is governed by an interfacial process and by the coalescence of reverse micelles at oil–water interface [7]. The addition of alkyl halides to CTAB reverse micelles will decrease the hydrophobic interaction between the hydrophobic region of protein and surfactant polar heads. The order of decrease of hydrophobic interaction caused by alkyl halides is R4 I > R8 Br > R4 Cl, resulting from the results of Figs. 3–5. The CTAB head groups will attract the hydrophilic regions of negative charge and repulse the hydrophilic regions of positive charge. The presence of alkyl halides in reverse micelles will decrease the electrostatic repulsion between the surfactant head groups and the positive regions of protein. These probably facilitate the protein molecules from getting into or out of the reverse micelles.
[3] [4]
[5]
[6]
[7]
[8]
4. Conclusions The addition of alkyl halide R8 Br to CTAB reverse micellar system could improve the BSA transfer from the aqueous phase to reverse micellar phase, while the addition of R4 Cl or R4 I had almost no effect on the BSA transfer. The mixed reverse micelles formed with CTAB and alkyl halides exhibit excellent backward extraction behavior for BSA. The mixed reverse micelles formed by CTAB and R4 I can realize maximum recovery of BSA in a wide pH region. The mixed reverse micelles formed with CTAB and R4 Cl, R8 Br and R4 I respectively can clearly enhance the BSA backward transfer at low ionic strength when KBr or KCl was used as salt. The mixed reverse micelles formed with CTAB and alkyl halides requires less time to reach mass transfer equilibrium for BSA in comparison with the reverse micelles formed with CTAB only. The mechanism of backward extraction may be suggested for the addition of alkyl halides to CTAB reverse micelles to decrease the hydrophobic interaction between reverse micelles and proteins extracted.
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17] [18]
Acknowledgements [19]
We acknowledge the financial support of the National Natural Science Foundation of China (No. 29836130) and the Outstanding Young Scientist Foundation of China (No. 29925617).
[20]
[21]
References [22] [1] M.J. Pires, M.R. Aires-Barros, J.M.S. Cabral, Liquid–liquid extraction of proteins with reversed micelles, Biotechnol. Progr. 12 (1996) 290–301. [2] T. Kinugasa, A. Hisamatsu, K. Watanabe, H. Takeuchi, A reversed micellar system using mixed surfactants of sodium bis-(2-ethylhexyl)
[23]
5
sulfosuccinate and di-(2-ethylhexyl) phosphoric acid for extraction of proteins, J. Chem. Eng. Jpn. 27 (1994) 557–562. M. Goto, T. Ono, F. Nakashio, T.A. Hatton, Reversed micelles recognize an active protein, Biotechnol. Tech. 10 (1996) 141–144. T. Zhang, H. Liu, J. Chen, Affinity extraction of BSA by mixed reversed micellar system with unbound triazine dye, Biochem. Eng. J. 4 (1999) 17–21. G. Spirovska, J.B. Chaudhuri, Sucrose enhances the recovery and activity of ribonuclease A during reversed micelle extraction, Biotechnol. Bioeng. 58 (1998) 374–379. K. Shiomori, Y. Kawano, R. Kuboi, I. Komasawa, Extraction of proteins and water with sodium bis-(2-ethylhexyl) sulfosuccinate/long chain alkyl amines mixed micellar system, J. Chem. Eng. Jpn. 32 (1999) 177–183. M. Dekker, K. Van’t Riet, S.R. Weijers, J.W.A. Baltussen, C. Lanne, B.H. Birjsterbosch, Enzyme recovery by liquid–liquid extraction using reversed micelles, Chem. Eng. J. 33 (1986) B27–B33. M. Dekker, R. Hilhorst, C. Lanne, Isolating enzymes by reversed micelles, Ann. Biochem. 178 (1989) 217–226. M. Dekker, K. Van’t Riet, B.H. Birjsterbosch, P. Fijneman, R. Hilhorst, Mass transfer rate of protein extraction with reversed micelles, Chem. Eng. J. 45 (1990) 2949–2957. S.R. Dungan, T.E. Bausch, T.A. Hatton, P.K. Plucinski, W. Nitch, Interfacial transport process in the reverse micellar extraction of proteins, J. Colloid Interf. Sci. 145 (1991) 30–45. T. Kinuguasa, S. Tanahshi, H. Takeuchi, Extraction of lysozyme using reversed micellar solution: distribution equilibrium and extraction rates, Ind. Eng. Chem. Res. 30 (1991) 2470–2476. A.N. Ermin, D.I. Metelitsa, Isolation of enzymes from mixed reversed micelles of surface-active agents, Prikl. Biokhim. Mikrobiol. 24 (1988) 42–49. J.M. Woll, T.A. Hatton, M.L. Yamrush, Bioaffinity separations using reversed micellar extraction, Biotechnol. Progr. 5 (1989) 57–62. M. Dekker, K. Van’t Riet, J.J. Van Der Pol, J.W.A. Baltussen, R. Hilhorst, B.H. Bijsterbosch, Effect of temperature on the reversed micellar extraction of enzymes, Chem. Eng. J. 46 (1991) B69–B74. J.B. Phillips, H. Nguyen, V.T. John, Protein recovery from reversed micellar solutions through contact with a pressurized gas phase, Biotechnol. Progr. 7 (1991) 43–48. A. Carlson, R. Nagarajan, Release and recovery of porcine pepsin and bovine chymosin from reverse micelles: a new technique based on isopropyl alcohol addition, Biotechnol. Progr. 8 (1992) 85–90. M.E. Leser, P.L. Luisi, Application of reversed micelles for the extraction of amino acids and proteins, Chimia 44 (1990) 270–282. R.B. Gupta, C.J. Han, K.P. Johnston, Recovery of proteins and amino acids from reverse micelles by dehydration with molecular sieves, Biotechnol. Bioeng. 44 (1994) 830–836. D.G. Hayes, C. Marchio, Expulsion of proteins from water-in-oil microemulsions by treatment with cosurfactant, Biotechnol. Bioeng. 59 (1998) 557–566. S. Jarudilokkul, L.H. Poppenborg, D.C. Stuckey, Backward extraction of reverse micellar encapsulated proteins using a counterionic surfactant, Biotechnol. Bioeng. 62 (1999) 593–601. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. T. Ono, M. Goto, F. Nakashio, T.A. Hatton, Extraction behavior of hemoglobin using reversed micelles by dioleyl phosphoric acid, Biotechnol. Progr. 12 (1996) 793–800. H.R. Rabie, T. Suyyagh, J.H. Vera, Reverse micellar extraction of proteins using dioctyldimethyl ammonium chloride, Sep. Sci. Technol. 33 (1998) 241–257.
Forward and backward extraction of BSA using mixed reverse micellar system of CTAB and alkyl halides Wei Zhang, Huizhou Liu∗ , Jiayong Chen Young Scientist Laboratory of Separation Science and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China Received 29 August 2000; accepted after revision 21 December 2001
Abstract The forward and backward extraction of bovine serum albumin (BSA) has been studied using mixed reverse micellar systems of cetyl trimethyl ammonium bromide (CTAB) and alkyl halides (including 1-chlorobutane (R4 Cl), 1-bromooctane (R8 Br) and 1-iodobutane (R4 I)). The addition of alkyl halide R8 Br to 50 mmol/l CTAB/20% (v/v) hexanol/petroleum ether reverse micellar system could improve the BSA transfer from the aqueous phase to reverse micellar phase, while the addition of R4 Cl or R4 I had almost no effect on the BSA transfer. The mixed reverse micelles formed with CTAB and alkyl halides exhibit excellent backward extraction behavior for BSA. The mixed reverse micelles formed with CTAB and R4 I can realize the recovery of BSA effectively in a wide range of pH up to or higher than the isoelectric point of BSA. The mixed reverse micelles formed with CTAB and R4 Cl, R8 Br or R4 I can obviously enhance the BSA backward transfer at low ionic strength with addition of KBr or KCl as electrolyte. The mixed reverse micellar system indicates that it requires less time to reach the mass transfer equilibrium in comparison with the reverse micellar system with CTAB only. The mechanism of backward extraction proposed that with the addition of alkyl halides to CTAB reverse micelles, the hydrophobic interaction between the reverse micelles decreased. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Reverse micelles; Backward extraction; Alkyl halide; BSA; CTAB
1. Introduction The separation of proteins using reverse micelles is rather easy to scale up and can be operated continuously [1]. Recently, synergism has been applied to the protein extraction by adding a secondary surfactant. Kinugasa et al. [2] have reported that the mixed reverse micelles formed by AOT (sodium bis-(2-ethylhexyl) sulfosuccinate) and DEHPA (di-2-ethylhexyl phosphoric acid) could be used to extract hemoglobin and the transfer can reach up to 80%. Goto et al. [3] reported that the mixed reverse micelles formed with AOT and DOLPA (dioleyl phosphoric acid) showed a high selectivity between the active ␣-chymotrypsin and denatured proteins. Zhang et al. [4] reported that the mixed reverse micelles formed with cetyl trimethyl ammonium bromide (CTAB) and TBP (tributyl phosphate) or TRPO (trialkyl phosphine oxide) show obvious advantage for the extraction of proteins over the reverse micelles formed with CTAB only. Spirovska and Chaudhuri [5] reported that the ∗ Corresponding author. Tel.: +86-10-6255-5005; fax: +86-10-6255-4264. E-mail address: [email protected] (H. Liu).
addition of sucrose in forward aqueous phase increased both the protein recovery and the activity of protein. Shiomori et al. [6] investigated the extraction of proteins using mixed reverse micellar systems of AOT and long chain alkyl amines, suggesting that the mixed micellar systems was a novel and functional system. It is clear that mixed reverse micelles formed by a surfactant with another surfactant or reagent exhibited some advantages over reverse micelles formed with a surfactant only for the extraction of proteins. It is well known that the backward transfer of protein from reverse micelles to the aqueous solution is relatively slow due to high interfacial resistance in mass transfer [7–11]. Conditions of high ionic strength and pH within a certain range, which would not allow the uptake of protein in forward transfer by reverse micelles, could not be used for the complete recovery of proteins solubilized in the forward extraction. A variety of alternative backward transfer methods has been studied. Ermin and Metelitsa [12] and Woll et al. [13] tested by addition of a second solvent to destabilize the reverse micelles to release the solubilized protein. Dekker et al. [14] studied by increasing the operating temperature of backward extraction to separate an excess aqueous phase
1369-703X/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 6 9 - 7 0 3 X ( 0 2 ) 0 0 0 0 6 - 2
2
W. Zhang et al. / Biochemical Engineering Journal 12 (2002) 1–5
that contained the protein in the aqueous phase of the reverse micelles during forward extraction. Phillips et al. [15] pressurized the reverse micelles with ethylene to convert the water pool to clathrate hydrates. Carlson and Nagarajan [16] added 10–50% isopropyl alcohol to facilitate the kinetics of protein release, while Leser and Luisi [17] added silica gel to adsorb the aqueous phase of reverse micelles. Recently, Gupta et al. [18] dehydrated the reverse micelles with molecular sieves. Hayes and Marchio [19] expelled proteins by adding a small portion (0.1 v/v or less) of cosurfactant (e.g., 1-alkanol) to w/o microemulsion solution, sometimes as solids, while most or all of the surfactant (AOT) remained in solution. Jarudilokkul et al. [20] obtained higher backward extraction yields by adding a counterionic surfactant at nearly neutral pH and low salt concentration. The drawback in adding alcohol or counterionic surfactant to the reverse micellar phase is recycling of the solvent for reuse. Ideally, in order to enhance the protein activity recovery during backward extraction, mild conditions such as neutral pH and low salt concentrations in the fresh aqueous phase would be preferred. Apparently, in most of the backward recovery methods studied, this is not feasible. Nevertheless, these may be possible if new mixed reverse micelles were used for extraction. In the present study, the mixed solution of CTAB and alkyl halides was used as a novel reverse micellar system for protein extraction. The forward and backward extraction of the bovine serum albumin (BSA) in the mixed reverse micelles was investigated.
2. Materials and methods BSA (molecular weight 68,000, isoelectric point 4.8) was purchased from Beijing Baitai Biochemical Reagents. CTAB (>99%, analytical grade), hexanol (>98%, chemical grade), petroleum ether (b.p.: 90–120 ◦ C, >99%, analytical grade), 1-chlorobutane (chemical grade), 1-bromooctane (>98%, chemical grade) and 1-iodobutane (chemical grade) were purchased from Beijing Chemical Reagent. Other reagents used in this work were all commercially available reagents of analytical grade and were used as received. Forward extraction procedures for BSA were conducted by mixing the petroleum ether solution containing 0.05 mol/l CTAB and 20% (v/v) hexanol with an aqueous solution in the desired pH containing 1.0 mg/ml BSA with desired amount of salt. Equal volumes (usually 5 ml each) of aqueous and organic solutions were poured gently into a 50 ml flask, which was stoppered to prevent the evaporation of the solvent. The flasks were shaken with the rate of agitation of 3.5 s−1 at room temperature (30 ± 0.5 ◦ C) for 20 min. After centrifugation (4000 rpm, 5 min) for phase separation, the protein concentration in the reverse micellar phase was determined by spectroscopy at 280 nm (UV-7542 spectrophotometer). The water concentration in the organic phase was determined by Karl–Fischer titration (WA-1A).
Backward extraction of BSA was conducted by mixing the reverse micellar solution that had extracted BSA in advance with a fresh aqueous phase of desired pH and ionic strength at room temperature (30 ± 0.5 ◦ C). Equal volumes (usually 5 ml) of aqueous and organic solutions were poured gently into a 50 ml stoppered flask. The flasks were shaken with the rate of agitation of 4.0 s−1 at room temperature (30±0.5 ◦ C) for 60 min. The mixture was then centrifuged (4000 rpm, 5 min), and the protein concentration of the stripped aqueous phase was determined by both Bradford method [21] and 280 nm spectroscopic method. The yield of the backward transfer of BSA was based on the percentage of BSA recovered in the aqueous phase relative to the amount of BSA extracted into the reverse micellar phase. The protein mass balances for a set of forward and backward extractions were generally within the experimental error of ±5%. 3. Results and discussion 3.1. Forward extraction The solubilization of water in the reverse micelles formed with CTAB and alkyl halides was examined with no protein extraction. The effect of the amount of alkyl halide addition on water content was shown in Fig. 1. The results indicated that the amount of water inside the reverse micelles did not change very much with the addition of alkyl halides, implying that there was not much of a change in the size of the reverse micelles. The effects of pH, ionic strength and the amount of alkyl halides added on BSA transfer were investigated. The addition of alkyl halide R8 Br to CTAB reverse micellar system could improve the BSA transfer from the aqueous phase to reverse micellar phase, while the addition of R4 Cl or R4 I had almost no effect on the BSA transfer. The results indicated that the formation of the mixed reverse micelles of CTAB and R4 Cl or R4 I had no obvious effect on protein
Fig. 1. Effect of concentration of alkyl halides added on the water content of CTAB reversed micelles. Aqueous phase: pH = 7.0, 0.1 mol/l KCl. Organic phase: 50 mmol/l CTAB/5% (v/v) alkyl halide/20% (v/v) hexanol/petroleum ether.
W. Zhang et al. / Biochemical Engineering Journal 12 (2002) 1–5
3
Fig. 2. Effect of the amount of alkyl halide added on BSA transfer. Aqueous phase: 1.0 mg/ml BSA, 0.1 mol/l KCl, pH = 7.0. Organic phase: 50 mmol/l CTAB with different amount of alkyl halide added/20% (v/v) hexanol/petroleum ether.
extraction, which might result from not much of change in the size of the reverse micelles. The effect of the amount of alkyl halides added to CTAB reverse micelles on the BSA transfer was shown in Fig. 2 at pH 7.0. The BSA transfer to reverse micelles formed by CTAB and R8 Br increased with the increase of the amount of R8 Br added. The BSA transfer was increased by 12% when 5% (v/v) R8 Br was added into CTAB reverse micelles. The BSA transfer increased by 4% to reverse micelles when 1.0% (v/v) R4 Cl was added into CTAB reverse micelles, but it did not increase with the further increase of the amount of R4 Cl. The BSA transfer of reverse micelles formed by CTAB and R4 I increased slightly by 2% when compared with the reversed micelles formed by CTAB only. The alkyl halides are weak Lewis alkali, while the CTAB head groups in the reverse micelles carry positive charge. Halogen atoms present in reverse micellar inner core could separate surfactant molecules such as CTAB. The addition of alkyl halides could decrease the electrostatic repulsion between the surfactant molecules and the positive region of proteins while the hydrophobic interaction between the surfactant head groups and the hydrophobic region of proteins could lead to the increase of BSA transfer. Although the electronegativity of the chlorine atom is highest, its size is very small, thus the improvement of R4 Cl addition for the BSA transfer is limited. Though the size of the iodine atom is highest, its electronegativity is the lowest, thus the improvement of R4 I addition for the BSA transfer is also very limited. The bromine atom has relatively strong electronegativity, and with large size, the improvement of R8 Br addition on the BSA forward transfer is obvious. Additionally, the alkyl chain of R8 Br is longer than that of R4 Cl or R4 I, which might influence the hydrophobicity of alkyl halides. 3.2. The backward transfer Effect of solution pH. The protein extracted into the reverse micelles can be recovered by the backward transfer
Fig. 3. Effect of backward transfer aqueous phase pH on BSA backward transfer. Forward transfer aqueous phase: 1.0 mg/ml BSA, pH = 9.10, 0.1 mol/l KCl. Organic phase: 50 mmol/l CTAB/4% (v/v) alkyl halide/20% (v/v) hexanol/petroleum ether. Backward transfer aqueous phase: 1.0 mol/l (a) KBr, (b) KCl. Backward transfer time is 60 min.
through the result of electrostatic repulsion by changing the solution pH, or through the size exclusion effect by increasing the salt concentration in the aqueous phase used for backward transfer [22]. The effect of pH of aqueous phase used for the backward transfer of the BSA was shown in Fig. 3. BSA could be recovered only under conditions with pH below pI using 1.0 mol/l KBr or KCl solution when using CTAB, CTAB–R4 Cl or CTAB–R8 Br to form reverse micelles. As shown in Fig. 3, the addition of R4 Cl or R8 Br to CTAB to form reversed micelles will lead to the recovery of BSA in a similar pH range as no alkyl halide was added. Surprisingly, BSA could be recovered with high back-transfer in a much wider pH range when using mixed reverse micelles formed by CTAB and R4 I. The result may be explained by the fact that the addition of alkyl halides to CTAB will decrease the hydrophobic interaction between the hydrophobic regions of protein and surfactant polar heads. The degree of decrease of hydrophobic interaction caused by the presence of R4 I is much higher in comparison with those caused by R4 Cl and R8 Br. BSA shows higher hydrophobicity when aqueous phase pH is closer to its isoelectric point (pI = 4.8), so the backward transfer of BSA is lower. The difference in the results obtained between using KBr and KCl (Fig. 3a and b) might be caused by the effect of anions on the protein
4
W. Zhang et al. / Biochemical Engineering Journal 12 (2002) 1–5
Fig. 4. Effect of ionic strength of aqueous phase on BSA backward transfer. Backward transfer aqueous phase: pH = 4.3, (a) KBr, (b) KCl. Other conditions are the same as given in Fig. 3.
Fig. 5. Effect of time of backward transfer on BSA backward transfer. Backward transfer aqueous phase: pH = 4.3, 1.0 mol/l (a) KBr, (b) KCl. Other conditions are the same as given in Fig. 3.
transfer, which could be explained by the competition between the anions acting as counterions of the surfactant [23]. Effect of solution ionic strength. The salt concentration in the aqueous phase can affect the efficiency of protein transfer through the size exclusion effect. The effect of ionic strength of aqueous phase in the backward extraction on BSA back-transfer was shown in Fig. 4. The mixed reverse micelles exhibit much better backward transfer behavior for BSA. Fig. 4a shows that the mixed reverse micelles formed with CTAB and by R4 Cl, R8 Br or R4 I obviously improved the BSA backward transfer at low ionic strength (0.2–1.0 mol/l KBr). For example, the BSA back-transfer of mixed reverse micelles formed with CTAB and by the addition of R4 Cl, R8 Br and R4 I at 0.4 mol/l KBr was 35, 68 and 70%, respectively. The BSA back-transfer from reverse micelles formed with CTAB only was 21% under the same conditions. Fig. 4b shows that the mixed reverse micelles formed by CTAB and R8 Br or R4 I clearly improved the BSA back-transfer at low ionic strength when KCl was used as salt, while the mixed reverse micelles formed with CTAB and R4 Cl give almost no effect on the BSA back-transfer. The mixed reverse micelles show very good protein back-transfer at low ionic strength that is very interesting, because low ionic strength of aqueous phase for backward transfer will be useful to protect protein and for recovery of enzyme activity.
Time required for backward extraction. It is well known that the rate of backward transfer of protein from reversed micelles to an aqueous phase is relatively slow. We examined the effect of the time of back-transfer on the BSA recovery as shown in Fig. 5. The mixed reverse micelles exhibited excellent backward extraction behavior for BSA. As shown in Fig. 5a, the mixed reverse micelles require less time to reach the maximum amount of protein recovery when KBr was used as salt. The improvement of CTAB–R4 I reverse micelles for BSA recovery was remarkable in comparison with those using CTAB–R4 Cl and CTAB–R8 Br reverse micelles. As shown in Fig. 5b, the mixed reverse micelles require less time to approach the mass transfer equilibrium when KCl was used as salt. The BSA back-transfer was remarkably enhanced when mixed reverse micelles was used for the same period of contact time. For example, the BSA back-transfer recovery from CTAB, CTAB–R4 Cl, CTAB–R8 Br and CTAB–R4 I reverse micelles in 10 min were 17, 46, 44 and 84%, respectively. The results indicated that the addition of alkyl halides to CTAB reverse micelles might decrease the hydrophobic interaction between the protein and the surfactant polar head groups. Mechanism of backward extraction of proteins with addition of alkyl halides. It is reasonable and widely acceptable that the backward transfer of protein from the reverse
W. Zhang et al. / Biochemical Engineering Journal 12 (2002) 1–5
micelles is governed by an interfacial process and by the coalescence of reverse micelles at oil–water interface [7]. The addition of alkyl halides to CTAB reverse micelles will decrease the hydrophobic interaction between the hydrophobic region of protein and surfactant polar heads. The order of decrease of hydrophobic interaction caused by alkyl halides is R4 I > R8 Br > R4 Cl, resulting from the results of Figs. 3–5. The CTAB head groups will attract the hydrophilic regions of negative charge and repulse the hydrophilic regions of positive charge. The presence of alkyl halides in reverse micelles will decrease the electrostatic repulsion between the surfactant head groups and the positive regions of protein. These probably facilitate the protein molecules from getting into or out of the reverse micelles.
[3] [4]
[5]
[6]
[7]
[8]
4. Conclusions The addition of alkyl halide R8 Br to CTAB reverse micellar system could improve the BSA transfer from the aqueous phase to reverse micellar phase, while the addition of R4 Cl or R4 I had almost no effect on the BSA transfer. The mixed reverse micelles formed with CTAB and alkyl halides exhibit excellent backward extraction behavior for BSA. The mixed reverse micelles formed by CTAB and R4 I can realize maximum recovery of BSA in a wide pH region. The mixed reverse micelles formed with CTAB and R4 Cl, R8 Br and R4 I respectively can clearly enhance the BSA backward transfer at low ionic strength when KBr or KCl was used as salt. The mixed reverse micelles formed with CTAB and alkyl halides requires less time to reach mass transfer equilibrium for BSA in comparison with the reverse micelles formed with CTAB only. The mechanism of backward extraction may be suggested for the addition of alkyl halides to CTAB reverse micelles to decrease the hydrophobic interaction between reverse micelles and proteins extracted.
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17] [18]
Acknowledgements [19]
We acknowledge the financial support of the National Natural Science Foundation of China (No. 29836130) and the Outstanding Young Scientist Foundation of China (No. 29925617).
[20]
[21]
References [22] [1] M.J. Pires, M.R. Aires-Barros, J.M.S. Cabral, Liquid–liquid extraction of proteins with reversed micelles, Biotechnol. Progr. 12 (1996) 290–301. [2] T. Kinugasa, A. Hisamatsu, K. Watanabe, H. Takeuchi, A reversed micellar system using mixed surfactants of sodium bis-(2-ethylhexyl)
[23]
5
sulfosuccinate and di-(2-ethylhexyl) phosphoric acid for extraction of proteins, J. Chem. Eng. Jpn. 27 (1994) 557–562. M. Goto, T. Ono, F. Nakashio, T.A. Hatton, Reversed micelles recognize an active protein, Biotechnol. Tech. 10 (1996) 141–144. T. Zhang, H. Liu, J. Chen, Affinity extraction of BSA by mixed reversed micellar system with unbound triazine dye, Biochem. Eng. J. 4 (1999) 17–21. G. Spirovska, J.B. Chaudhuri, Sucrose enhances the recovery and activity of ribonuclease A during reversed micelle extraction, Biotechnol. Bioeng. 58 (1998) 374–379. K. Shiomori, Y. Kawano, R. Kuboi, I. Komasawa, Extraction of proteins and water with sodium bis-(2-ethylhexyl) sulfosuccinate/long chain alkyl amines mixed micellar system, J. Chem. Eng. Jpn. 32 (1999) 177–183. M. Dekker, K. Van’t Riet, S.R. Weijers, J.W.A. Baltussen, C. Lanne, B.H. Birjsterbosch, Enzyme recovery by liquid–liquid extraction using reversed micelles, Chem. Eng. J. 33 (1986) B27–B33. M. Dekker, R. Hilhorst, C. Lanne, Isolating enzymes by reversed micelles, Ann. Biochem. 178 (1989) 217–226. M. Dekker, K. Van’t Riet, B.H. Birjsterbosch, P. Fijneman, R. Hilhorst, Mass transfer rate of protein extraction with reversed micelles, Chem. Eng. J. 45 (1990) 2949–2957. S.R. Dungan, T.E. Bausch, T.A. Hatton, P.K. Plucinski, W. Nitch, Interfacial transport process in the reverse micellar extraction of proteins, J. Colloid Interf. Sci. 145 (1991) 30–45. T. Kinuguasa, S. Tanahshi, H. Takeuchi, Extraction of lysozyme using reversed micellar solution: distribution equilibrium and extraction rates, Ind. Eng. Chem. Res. 30 (1991) 2470–2476. A.N. Ermin, D.I. Metelitsa, Isolation of enzymes from mixed reversed micelles of surface-active agents, Prikl. Biokhim. Mikrobiol. 24 (1988) 42–49. J.M. Woll, T.A. Hatton, M.L. Yamrush, Bioaffinity separations using reversed micellar extraction, Biotechnol. Progr. 5 (1989) 57–62. M. Dekker, K. Van’t Riet, J.J. Van Der Pol, J.W.A. Baltussen, R. Hilhorst, B.H. Bijsterbosch, Effect of temperature on the reversed micellar extraction of enzymes, Chem. Eng. J. 46 (1991) B69–B74. J.B. Phillips, H. Nguyen, V.T. John, Protein recovery from reversed micellar solutions through contact with a pressurized gas phase, Biotechnol. Progr. 7 (1991) 43–48. A. Carlson, R. Nagarajan, Release and recovery of porcine pepsin and bovine chymosin from reverse micelles: a new technique based on isopropyl alcohol addition, Biotechnol. Progr. 8 (1992) 85–90. M.E. Leser, P.L. Luisi, Application of reversed micelles for the extraction of amino acids and proteins, Chimia 44 (1990) 270–282. R.B. Gupta, C.J. Han, K.P. Johnston, Recovery of proteins and amino acids from reverse micelles by dehydration with molecular sieves, Biotechnol. Bioeng. 44 (1994) 830–836. D.G. Hayes, C. Marchio, Expulsion of proteins from water-in-oil microemulsions by treatment with cosurfactant, Biotechnol. Bioeng. 59 (1998) 557–566. S. Jarudilokkul, L.H. Poppenborg, D.C. Stuckey, Backward extraction of reverse micellar encapsulated proteins using a counterionic surfactant, Biotechnol. Bioeng. 62 (1999) 593–601. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. T. Ono, M. Goto, F. Nakashio, T.A. Hatton, Extraction behavior of hemoglobin using reversed micelles by dioleyl phosphoric acid, Biotechnol. Progr. 12 (1996) 793–800. H.R. Rabie, T. Suyyagh, J.H. Vera, Reverse micellar extraction of proteins using dioctyldimethyl ammonium chloride, Sep. Sci. Technol. 33 (1998) 241–257.