Znd. Eng. Chem. Res. 1992,31, 1827-1829
1827
Activity and Conformation of Lysozyme in Reversed Micellar Extraction A study has been made on the activity and conformation of lysozyme in reversed micellar extraction using a spectrophotometer and spectropolarimeter, respectively. Lysozyme extracted in Aerosol OT/isooctane reverse micelle could be recovered into an aqueous solution in the pH range of 10.5-12 under conditions of high ionic strength. The enzyme activity of the lysozyme stripped to the aqueous solution of pH lower than 12 was retained as compared with the native enzyme. Circular dichroism spectra of lysozyme entrapped in the micelle showed that no significant change is observed in the structure of the main chain, although the fine structure originated from the aromatic residues is influenced by the surfactant molecules. In the stripping of lysozyme from the reverse micelle to aqueous solution, the enzyme is returned to the native conformation and thus remains the initial value in the activity.
Introduction Recently, an extraction using reverse micelles has been attractively studied for a separation and purification of bioproducta, such as proteins (including enzymes) (Luisi et al., 1988; Dekker et al., 1989; Hatton, 1989) and amino acids (Hatton, 1987; Furusaki and Kishi, 1990). The water pool within the reverse micelle is shielded from organic medium, and then bioproducta can be solubilized into ita core water without loss in activity (Luisi, 1985; Martinek et al., 1986). There are, however, several reports on the denaturation of proteins solubilized in reversed micellar solution. Gonnelli and Strambini (1988) examined enzyme activity and phosphorescence of alkaline phosphatase in Aerosol OT (AOT)/isooctane reverse micelle; they found that at lower water content of the reverse micelle the Pstructure is unfolded and then the enzyme is denatured. With increasing water content such a reverse micelle also reduced the stability of solubilized lipase (Han and Rhee, 1986). In a previous paper (Kinugasa et al., 1991),we reported the partitioning and extraction rate of lysozyme in an AOT/isooctane reverse micelle. It is crucial to know whether the enzyme activity remains during the operation or not. However, no detailed data have been published concerning the activity of lysozyme. The present Note has described the behavior of activity and conformation of lysozyme in the reversed micellar extraction and stripping processes. Experimental Procedure An aqueous solution was prepared by dissolving a typical enzyme, lysozyme from chicken egg white obtained from Sigma Chemical Co., in phosphate buffer solution containing 0.3 M KC1 for the adjustment of ionic strength. The pH value of the solution was adjusted at 7. An organic solution was prepared by dissolving anionic surfactant, 0.05 M sodium bis(2-ethylhexyl)sulfosuccinate (AOT) obtained from Wako Pure Chemical Ind. Ltd., in isooctane of commercial GR grade. Equal volumes of the aqueous enzyme and AOT/isooctane solutions were placed in a flask and shaken for 60 min at 298 K. Both phases were centrifuged to separate from each other. The enzyme extracted into the organic phase was further stripped into an aqueous solution adjusted at high pH with NaOH, and the mixture was also separated on a centrifuge. The concentration of lysozyme in each solution was determined by UV spectrophotometry at 280 nm. The recovery ratio R was obtained from R = CwVw/(Co,iVo,i) (1) where C, and Co,iare the lysozyme concentrations in the aqueous and the organic phases after and before the stripping (back extraction), respectively, and Vw and Vo,i are the respective solution volumes.
The enzyme activity of lysozyme in the aqueous solution was determined at 313 K by means of ethylene glycol chitin (Sigma Chemical Co.) as the substrate. The details of the assay are available elsewhere (Imoto and Yagishita, 1971). The activity measurements were conducted at the optimal pH value, 5.5, for the enzyme solution. The specific activity of the native lysozyme was about 0.8 unit/mg of enzyme; then, the relative specific activity a can be defined as the ratio of the specific activity of lysozyme after the back extraction to that before the forward extraction. Circular dichroism (CD) spectra of lysozyme were obtained for both aqueous and organic phases at 298 K on a Jasco J-500E spectropolarimeter, the molar ellipticity, [e], obtained being expressed in terms of amino acid residue. Most samples of reversed micellar solution containing proteins were prepared by the extraction (phase transfer) method, as mentioned above. Also, a few samples were prepared by the injection method whereby after addition of a small amount of a concentrated aqueous solution of the protein into a micellar solution, its mixture was intensively shaken to reach an optically transparent state.
Results and Discussion Figure 1shows the effect of pH value in the strip solution on the recovery ratio, R, and the relative specific activity, a, of lysozyme, represented by open symbols, indicating that there is a maximum value of R in the pH region of 10.5-12. In the strip solution of a lower pH than the enzyme has positive net charge; thus, it ita PI(=ll), is hard for the reverse micelle to release the enzyme into the aqueous phase and the enzyme remains entrapped there owing to electrostatic attraction. At pH values above 12, insoluble aggregates composed of lysozyme and AOT molecules were formed at the oil-water interface, resulting in a decrease in the recovery ratio. On the other hand, it can be seen in Figure 1that the specific activity of lysozyme remains the native one even after the back extraction by use of the solution with pH up to 12. For pH above 12, however, the enzyme activity was drastically reduced. The relative specific activity after allowing lysozyme to be in high pH solutions for 1h is also plotted against pH value in Figure 1with closed circles. From a comparison of these activity data, a conclusion can be drawn that the reduction in the activity of the enzyme stripped is attributed not to reversed micellar extraction itself but to contact with alkaline medium. Consequently, for the enzyme recovery from the micellar solution, an optimal pH of the strip solution is in the range of 11-12, where also the back extraction rate has proved to be favorable to recovery (Kinugasa et al., 1991). Figure 2 shows the effect of KC1 concentration in the strip solution on the recovery ratio and the relative specific activity of lysozyme. In the lower KC1 concentration region, it is considered that the electrostatic repulsion between the enzymes and AOT head groups is enhanced,
Q888-5885/92/2631-1827$03.QO/Q0 1992 American Chemical Society
1828 Ind. Eng. Chem. Res., Vol. 31, No. 7,1992 I
1
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pH [-I Figure 1. Effect of pH in strip solution on recovery ratio, R, and relative specific activity, a, of lysozyme. Closed symbols refer to relative specific activity of lysozyme allowed to be in different pH solutions for 1 h. 200
o1 a°
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Figure 2. Effect of ionic strength in strip solution on recovery ratio and relative specific activity of lysozyme.
whereas the diameter of the reverse micelle becomes larger as found in the previous paper. The effect of the former leads to increase in the recovery ratio with lowering ionic strength; on the other hand, the size effect is to act inversely. Hence, this implies that the exclusion effect due to the size of the reverse micelle is significant in the stripping of lysozyme at a pH near ita PI,compared with the electrostatic interaction. The activity characteristics remained regardless of the KC1 concentration. Moreover, we confirmed that the native activity is preserved in the reverse micelle even for 20 h. This implies that the AOT/isooctane reverse micelle does not affect the active site of lysozyme. Further examination was made of the behavior of the conformation of lysozyme in reversed micellar extraction by obtaining the CD spectrum. Figure 3 shows the CD spectra for lysozyme in aqueous solution with different pH values. A positive CD band in the near-UV region may be ascribed to the aromatic amino acid residues (tryptophan, tyrosine, and phenylalanine residues), whereas a negative band in the far-UV region may be ascribed to the peptide groups. The appearance of a maximum ellipticity a t 253 nm for the high solution pH (lines 2 and 3) is responsible for the ionization of two tyrosine residues of lysozyme: Tyr-20and -23(Ikeda and Hamaguchi, 1969). In the solutions with pH above 13,the lysozyme was denatured, and thus the CD spectrum irreversibly changes, while, below 12 it reversibly changes. The CD spectra for lysozyme in the reversed micellar extraction are shown in Figure 4. A significant difference (line 2) is observed for the ellipticity in the near-W region in comparison to that (line 1) in the aqueous solutions.
-2.0 4- 200
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X hml Figure 4. CD spectra for lysozyme in reversed micellar extraction. 1, in aqueous solution (pH 7.3) before extraction; 2, in reversed micellar solution, [H,O]/[AOT] = 10.5; 3, in aqueous solution (pH 7.5) after stripping (neutralii with HC1 after stripping at pH 11.5).
This suggests that a change in fine structure of the aromatic amino residues took place from the electrostatic or hydrophobic interaction with the surfactant molecules. Especially for lysozyme in the reverse micelle, the appearance of a maximum ellipticity at 255 nm seems to come from a change in environment around the tyrosine residue, as observed in the high-pH solution (see Figure 3). On the high-pH condition of aqueous solution (pH 8.2-10.5 and 9.4-12.2 for the extraction and injection method, respectively), however, no significant difference in CD spectra was observed as compared with line 2 in Figure 4 (pH 7.3 by the extraction method). It is implied that the level of dissociaton of tyrosine residues is independent of pH in the water pool, especially of local pH at its periphery close to the surfactant head groups, due to
Ind. Eng. Chem. Res., Vol. 31, No. 7,1992 1829 an electrostatic interaction with the polar head. According to a previous study on the conformation of lysozyme in a reverse micelle (Steinmann et al., 19861, lysozyme is protected against denaturation by binding with ita substrate or inhibitor, and therefore tryptophan residues at the active site may be interacted with the surfactant molecules in the AOT/isooctane system. For the lysozyme (line 3) stripped into an aqueous solution, however, the CD spectrum returned to the native one, corresponding to the result of the activity measurement. It is to be noted that, in the far-UV region, the CD spectra are close to each other. This indicates that the secondary structure of the enzyme, a-helix and @structure, almost retains the native structure. The helical content of lysozyme, estimated from the ellipticity at 208 nm by the method of Greenfield and Fasman (1969), is 29% in aqueous solution and 36% in reversed micellar solution. Thus, lysozyme appears to become slightly more rigid in the reverse micelle as pointed out by Grandi et al. (1981). Such conformation characteristics have been also reported for a-chymotrypsin (Barbaric and Luisi, 1981). In conclusion, lysozyme is affected by the specific environment of the water pool within AOT/isooctane reverse micelles; however, its interaction with the surfactant molecules is comparatively weak. Being recovered from the micellar solution into an aqueous phase, the enzyme gets back ita native activity. Conclusions The activity and conformation of lysozyme were examined in reversed micellar extraction. Lysozyme can be extracted in the micellar solution and stripped in recovering aqueous solution without denaturation. In the reversed micellar extraction, aromatic amino residues of the enzyme entrapped in the micelle are affected by the surfactant molecules electrostatically or hydrophobically. Being stripped from the reverse micelle into an aqueous solution having pH below 12, the enzyme is returned to ita native conformation and remains in the original activity. Therefore, the reversed micellar extraction might be available for a separation and purification of lysozyme without denaturation. Nomenclature a = relative specific activity, dimensionless C = concentration of lysozyme, kg/m3 R = recovery ratio, dimensionless V = volume, m3
Greek Symbols [e] = molar ellipticity, degcm*/dmol X = wavelength, nm Subscripts
i = initial value o = reversed micellar phase w = aqueous phase
Literature Cited Barbaric, S.; Luisi, P. L. Micellar Solubilization of Biopolymers in Organic Solvents. 5. Activity and Conformation of a-chymotrypsin in Isooctane-AOT Reverse Micelles. J . Am. Chem. SOC. 1981,103,4239-4244. Dekker, M.; Hilhorst, R.; Lame, C. Isolating Enzymes by Reversed Micelles. Anal. Biochem. 1989,178,217-226. Furusaki, S.;Kishi, K. Extraction of Amino Acids by Reversed Micelles. J . Chem. Eng. Jpn. 1990,23,91-93. Gonnelli, M.; Strambini, G. B. Protein Dynamical Structure by Tryptophan Phosphorescence and Enzymatic Activity in Reverse Micelles: 2. Alkaline Phosphatase. J. Phys. Chem. 1988,92, 2854-2857. Grandi, C.; Smith, R. E.; Luisi, P. L. Micellar Solubilization of Biopolymers in Organic Solvents: Activity and Conformation of Lysozyme in Isooctane Reversed Micelles. J . Biol. Chem. 1981,256, 837-843. Greenfield, N.; Fasman, G. D. Computed Circular Dichroism Spectra for the Evaluation of Protein Conformation. Biochemistry 1969, 8,4108-4116. Han, D.; Rhee, J. S. Characteristics of Lipase-Catalyzed Hydrolysis of Olive Oil in AOT-Isooctane Reversed Micelles. Biotechnol. Bioeng. 1986,28,1250-1255. Hatton, T. A. Extraction of Proteins and Amino Acids Using Reversed Micelles. In Ordered Media in Chemical Separations; Hinze, W. L., Armstrong, D. W., Eds.; ACS Symposium Series 342;American Chemical Society: Washington, DC, 1987;Chapter 9. Hatton, T. A. Reversed Micellar Extraction of Proteins. In Surfactant-Based Separations; Scamehorn, J. F., Harwell, J. H., Eds.; Marcel1 Dekker: New York, 1989;Chapter 3. Ikeda, K.; Hamaguchi, K. The Binding of N-Acetylglucosamine to Lysozyme. Studies on Circular Dichroism. J . Biochem. 1969,66, 513-520. Imoto, T.; Yagishita, K. A Simple Activity Measurement of Lysozyme. Agric. Biol. Chem. 1971,35,1154-1156. Kinugasa, T.; Tanahashi, S.; Takeuchi, H. Extraction of Lysozyme Using Reversed Micellar Solution: Distribution Equilibrium and Extraction Rates. Znd. Eng. Chem. Res. 1991,30, 2470-2476. Luisi, P. L. Enzymes Hosted in Reversed Micelles in Hydrocarbon Solution. Angew. Chem., Int. Ed. Engl. 1986,24,439-450. Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Reverse Micelles as Hosta for Proteins and Small Molecules. Biochim. Biophys. Acta 1988,947,209-246. Martinek, K.;Levashov, A. V.; Klyachko, N.; Khmelnitski, Y. L.; Berezin, I. V. Micellar Enzymology. Eur. J. Biochem. 1986,155, 453-468. Steinmann, B.; Jackle, H.; Luisi, P. L. A Comparative Study of Lysozyme Conformation in Various Reverse Micellar Systems. Biopolymers 1986,25, 1156.
Takumi Kinugasa, Kunio Watanabe Department of Industrial Chemistry Niihama National College of Technology Niihama 792, Japan
Hiroshi Takeuchi* Department of Chemical Engineering Nagoya University Chikusa-ku, Nagoya 464-01, Japan Received for review December 3, 1991 Revised manuscript received April 21, 1992 Accepted May 8, 1992
