184 J, A. Asenjo and J B Chaudhuri PEG/Dextran systems with addition of small concentration of salts whose charged ions will partition between the phases (e. g. 0. 1 M NaCl or 0.05 M Na2SO4) and also by manipulating the pH of the system. In PEG/salt systems an increase in the pH usually increases the value of K. Figure 7.3 shows the increase in K with pH for a-amylase in a PEG/phosphate system. As the bottom phase has a high concentra tion of salt, it is the charges in the top phase that affect partitioning; thus the top (4) Biospecific affinity. The affinity between sites on the proteins and ligands attached to one of the phase polymers is used for separation. Dyes, inhibitors, fatty acids glutathione, Protein A and several other ligands have been used. Particularly impressive results for selective partitioning have been obtained with metal ions (e.g. Cu**)both in PEG/Dextran but also in PEG/salt systems(Wuenschell et al (5) Solubility dependent. In addition it is important to know the actual concentration of the protein in the extractant phase. In typical PEG/salt systems, protein solubility tends to be higher in the PEG and lower in the salt phase. It is clear that, in the region near saturation of the protein in one of the phases, a constant partition Fig. 7.3. Partition of pure a-amylase()in PEG 4000(10% w/w)/phosphate(11.5% w/w) According to this, it is possible to split the partition coefficient into different terms K=KhfobKe kmw ksol where hfob, el, mw, aff and sol stand for hydrophobicity, electrostatic, size(molecular weight), affinity and solubility contributions to the partition coefficient In practical terms partitioning in aqueous two-phase systems is influenced by many ystem variables. Generally, the higher the molecular weight of the polymers the lower the concentration needed for the formation of two phases. Also, the larger the molecular weight of the PEG, the lower the value of K. Work is presently being carried out on elucidating how the different physicochemical properties of individual proteins determine
1.0 Y g 0.5 - 0 - - I I
Innovative separation methods in bioprocessing 185 their partition behaviour in two phase systems(Hachem, 1992, Asenjo et al., 1994 Schmidt, 1994). It has been possible to correlate the partition coefficient of a representative number of proteins to their hydrophobicity measured by precipitation. This correlation was not as ood if the hydrophobicity was evaluated by hydrophobic interaction chromatography HIC)or also by reverse phase -HPLC (RP-HPLC)(Hachem, 1992; Asenjo et al., 1994 Hachem et al., 1994). In a typical protein precipitation graph as that shown in Fig. 7.4 (S=protein in solution), the proteins solubility (and thus 'hydrophilicity) can be expressed by point m, which is oint at which the protein starts precipitating, Thus the hydrophobicity, P, was evaluated as 1/m. The correlation found between hydrophobicity and partition in PEG /salt systems with a high concentration of NaCl is shown in Fig. 7.5. This can be represented by the equati gK=D△ w log P-D△ w log Po co AW corresponds to the tie- line length of a system and is evaluated by the difference in oncentration of one component(e. g. PEG or salt) between the phases which is constan for one particular system. D is the'discrimination factor'and thus D Aw is the slope in Fig.7.5 which corresponds to the resolution of a particular system to exploit differences in hydrophobicity between proteins. Table 7. 2 gives values of resolution (D Aw) and trinsic hydrophobicity'(Po) found for PEG/phosphate systems with different concen trations of NaCl. Clearly the systems with higher concentrations of Nacl give a higher resolution to exploit the protein s hydrophobicity in partitioning 7.3.2 Reverse micelle extraction Water-in-oil microemulsions, or reverse micelles, are stable, monodisperse aggregates of surface-active molecules (1-10 nm diameter) in an organic solvent. Typically, the =-012330+0.8431x-8.83470e-2x22=0.972 y=87919-24846XP=0.985 08L Concentration of ammonium sulphate(M) Fig. 7. 4. The fitted curves o oulin a at 25c in a solution containing added ammonium the first represents the represe salting-in region and the equati
0.4 0.2 0.0 E . 9 v) -0.2 0, - -0.4 -0.6 -0.8 - - I - - - - - I I
LA.A y=10043+22986XP2=0.915 Fig. 7. 5. The relationship between log K of the model proteins partitioned in two-phase syste made of 8% PEG and 12% PO!"(pH 7.0)to which 9.6%(w/w) NaCl was added and their log(1/m). Experiments were carried out at room temperature, Abbreviations are lysozyme Lys), a-lactalbumin Lac), B-lactoglobulin A, conalbumin (Conal), and bovine serum albumin(BSa) Table 7.2. The calculated values of DAw and the intrinsic hydrophobicity, log Po, of the aqueous two-phase systems used at 20°C Two-phase system(wt %) D△W log Po PEG, 12% PO 0.23 8%PEG,12%PO4+0.48%NaC %o Peg. 12% PO+4.8% NaCl 14.9 0.38 80 PEG, 12%0 POA+9.6%NaCl 0.45 8%0 PEG. 12% PO+17.6%NaCl 228 0.45 surfactant Aerosol-OT(AOT) in isooctane is used because AOT can solubilise a large amount of water in isooctane and similar hydrocarbons, thus forming reverse micelles without the use of co-surfactants. Figure 7.6 shows a diagram of a protein partitioni into reverse micelles. Water pools exist within these microemulsions, and are stabilised y the surfactant. This system can be manipulated so that certain protein species will partition into and out of the water pools, which are a suitable environment in which proteins may exist shielded from the denaturing organic phase(luisi et al., 1988). Thus, reverse micelle solutions have the potential to be used as an extractant phase in a paration process for proteins, offering the following advantages of conventional liquid- liquid extraction: already established continuous processes; use of inexpensive solvents and high volumetric capacities, but with greater selectivity than solvent extraction
-4 -
Innovative separation methods in bioprocessing 187 Reverse ·““ Protein Fig. 7.6. Protein partition into reverse There are two techniques for transferring proteins into the micellar phase. The m widely used method involves extraction of the protein with a biphasic liquid system,i.e iquid-liquid extraction. One phase is the aqueous solution of the protein, and the other the organic micellar solution, usually in equal volume. By gently shaking the two phases, he protein partitions from the aqueous into the micellar phase. In the second method solid state extraction of the protein, the protein powder is suspended in the micellar phase and gently stirred The protein solubilised in the reverse micellar solution can be transferred back into an aqueous solution, by contacting the micellar solution with an aqueous solution containing a high concentration of a particular salt(KCl, CaCl2), which has the capability to ex change with the protein in the micelles The basic idea is that the process of protein extraction by reverse micelles can be made pecific (i.e. tailored to a specific protein) and efficient (i.e. high extraction yield) by ontrolling the micellar parameters such as the water content, the type and concentration of surfactant, the type and concentration of salt, and the pH Leser et al.(1986)examined the transfer of ribonuclease-A, lysozyme, trypsin an pepsin, monitoring the protein concentration and the concentration of water found in the organic phase. It was observed that the transfer of water is generally moderate(beloy 4%), whereas, under certain conditions, the protein is quantitatively transferred. This fact demonstrated that the transfer of the protein into the micellar phase is not a passive process, i.e. is not simply due to the fact that water is transferred and with it the protein The conclusion was that there is a thermodynamic driving force for the hydrophilic protein to leave the aqueous environment and to transfer into the reverse micelles. In other words, it seems that under certain conditions the protein-reverse micelle complex is energetically favoured above the free protein and empty reverse micelles. Interactions can be electrostatic, when surfactants with charged head groups are used, or hydrophobic with the surfactant interface or the apolar solvent
Innovative separation methods in bioprocessing 187 Organic phase 4V& - .t nv Reverse 4 ' L micelle ?fiC%4k/ h 3 nc L.UVVaUL. V UdU UU!iiCUY u Protein 0 a a Aqueous phase Fig. 7.6. Protein partition into reverse micelles. There are two techniques for transferring proteins into the micellar phase. The most widely used method involves extraction of the protein with a biphasic liquid system, i.e. liquid-liquid extraction. One phase is the aqueous solution of the protein, and the other the organic micellar solution, usually in equal volume. By gently shaking the two phases, the protein partitions from the aqueous into the micellar phase. In the second method, solid state extraction of the protein, the protein powder is suspended in the micellar phase and gently stirred. The protein solubilised in the reverse micellar solution can be transferred back into an aqueous solution, by contacting the micellar solution with an aqueous solution containing a high concentration of a particular salt (KC1, CaC12), which has the capability to exchange with the protein in the micelles. The basic idea is that the process of protein extraction by reverse micelles can be made specific (Le. tailored to a specific protein) and efficient (Le. high extraction yield) by controlling the micellar parameters such as the water content, the type and concentration of surfactant, the type and concentration of salt, and the pH. Leser et al. (1986) examined the transfer of ribonuclease-A, lysozyme, trypsin and pepsin, monitoring the protein concentration and the concentration of water found in the organic phase. It was observed that the transfer of water is generally moderate (below 4%), whereas, under certain conditions, the protein is quantitatively transferred. This fact demonstrated that the transfer of the protein into the micellar phase is not a passive process, i.e. is not simply due to the fact that water is transferred and with it the protein. The conclusion was that there is a thermodynamic driving force for the hydrophilic protein to leave the aqueous environment and to transfer into the reverse micelles. In other words, it seems that under certain conditions the protein-reverse micelle complex is energetically favoured above the free protein and empty reverse micelles. Interactions can be electrostatic, when surfactants with charged head groups are used, or hydrophobic with the surfactant interface or the apolar solvent
188 J. A. Asenjo and J B Chaudhuri The fact that electrostatic interactions play an important role in the distribution of oteins over reverse micellar and aqueous phase is shown by the dependence of the aqueous phase pH and ionic strength The ph of the solution will affect the solubilisation characteristics of a protein prima rily in the way in which it modifies the charge distribution over the protein surface. With creasing pH the protein becomes less positively charged until it reaches its isoelectric point (pl). At pHs above the pI the protein will take on a net negative charge. If electro- static interactions play a significant role in the solubilisation process, partition with anionic surfactants should be possible only at pHs below the pl of the protein, where the protein is positively charged and electrostatic attractions between the protein and the surfactant head groups are favourable. At pHs above the pl, electrostatic repulsions ould inhibit protein solubilisation Goklen and Hatton(1987) have presented results on the effect of ph on solubilisation tochrome-c, lysozyme, and ribonuclease-A, in AOT/isooctane reverse micelle solu tions. The results were presented as the percentage of the protein transferred from a I mg/ml aqueous protein solution to an equal volume of isooctane containing 50 mM of the anionic surfactant AOT. A summary of their results is presented in Table 7.3 Table 73. Effect of pH on solubilisation Protel pH range of maximum solubilisation 10 5-10 l1,.1 6-11 As anticipated, only at pHs lower than the pI was there any appreciable solubilisation of a given protein, while above the pl the solubilisation appears to be totally suppressed However, at extremes of pH there is a drop in the degree of solubilisation of the proteins due to protein denaturation, observed as precipitate formation at the interface( Chaudhuri eral.,1993) Luisi et al.( 1979)used the quaternary ammonium salt methyl-trioctylammonium chloride(TOMAC) for the transfer of a-chymotrypsin from water to cyclohexane. It was found that the pH had to be reduced to values significantly below the pI (pI =8.)for there to be any appreciable solubilisation. The solubilisation occurred only over a very narrow ph range before decreasing rapidly again with further decreases in the ph of the aqueous feed phase, accompanied by precipitation at the interface Similar results have been obtained by Dekker et al. (1986) for the enzyme a-amylase Significant solubilisation of the enzyme was observed over a narrow pH range in the vicinity of 10-10.5(pI=5.1). In this pH range, all basic residues will be deprotonated d the only charged residues being the carboxyl groups bearing a negative charge
188 J. A. Asenjo and J. B. Chaudhuri The fact that electrostatic interactions play an important role in the distribution of proteins over reverse micellar and aqueous phase is shown by the dependence of the aqueous phase pH and ionic strength. The pH of the solution will affect the solubilisation characteristics of a protein primarily in the way in which it modifies the charge distribution over the protein surface. With increasing pH the protein becomes less positively charged until it reaches its isoelectric point (PI). At pHs above the PI the protein will take on a net negative charge. If electrostatic interactions play a significant role in the solubilisation process, partition with anionic surfactants should be possible only at pHs below the PI of the protein, where the protein is positively charged and electrostatic attractions between the protein and the surfactant head groups are favourable. At pHs above the PI, electrostatic repulsions would inhibit protein solubilisation. Goklen and Hatton (1987) have presented results on the effect of pH on solubilisation of cytochrome-c, lysozyme, and ribonuclease-A, in AOT/isooctane reverse micelle solutions. The results were presented as the percentage of the protein transferred from a 1 mg/ml aqueous protein solution to an equal volume of isooctane containing 50 mM of the anionic surfactant AOT. A summary of their results is presented in Table 7.3. Table 7.3. Effect of pH on solubilisation Protein PI pH range of maximum solubilisation cy tochrome-c 10.6 5-10 ribonuclease-A 7.8 1-7 lysozyme 11.1 6-1 1 As anticipated, only at pHs lower than the pl was there any appreciable solubilisation of a given protein, while above the PI the solubilisation appears to be totally suppressed. However, at extremes of pH there is a drop in the degree of solubilisation of the proteins due to protein denaturation, observed as precipitate formation at the interface (Chaudhuri et al., 1993). Luisi et al. (1979) used the quaternary ammonium salt methyl-trioctylammonium chloride (TOMAC) for the transfer of a-chymotrypsin from water to cyclohexane. It was found that the pH had to be reduced to values significantly below the PI (PI = 8.3) for there to be any appreciable solubilisation. The solubilisation occurred only over a very narrow pH range before decreasing rapidly again with further decreases in the pH of the aqueous feed phase, accompanied by precipitation at the interface. Similar results have been obtained by Dekker et al. (1986) for the enzyme a-amylase. Significant solubilisation of the enzyme was observed over a narrow pH range in the vicinity of 10-10.5 (PI = 5.1). In this pH range, all basic residues will be deprotonated and the only charged residues being the carboxyl groups bearing a negative charge