WO2003080651A1

WO2003080651A1 - Purification of proteins using ionic surfactants and polar solvents

Info

Publication number: WO2003080651A1
Application number: PCT/CA2003/000429
Authority: WO
Inventors: Youn-Ok Shin; Martin E. Weber; Juan H. Vera
Original assignee: Mcgill University
Priority date: 2002-03-22
Filing date: 2003-03-24
Publication date: 2003-10-02
Also published as: AU2003212169A1

Abstract

The invention relates to a method for purifying proteins from various aqueous solutions, extracts and other sources, and comprises: a) contacting a protein solution with an ionic surfactant; b) precipitating the proteins by reaction with the ionic surfactant to yield an insoluble complex; and c) recovering the protein from the insoluble complex by contacting the complex with a ketone, an alcohol, an ester, their mixtures, their aqueous solutions or other suitable polar solvents which dissociates the complex to yield a solid protein product and a solution containing the ionic surfactant. In the case of enzymes, the recovered protein, retains its original activity. The selection of the ionic surfactant, either cationic or anionic, is made based on the pI of the protein. Advantageously, this method can be used as an initial, intermediate, and/or final purification process.

Description

PURIFICATION OF PROTEINS USING IONIC SURFACTANTS AND POLAR SOLVENTS

BACKGROUND OF THE INVENTION

TECHNICAL FIELD

This invention relates to the purification of proteins from an aqueous phase. More specifically the invention relates to the use of ionic surfactants to selectively precipitate proteins in solution and to recover the said proteins, as a solid product, from the protein-surfactant complex using a polar organic solvent such as a ketone, an alcohol, an ester, their mixtures, their aqueous solutions, or other suitable polar solvents.

DESCRIPTION OF THE PRIOR ART

Industrial methods to purify proteins are direct extensions of laboratory scale analytical techniques. They are well described in standard bioseparation textbooks [A. Sadana, Bioseparation of Proteins: Unfolding / Folding and Validation; Ahuja, S., Ed.; Academic Press: New York, 1998][M. Ladisch, Bioseparations engineering: principles, practice, and economics, Wiley-Interscience, New York, 2001]. Crystallization of proteins using inorganic salts with a polar organic solvent is one the most commonly known methods of purification [C.E. Brothers and CY. Kim, Process to solubilize enzymes and an enzyme liquid product produced thereby, U.S. Patent, 4,673,647, June 16, 1987] [P.L. Joergensen, P.E. Pedersen, J. Petersen, T.K. Nielsen and J.M Mikkelsen, Method for protection of proteolysis- susceptible protein during protein production in a fluid medium, U.S. Patent, 5,623,059, April 22, 1997].

It has been known that surfactants precipitate proteins from an aqueous phase [R. Johnson and N. Lloyd, Enzyme purification Process, U.S. Patent 4,634,673, January 6, 1987]. However, it has also been a common belief that ionic surfactants denature proteins [G. Marcozzi, CD. Domenico and N. Spreti, Effects of surfactants on the stabilization of the bovine lactoperoxidase activity. Biotechnol. Prog., 1998, 14, 653-656].

The use of commercial ionic surfactants to purify proteins in solution has been limited to the solvent extraction processes [E.L. Braunstein, N.T. Becker, G.C. Ganshaw and T.P. Graycar, Surfactant-based extraction process, US Patent 6,105,786, August 22, 2000]. Commercial surfactants have also been used to extract proteins by formation of reverse micelles in an organic phase. Surfactants most commonly used to form a reverse micellar phase are; sodium di-(2- ethylhexyl) sulfosuccinate, aerosol OT (AOT), as an anionic surfactant, and trioctylmethyl ammonium chloride (TOMAC), cetyltrimethylainmonium bromide (CTAB) or dioctyldimethyl ammonium chloride (DODMAC) as cationic surfactants [R. Wolbert, R. Hilhorst, G. Voskuilen, H. Nachtegaal, M. Dekker, K. Nan't Riet and B. Bijsterbosch, Protein transfer from an aqueous phase into reversed micelles: The effect of protein size and charge distribution, Enr. J. Biochem., 1989, 184, 627-633][G. Krei and H. Hustedt, Extraction of enzymes by reverse micelles, Chem. Eng. Set, 1992, 47(1), 99-l l l][H.R. Rabie, T. Suyyagh, and J.H. Vera, Reverse micellar extraction of proteins using dioctyldimethylammonium chloride. Sep. Sci. Technol., 1998, 33(2), 241-257]

The recovery of proteins from the reverse micellar phase by contact with an aqueous phase with high ionic strength or extreme pH, is often complicated due to a slow kinetics, protein denaturing, or loss of enzymatic activity [Y. Sun, S. Ichikawa, S. Sugiura and S. Furusaki. Affinity extraction of proteins with a reversed micellar system composed of cibacron blue-modified lecithin. Biotechnol. Bioeng., 1998, 58(1), 58-64]. The change of temperature [V.M. Paradkar and J.S. Dordick, Affinity-based reverse micellar extraction and separation (ARMES): A facile technique for the purification of peroxidase from soybean hulls. Biotechnol. Prog., 1993, 9, 199-203] or addition of alcohol to the reverse micellar phase [M.R. Aires-Barros and J.M. Cabral, Selective separation and purification of two lipases from Chromobacterium viscosum using AOT reversed micelles. Biotechnol. Bioeng., 1991, 38, 1302-1307] have been suggested to improve the recovery of the proteins from the reverse micellar phase. Instead of using an aqueous phase, the use of acetone directly added to the organic phase containing reverse micelles and protein is suggested as a tool to break the reverse micellar structure and to recover the protein from the organic phase [A.N. Kabanov, S.Ν. Νametkin, G.Ν. Evtushenko, Ν.Ν. Chernov, Ν.L. Klyachko, A.N. Levashov and K. Martinek, A new strategy for the study of oligomeric enzymes: γ-glutamyltransfererase in reversed micelles of surfactants in organic solvents, Biochim. Biophys. Ada, 1989, 996, 147-152][M. Goto, Y. Hashimoto, T. Fujita, T. Ono and S. Furusaki, Important parameters affecting efficiency of protein refolding by reverse micelles, Biotechnol. Prog., 2000, 16, 1079-1085].

One inevitable loss of protein, always quoted in the study of reverse micellar extraction processes, is a formation of white water insoluble protein-surfactant complex precipitated at the interface of an aqueous and an organic phase [G. Lye, J. Asenjo and D. Pyle, Extraction of lysozyme and ribonuclease-a using reverse micelles: Limits to protein solubilization. Biotechnol. Bioeng., 1995, 47, 509- 519] [P. Jauregi and J. Narley, Colloidal Gas Aphrons: A Novel Approach to Protein Recovery, Biotechnol. Bioeng., 1998, 59, 471-481]. The white precipitate of protein-surfactant complex at the interface was considered undesirable, since the protein appeared to be severely denatured [M. Adachi and M. Harada, Solubilization mechanism of cytochrome c in sodium bis-(2-hexyl) sulfosuccinate water/oil microemulsion, J. Phys. Chem.,1993, 97, 3631-3640]. Thus, efforts were made to avoid the formation of said complex by increasing the surfactant concentration in the organic phase or by developing a different surfactant to form a new reverse micellar phase [G.J. Lye, J.A. Asenjo and D.L Pyle. Extraction of lysozyme and ribonuclease-a using reverse micelles: Limits to protein solubilization. Biotechnol. Bioeng., 1995, 47, 509-519] [T. Ono, M. Goto, F. Nakashio, and T.A. Hatton, Extraction Behavior of Hemoglobin using Reversed Micelles by Dioleyl Phosphoric Acid, Biotechnol. Prog., 1996, 12, 793-800]. Further research to develop protein- surfactant combinations, which do not lead to protein precipitation at the aqueous-organic interface, is advocated as the only a solution to prevent the loss of proteins due to the formation of white precipitate [P. Jauregi and J. Varley, Colloidal Gas Aphrons: A Novel Approach to Protein Recovery, Biotechnol. Bioeng., 1998, 59, 471-481].

The formation of an insoluble complex between protein and surfactant was proposed as an initial stage of a purification process to remove various impurities from the desired protein solution. A quaternary ammonium compound was used to form a precipitate with unwanted impurities [G.B. Borglum, Enzyme Purification Process, U.S. Patent 3,728,224, April 17, 1973].

Only in one case, the use of a surfactant-protein precipitate has been described as the desirable product, for the particular case of glucose isomerase with ternary and quaternary ammonium salts, since the enzymes in the enzyme-surfactant complex retained its enzymatic activity [R. Johnson and N. Lloyd, Enzyme purification Process, U.S. Patent 4,634,673, January 6, 1987]. The glucose isomerase was recovered from the enzyme-surfactant complex using a strongly ionic solution wherein the complex dissociates to produce a soluble product. Ultrafiltration or ion exchange resins were used as an additional step to separate the protein from the surfactant.

Surprisingly the use of generic ionic commercial surfactants has not been proposed for the purification of proteins by precipitation. Furthermore the subsequent recovery of the protein from the precipitate produced with the surfactant, by means of a polar organic solvent, such as ketone, an alcohol, their mixtures, their aqueous solutions, or other, is also an unexpected result. Particularly because it has been reported that the proteins are denatured when they form an insoluble complex with surfactants, this invention clearly indicates that it is possible to recover the protein from the protein- surfactant complex with near fully recovered properties after being treated with a polar organic solvent or their aqueous solutions. In the case of enzymes, the enzymatic activity is restored. Thus, it is an object of this invention to provide a novel process for precipitation of proteins from aqueous streams using ionic surfactants and to recover the said proteins, as a solid product, from the surfactant-protein water insoluble complex comprising a ketone, an alcohol, an ester, their mixtures, their aqueous solutions, or other suitable polar solvents. The recovery and recirculation of the surfactant and the organic solvent make this a cost efficient process.

SUMMARY OF THE INVENTION

The main object of the present invention is to provide a process for purifying protein from a protein containing aqueous solution, which includes the following steps;

a) mixing the solution with an ionic surfactant;

b) precipitating the protein through a reaction with the ionic surfactant to yield an insoluble complex; and,

c) recovery of the protein from the insoluble complex by contacting the complex with a polar solvent which dissociates the complex to yield, a solid protein product and a solution containing the ionic surfactant; whereby,

the solid protein product retains a majority of the original properties of the protein. BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present invention will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

Figure 1: Percent mass of lysozyme removed via formation of insoluble complex with AOT versus R (Molar Ratio of surfactant to Protein).

Figure 2: Effect of salt on the percent removal of lysozyme forming insoluble complex with AOT: C_p = 1 g/L versus R (Molar Ratio of surfactant to Protein).

Figure 3: Percent mass of ribonuclease removal via formation of insoluble complex with AOT: Cp = 0.1 - 1.0 g/L versus R (Molar Ratio of surfactant to Protein).

Figure 4: Percent mass of cytochrome removal via formation of insoluble complex with AOT: C° = 0.1 - 1.0 g/L versus R (Molar Ratio of surfactant to Protein).

Figure 5: Percent mass of chymotrypsin removal via formation of insoluble complex with AOT: C° = 0.1 - 1.0 g/L versus R (Molar Ratio of surfactant to Protein).

Figure 6: Block Diagram of the Process for the Purification of Proteins using Ionic Surfactants and Polar Solvents.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It has been found that proteins precipitated with ionic surfactants, such as di-(2- ethylhexyl) sulfosuccinate (AOT), trioctylmethyl ammonium chloride (TOMAC), cetyltrimethylammonium bromide (CTAB)and dioctyldimethyl ammonium chloride, (DODMAC), among others, can be recovered from the precipitate as a surfactant-free pure solid after being treated with a polar organic solvent, such as acetone, ethanol, isopropanol and methyl acetate, with acetone and ethanol, and their aqueous solutions, being preferred embodiments. Thus, the present invention provides a method for the purification of proteins in an aqueous solution, either from fermentation, from cell extract or other sources, the method (Represented in Figure 6) comprising the steps of:

1) contacting the aqueous protein solution with a selected ionic surfactants, such as di-(2-ethylhexyl) sulfosuccinate (AOT), cetyltrimethylammonium bromide (CTAB) dioctyldimethyl ammonium chloride, (DODMAC) and or other suitable ionic surfactants (through a simple vortexing for 5 to 10 seconds),

2) achieving precipitation of an insoluble protein-surfactant complex of said soluble protein species by reaction with said ionic surfactant to yield a precipitate (through centrifugation); and

3) recovering the protein from the insoluble complex by contacting (vortexing for 5 to 10 seconds) the said complex with a ketone, an alcohol, an ester, their mixtures, their aqueous solutions, or other suitable polar solvents, possibly including NaCl solutions which dissociate the complex to yield a final solid protein product (by centrifugation) and a solution containing the ionic surfactant. An optional drying step may also be performed to obtain a dried final solid protein product. The ionic surfactant and the polar solvent can be separated by distillation of the solvent or precipitation of the surfactant, or by some other method.

The solvent and solvent mixtures tested were from pure acetone; and pure ethanol; to 50 mole % of ethanol in a binary mixture of acetone; and to 50 mole % of acetone in a binary mixture of ethanol; as well as, up to 60 mole % of ethanol in a binary mixture with water; to 60 mole % of acetone in a binary mixture with water. The selection of surfactants, either anionic or cationic, is made based on the pi (isoelectric pH) of the said protein. For example, if the pi of the said protein is below pH 5, a cationic surfactant is recommended as a reactant to form an insoluble complex with the said protein. Anionic surfactants are to be used for all other proteins. More specifically, the pH of the protein solution should be greater than the pi of targeted protein when cationic surfactant is chosen, and less than the pi of the said protein when anionic surfactant is chosen. Advantageously, this method can be used as an initial, intermediate, and/or final purification process and also to selectively separate two proteins of different pi values.

In step 3), the said insoluble protein- surfactant complex is dissolved in a polar solvent such as acetone or an aqueous solution of ethanol, and surfactant-free protein precipitates out of the said solvent, while surfactant remains in the solution. The polar solvents tested include acetone; methylethyl ketone; methyl acetate; formaldehyde; acetonitrile; methanol; ethanol; isopropanol; pentanol; mixtures of acetone and ethanol; mixtures of acetone and water; and mixtures of ethanol and water. Where the preferred embodiments of the invention are acetone, ethanol, their mixtures with water and ethanol and acetone mixtures. Acetone was found to provide the best recovery of lysozyme, at more than 70%. The recovery of lysozyme with isopropanol was 20%. Aqueous solutions of ethanol and acetone, as well as mixtures of acetone/ethanol were found to provide the best recoveries of xylanase with recoveries of 70%.

If no precipitation of surfactant- free protein is obtained in the polar solvent phase, an addition of small amount of NaCl or other inorganic salt solution (for example, less than lOμL of 0.1 M NaCl solution for 40 mL acetone) can be required. This procedure should not be necessary if the initial protein solution contained ions from any inorganic salt, acid or base. If the initial concentration of inorganic salt in the aqueous solution is less than 10^"3 M, more inorganic salt is added to improve the recovery of the protein from the insoluble complex. The protein containing aqueous solution also contains inorganic salts in a range of concentration that may reach 1 M, but is preferably between 10^"6 to 0.3 M and most preferably between 10^"3 and 0.1 M.

It is to be understood that the invention is not limited in its application to the ionic surfactants, solvents and proteins mentioned herein. The invention is capable of other combinations and of being practiced in various ways. It is also to be understood that the phraseology or terminology used herein is for the purpose of description and not limitation.

The following Examples are for illustrative purposes only and should not be interpreted as limiting the scope of the present invention thereof. Due to the formation of a protein- surfactant complex, the surfactant is sometimes referred to as ligand.

Glossary of Terms

As used herein, the flowing symbols refer to:

R : Molar ratio of surfactant to protein.

Cp : Initial concentration of protein in aqueous solution.

n_PL : Moles of protein in the protein-surfactant precipitate

n_PR : Moles of protein recovered as a solid from the polar organic solvent

N : Total volume of initial aqueous phase containing protein and surfactant EXAMPLE 1

This example illustrates the formation of an insoluble lysozyme- AOT complex and then dissociation of the complex in acetone to produce a purified lysozyme.

An aqueous solution containing AOT (di-(2-ethylhexyι) sulfosuccinate) was directly added to the lysozyme containing aqueous phase. The lysozyme concentration was varied from 0.01 to 0.36 mM (0.15 to 5 g/L). No pH or salt concentration adjustment was made to the solution. The volume of AOT (di-(2- ethylhexyl) sulfosuccinate) solution added to the lysozyme solution was set so that the molar ratio between AOT to lysozyme was from 0.1 to 40. The concentration of AOT in the aqueous phase was below the critical micellar concentration. As soon as AOT was added, the insoluble lysozyme-ligand complex formed instantaneously. The mixture was vortexed for 10 seconds and centrifuged. The lysozyme-ligand complex was then collected, washed with distilled water. The lysozyme-ligand complex was dissolved in acetone, and the surfactant-free lysozyme was recovered as solid.

FIG. 1 shows the percentage removal of lysozyme as a function of the molar ratio between AOT and lysozyme initially added to the aqueous solution, denoted as R. The percentage removal is defined as:

% remo al = _?-?--_ x 100

All symbols of this, and subsequent equations, are defined in the nomenclature. As shown in FIG. 1, for all AOT concentrations tested, an increase in the AOT concentration in the aqueous phase increased the percent removal until it reached a mole ratio of around 10. A 100% removal was obtained at this value of the ratio R. The concentration of AOT remaining in the solution was below the detection limit when R was less than 10, indicating that all the ligand added to the solution formed lysozyme-ligand complex and was removed from the aqueous phase. When R was greater than 10, as all protein was already removed, the excess surfactant remained in the aqueous phase.

The percent recovery of protein, obtained as a solid from the said insoluble lysozyme-surfactant complex using acetone, is defined as:

% recovery = — ^ x 100 ⁿp_L

The percent recovery of the solid lysozyme from the precipitate, for the case depicted in FIG. 1, was found to be 70 ± 18 %. The loss of the mass of lysozyme most probably occurred during washing. The recovered solid lysozyme, when dissolved in an aqueous phase, retained its original enzymatic activity. An activity of 48,000 units per mg protein in solution, was obtained for a purchased enzyme and for the same purchased enzyme recovered from water solution after the treatment by the process of the invention. The' enzymatic activity of lysozyme was measured by the standard assay described by Davies R.C., A. Neuberger and B.M. Wilson, "The dependence of lysozyme activity on pH and ionic strength", Biochim. Biophys. Acta, volume 178, pages 294-305 (1969). The apparatus used was a Gary Varian l/3UV/visible espectrophotometer (Varian Techtron, Pty Ltd., Victoria, Australia). Three replicate samples were used.

EXAMPLE 2

This example illustrates the formation of an insoluble lysozyme-AOT complex when the initial lysozyme solution contained NaCl.

FIG. 2 shows the percent removal of lysozyme with AOT (di-(2-ethylhexyl) sulfosuccinate) when sodium chloride was present the initial lysozyme solution. As shown in FIG. 2, as more salt was added to the lysozyme solution, the amount of lysozyme forming insoluble complex decreased. At 0.3 M NaCl, when R=10, the percent mass of lysozyme precipitated was about 60 % compared with 100% without salt present. About 95 % removal was obtained at R = 20 at 0.3 M NaCl. Further increase in the salt concentration in the initial lysozyme solution to 1 M resulted in further decrease in the percent removal. At 1 M, the efficiency was found to be in a range of 30 to 35 %, and did not have a strong dependence on the molar ratio between the ligand and the protein. This indicates that the lysozyme undergoes not only the formation of insoluble lysozyme-ligand complex, but also that there is salt precipitation at the said salt concentration.

EXAMPLE 3

This example illustrates the formation of an insoluble ribonuclease A -AOT complex and then dissociation of the complex in acetone to produce a purified solid ribonuclease A. Similar to the experimental procedure for Examples 1 and 2, an aqueous solution containing ribonuclease A was prepared without any pH adjustment. The protein concentration was at 0.1 to 1.0 g/L and the salt concentration was at 0 to 0.6 M NaCl in the aqueous phase. An aqueous phase containing 5 g/L AOT was directly added to the initial ribonuclease A solution so that the molar ratio of AOT to the protein, R, to be from 5 to 30.

FIG. 3 shows the percentage removal of ribonuclease as a function of the molar ratio between AOT and ribonuclease initially added to the aqueous solution, R. The percentage removal was defined as in Example 1. As shown in FIG. 3, the percent removal of ribonuclease increased as R increased, and when no salt was added to the initial protein solution, 100 % of the protein in the solution formed an insoluble complex with AOT and precipitated at R= 17. The percent recovery of the solid ribonuclease obtained from the said insoluble complex was found to be 75 ± 25 %. The recovered ribonuclease retained its original enzymatic activity. The activity of ribonuclease A was measured following the method described by E.M. Crook, A.P. Mathias and B.R. Robin, Spectrophotometric assay of bovine pancreatic ribonuclease by use of cytidine -2 ',3 '-phosphate, Biochem. J., 74, (1960) 234-238. The measured activities of the enzyme before and after the process of the invention were 41 + 7 and 42 + 5 units per mg of protein. The said percentage of recovery of protein depends largely on the mass loss during washing.

An increase in the salt concentration in the initial protein solution resulted in a decrease in the percentage removal of protein at a given R. For example, at R= 22, the percentage removal of ribonuclease was about 95 % and 40 % when the salt concentration was set at 0.1 M and 0.3 M NaCl. At 0.6 M NaCl, the percentage of ribonuclease precipitated was found to be around 20 % independent of R, which indicates that at this salt concentration, the protein undergoes precipitation due to salt instead of formation of an insoluble complex with AOT.

EXAMPLE 4

This example illustrates the formation of an insoluble cytochrome c - AOT complex and then dissociation of the complex in acetone to produce a purified solid cytochrome C. The experimental procedure explained in Example 3 was followed in the experiments.

FIG. 4 shows the percentage removal of cytochrome c as a function of the molar ratio between AOT and cytochrome c initially added to the aqueous solution, R. The percentage removal was defined as the sample as in Example 1. As shown in FIG. 4, the percent removal of cytochrome c increased as R increased. A complete removal of cytochrome c via formation of an insoluble complex with the surfactant was at R = 15, when no salt was added to the initial protein solution. The percent recovery of the solid cytochrome c obtained from the said insoluble complex was found to be 65 + 26 %. A loss of mass is expected to occur during washing. The decrease in the percent removal due to the presence of salts, as shown in Examples 2 and 3 is also observed in this example. The percentage of cytochrome c precipitated at a given R decreased as the salt concentration increased in the initial protein solution.

EXAMPLE 5

This example illustrates the formation of an insoluble α-chymotrypsin - AOT complex and then dissociation of the complex in acetone to produce a purified solid α-chymotrypsin. The experimental procedure explained in Example 3 was followed in the experiments.

FIG. 5 shows the percentage removal of α-chymotrypsin as a function of R, the molar ratio between AOT and α-chymotrypsin initially added to the aqueous solution. The percentage removal was defined for the sample as in Example 1. As shown in FIG. 5, the percent removal of α-chymotrypsin increased as R increased, and complete removal of α-chymotrypsin via formation of an insoluble complex with the surfactant was at R = 7. The percent recovery of the solid α-chymotrypsin obtained from the said insoluble complex was found to be 37 + 18%. The recovered protein retained its original enzymatic activity, when dissolved in an aqueous solution. The activity of α-chymotrypsin was measured using the method of B.C. Hummel, "A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin", Can. J. Biochem., 37 (1959) 1393-1399. The recovered enzyme retained its original activity of 43 ± 5 units/mg protein. At 0.3 M NaCl, the percentage removal of protein ranged from 20 to 40 %.

It should be noted, that a decrease in the percent removal due to an increase in the salt concentration in the initial protein solution was found with all the proteins studied (FIGs. 2, 3, 4 and 5). EXAMPLE 6

This example illustrates the formation of an insoluble α-amylase - CTAB complex and then dissociation of the complex in acetone to produce a purified solid α- amylase.

An aqueous solution containing 0.5 g/L α-amylase was prepared. Unlike AOT, CTAB is sparingly soluble in pure aqueous or organic solvents such as isooctane. Thus, CTAB was solubilized by adding known amount of the surfactant in an aqueous phase contacted with an organic solvent, isooctane in this example. The salt concentration in the aqueous phase was set at 0.1 M NaCl to induce the reverse micellar formation by the surfactant in the said organic solvent. The surfactant concentration in the organic solvent was set at 0.1 M. A volume of 0.1 to 1 mL of the CTAB solution was then directly contacted with a 20 mL volume of the initial α-amylase solution (the molar ratio between CTAB and α-amylase complex from 50 to 500). The mixture was centrifuged, and the precipitated α-amylase - CTAB was collected. The best precipitation was obtained when the pH of the protein solution was at pH = 9. An addition of 0.05 to 0.1 M NaCl in the initial protein solution was necessary in order to prevent micellar formation of CTAB in the said phase. When micelles are formed in the aqueous phase, no formation of an insoluble α-amylase - CTAB complex is obtained.

The percent removal, defined as Example 1, was found to be 74 ± 7 %, independent of R, the molar ratio between surfactant and the protein, tested. The protein concentration remaining after a contact with CTAB was found to be 0.13 + 0.03 g/L, indicating that the α-amylase - CTAB complex may be slightly more soluble in water than other protein tested, as described in Examples 1, 3, 4, and 5. The percent recovery of solid α-amylase obtained from the precipitated α-amylase - CTAB complex was found to be 49 + 1 %. EXAMPLE 7

This example illustrates the selective separation of lysozyme from other proteins contained in hen egg-white, mainly albumin, using AOT. The pi of other major proteins in the hen egg white are ovomucoid pi = 4.5 and ovotransferin, pi = 6.

Hen egg-white was separated from the yolk and diluted 50 fold with 0.02M phosphate buffer solution. Analysis of the solution indicated about 0.1 g/L of lysozyme and 1.0 g/L albumin. Similarly, a synthetic mixture was prepared from the pure (purchased) enzyme and pure albumin to obtain 0.1 g/L of lysozyme and 1.0 g/L albumin. Aqueous solutions containing 5 g/L of AOT were added to 20 mL of the aqueous egg-white solutions. The total volume of AOT solution was in the range from 0.1 to 0.6 mL. The highest precipitation of lysozyme obtained was 80% with a pH between 4 and 6. Acetone was used as the organic solvent to recover the lysozyme from the ligand-lysozyme complex. No other egg white proteins were detected when the recovered lysozyme was analyzed. The activity of the recovered lysozyme was only slightly lower than the activity of pure lysozyme purchased from a chemical supplier. The activities obtained at pH 9 were: 40350 ± 2550 units / mg protein for the synthetic egg mixture and 32,520 ± 2580 units per unit protein prepared from egg . The enzymatic activities were obtained using the method cited in Example 1 by Davies et al.

EXAMPLE 8

This example illustrates the selective separation of xylanase from an aqueous solution containing xylanase and cellulase, using AOT.

A volume of 10 mL of an aqueous solution containing 1.68 g/L cellulase and 0.4 g/L xylanase was mixed with 1 mL of a 5 g/L solution of AOT. Upon addition of the AOT solution, a precipitate formed instantaneously. About 50% of the xylanase present in the mixture was recovered in the precipitate. The precipitate was separated and treated with different solvents. Analytical quality acetone and analytical quality ethanol, together with their mixtures, and also mixtures of acetone with water and of ethanol with water were tested as solvents. The largest recoveries of xylanase were obtained using ethanol-water mixtures and acetone- ethanol mixtures. No cellulase was detected in the recovered xylanase. The activities of xylanase and cellulase were controlled following assay protocols obtained from industrial sources known in the art. The percent xylanase activity recovered from the precipitate enzyme, was about 90% of the initial activity phase at pH 4.5. The cellulase activity in the recovered xylanase was below the detection limit.

The embodiments of the invention described above are intended to be exemplary only. The scope of the invention is therefore intended to be limited solely by the scope of the appended claims.

Claims

WHAT IS CLAIMED IS

1. A process for purifying protein from a protein containing aqueous solution, which includes the following steps;

a) mixing the solution with an ionic surfactant;

c) recovery of the protein from the insoluble complex by contacting the complex with a polar solvent which dissociates the complex to yield, a solid protein product and a solution containing the ionic surfactant; whereby

the solid protein product retains a majority of the original properties of the protein.

2. A process according to claim 1, wherein the ionic surfactant and the polar solvent are separated from one another and recycled.

3. A process according to claim 1 or 2, wherein the protein containing aqueous solution contains an inorganic salt in a concentration up to 1 M but preferably between 10^"6 and 0.3 M, and most preferably between 10^"3 and 0.1 M.

4. A process according to any one of claims 1 to 3, wherein the protein containing aqueous solution contains less than 10^"3 M of the inorganic salt and a secondary salt is added to improve the recovery of the protein from the insoluble complex.

5. A process according to any one of claims 1 to 4, wherein the protein containing aqueous solution is obtained from a fermentation broth.

6. A process according to any one of claims 1 to 4, wherein the protein containing aqueous solution is obtained from a cell extract.

7. A process according to any one of claims 1 to 6, wherein the polar solvent is selected from the group consisting of acetone, ethanol, isopropanol, methyl acetate and their aqueous mixtures.

8. A process according to any one of claims 1 to 7, wherein the polar solvent is selected from the group consisting of acetone, ethanol, their mixtures and their aqueous mixtures.

9. A process according to any one of claims 1 to 8, wherein the ionic surfactant is an anionic surfactant used to precipitate proteins with a pi (isoelectric pH) higher than 5.

10. A process according to any one of claims 1 to 8, wherein the ionic surfactant is a cationic surfactant used to precipitate proteins with a pi (isoelectric pH) lower than 5.

11. A process according to claim 9, wherein the anionic surfactant is sodium di- (2-ethylhexyl) sulfosuccinate, aerosol OT (AOT).

12. A process according to claim 10, wherein the cationic surfactant is selected from the group consisting of trioctylmethyl ammonium chloride (TOMAC), cetyltrimethylammonium bromide (CTAB) and dioctyldimethyl ammonium chloride, (DODMAC).

13. A process according to claim 12, wherein the cationic surfactant is trioctyhnethyl ammonium chloride (TOMAC).

14. A process according to any one of claims 1 to 13, where a Molar Ratio of the protein to the ionic surfactant, defined as R, has a value between 0.01 and 30, and preferably between 5 and 20 and most preferably between 7 and 15.

15. A process according to claim 9, wherein the pH of the protein solution is less than the pi of the protein, the anionic surfactant is used.

16. A process according to claim 10, wherein the pH of the protein solution is greater than the pi of the protein, the cationic surfactant is used.

17. A process according to any one of claims 1 to 16, wherein the purification process can be used at any one of following process sequence points; initial, intermediate and final purification.

18. A process according to claim 4, wherein the secondary salt added is NaCl.

19. A process according to claim 2, wherein the ionic surfactant and the polar solvent are separated by distillation of the solvent or by precipitation of the surfactant.