JPH07171303A - Aqueous two-phase separation method - Google Patents

Abstract

PURPOSE:To enable industrial utilization and to enhance separation efficiency, in performing separation by utilizing at least one kind of a water-soluble high polymer and other substance forming a two-phase distribution system in combination with the water-soluble high polymer, by allowing a cationic surfactant to coexist in the two-phase distribution system. CONSTITUTION:As a substance system to be separated in an aq. two-phase distribution system, a mixture of a bacterial cell and protein or monomeric matter produced from the bacterial cell or a mixture of two or more kinds of proteins can be designated. The use of a cationic surfactant in the aq. two- phase distribution system is also useful in the improvement of the hue of a liquid wherein a high polymer such as enzyme or protein separated from an organism is dissolved or dispersed in an aq. solvent. For example, when a culture soln. containing enzyme is treated with the aq. two-phase distribution system containing the cationic surfactant, effect is developed in the reduction of coloration or decoloration.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to an improved aqueous two-phase separation process.

[0002]

2. Description of the Related Art As a method for separating a target substance from a mixture of two or more kinds of substances, an aqueous solvent or an aqueous solution and an organic solvent are used, and the difference in distribution coefficient of each substance to those solvents is utilized. A method of separating (or extracting) is generally used. However, this method using an organic solvent cannot often be used particularly for separating biopolymers such as proteins. That is, many biopolymers undergo denaturation upon contact with an organic solvent. Therefore, a water-based polymer that uses a biopolymer such as a protein only as water as a partitioning solvent and utilizes a water-soluble polymer and another substance that is combined with the water-soluble polymer to form a two-phase partition system. A phase separation method (also called an aqueous two-phase partition method or simply two-phase separation method) has already been proposed and is actually used at the laboratory level. Examples of the distribution system include a combination of two types of non-electrolytic polymers, a combination of non-electrolytic polymers and electrolytic polymers, a combination of two types of electrolytic polymers, a polymer and a low-molecular substance. Combinations and the like are known, and they are used in various ways depending on the object to be separated. Specifically, a combination of polyethylene glycol and dextran, a combination of polyethylene glycol and a phosphate (sodium salt, potassium salt, etc.), a combination of polyethylene glycol and citrate (sodium salt, potassium salt, etc.), polyethylene For various substances such as a combination of glycol and sulfate (sodium salt, potassium salt, etc.), a combination of polyethylene glycol and sodium carboxymethyl cellulose, a combination of polyethylene glycol and dextran sulfate, a combination of polyethylene glycol and polyvinyl alcohol. A combination is used.

[0003]

The known aqueous two-phase separation methods are not sufficient in terms of separation efficiency (separation rate, distribution ratio, etc.) and are hardly used industrially. The main object of the present invention is to provide various highly efficient aqueous two-phase separation methods that can be industrially used.

The present invention also provides an aqueous two-phase separation method capable of efficiently separating bacterial cells and proteins or low-molecular substances produced from the bacterial cells.

The present invention also provides an aqueous two-phase separation method capable of efficiently separating two or more proteins from each other.

[0006]

In carrying out an aqueous two-phase separation method utilizing at least one water-soluble polymer and another substance which is combined with the water-soluble polymer to form a two-phase distribution system. An aqueous two-phase separation method, characterized in that a cationic surfactant is allowed to coexist in the two-phase distribution system.

[0007] A bacterium and a protein or a low molecular weight substance produced from the bacterium are combined with at least one water-soluble polymer and the water-soluble polymer in the presence of a cationic surfactant. A method of separation using an aqueous two-phase separation system formed from other substances that form a two-phase distribution system.
Forming two or more kinds of proteins from at least one water-soluble polymer in the presence of a cationic surfactant and another substance which is combined with the water-soluble polymer to form a two-phase partition system. A method of separating from each other using an aqueous two-phase separation system.

Next, the present invention will be described in detail. Examples of the distribution system used in the two-phase distribution system of the present invention include a combination of two known non-electrolytic polymers described above, a combination of a non-electrolytic polymer and an electrolytic polymer, and two types of electrolytic high Examples thereof include a combination of molecules and a combination of a polymer and a low molecular weight substance. Specifically, the above-mentioned combination of polyethylene glycol and dextran, combination of polyethylene glycol and phosphate (sodium salt, potassium salt, etc.), combination of polyethylene glycol and citrate (sodium salt, potassium salt, etc.) , A combination of polyethylene glycol and sulfates (sodium salt, potassium salt, etc.),
A combination of polyethylene glycol and sodium carboxymethyl cellulose, a combination of polyethylene glycol and dextran sulfate, a combination of polyethylene glycol and polyvinyl alcohol, and the like can be mentioned, and these are properly used according to the purpose. Further, as the salts, other alkali metal salts, alkaline earth metal salts, halides, thiocyanates and the like can be used.

Examples of the cationic surfactant used in the aqueous two-phase distribution system in the present invention include the following cationic surfactants. Hexadecyltrimethylammonium salt, tetradecyltrimethylammonium salt, dodecyltrimethylammonium salt, hexadecylpyridinium salt and other quaternary amine salts, dodecylamine salt, octylamine salt, octadecylamine salt and other primary amine salts, and various other types. Secondary amine salts and tertiary amine salts of. In the present invention, the cationic surfactant is usually added in an amount of 0.001 to 5% by weight based on the component (mixture containing water) forming the aqueous two-phase distribution system.

The substance system to be separated by the aqueous two-phase partitioning system of the present invention includes a mixture of bacterial cells and a protein or low molecular weight substance produced from the bacterial cells, a mixture of two or more kinds of proteins, etc. Can be mentioned. Specifically, it is particularly useful for separating proteins (enzymes and the like) from the culture liquid of various bacteria such as protein-producing bacteria and separating the product from the disrupted liquid of bacterial cells.

The use of the cationic surfactant in the aqueous two-phase partition system in the present invention also improves the hue of a liquid in which a polymer such as an enzyme or a protein separated from a living organism is dissolved or dispersed in an aqueous solvent. It is useful. That is, according to the present invention, for example, when a culture solution containing an enzyme is treated with an aqueous two-phase partitioning system containing a cationic surfactant, the hue of the liquid phase in which the enzyme is preferentially distributed is improved (coloration reduction or It also has the advantage of being decolorized.

[0012]

【Example】

[Example 1] Separation of bacterial cells and recovery of protease by aqueous two-phase separation method Extracellular protease producing Bacillus sp. KSM-K1
6 strains (described in JP-A-4-349882)
Was inoculated into a 50 mL liquid medium (comprising the following medium composition) in a 500 mL capacity = Sakaguchi flask and cultured at 30 ° C. for 48 hours to obtain a culture solution. Medium composition Glucose 2.0% by weight Polypeptone S 1.0% by weight Yeast extract (Difco) 0.05% by weight Monopotassium phosphate 0.1% by weight Magnesium sulfate · 7H ₂ O 0.02% by weight Sodium carbonate 1.0 weight%

Separately, five test tubes were prepared, and a light liquid side phase forming substance (polyethylene glycol (PEG) 4000) and a heavy liquid side phase forming substance (sodium citrate) for aqueous two-layer separation were prepared in each test tube. Dihydrate) and 0.7 g in the form of powder. Cationic surfactant (hexadecyltrimethylammonium bromide, HD
AB) was added at different concentrations so as to reach the concentrations shown in Table 1 (the one without addition is for comparison). The above culture solution was added to these test tubes, and the mixture was stirred and mixed to dissolve the powder in the test tubes to prepare a mixed solution of 5 mL. These test tubes were allowed to stand at 15 ° C for 1 hour to separate the cells into the lower layer, and then the upper layer PEG-protease solution was collected using a Pasteur pipette. The amount of the upper layer liquid taken out was 2.0 mL and the amount of the lower layer liquid was 3.0 mL in each test tube.
In addition, about 3 PEG4000 was contained in the extracted upper layer liquid.
5% by weight, 5% by weight of citrate and 0.1% by weight of HDAB.

The protease enzyme activity present in the upper layer liquid and the lower layer liquid obtained from each test tube was determined by the following casein method, and the distribution coefficient (upper layer / lower layer) of the protease in the mixed solution in each test tube was determined. It was Protease enzyme activity measurement method (casein method) Activity measurement method using casein substrate: 50 mM boric acid-NaOH buffer solution (pH 10.0) containing 1% by weight of casein 1
Mix mL with 0.1 mL of protease containing solution,
After reacting at 0 ° C for 10 minutes, 2 mL of a reaction stop solution (0.123M trichloroacetic acid-0.246M sodium acetate-0.369M acetic acid) was added to this, and the mixture was stirred at 30 ° C for 2 minutes.
It was left for 0 minutes. The reaction liquid left to stand was then filtered using a filter paper (Whatman, No. 2), and the protein degradation product in the filtrate was measured by an improved method of the Foreign Lowry method. In addition, 1P. U. Is 1 under the above reaction conditions.
It was defined as the amount of enzyme that liberates 1 mmol of tyrosine per minute.

Table 1 shows the distribution coefficient of protease and the recovery rate of protease in the mixed solution in each test tube, which was obtained from the measurement results . The protease recovery rate was measured by the following method. Protease recovery rate The extracted upper layer liquid was subjected to a molecular weight cut-off of 6000 to 1300.
Using an ultrafiltration membrane of 0, PEG, citrate, and HDAB are permeated by the ultrafiltration membrane method to recover the protease. Furthermore, the relationship between the bacterial turbidity on the light liquid phase (upper layer) side and the concentration of added HDAB is shown in FIG.

Table 1 ──────────────────────────────────── HDAB concentration (mM) 0 25 50 75 100 ──────────────────────────────────── Protease partition coefficient 35 55 64 77 77 117 ───── ─────────────────────────────── Protease recovery (%) 95.8 97.3 97.7 98.1 98 . 7 ─────────────────────────────────────

From the above results, the presence of the cationic surfactant hexadecyltrimethylammonium bromide in the aqueous two-phase partition system increases the partition coefficient of the protease and thus improves the recovery rate of the protease. It turns out that it will be possible.

Example 2 Separation of Protein by Aqueous Two-Phase Separation Method A large number of test tubes were prepared, and a light liquid side phase forming substance (polyethylene glycol (PEG) 4 for aqueous two-layer separation was provided in each test tube.
000) and a heavy liquid side phase forming substance (sodium citrate dihydrate) were added in the form of powder in an amount of 0.7 g. A cationic surfactant (hexadecyltrimethylammonium bromide, HDAB) was sequentially added to each test tube at a concentration of 10 m.
M, 20 mM, 30 mM, 50 mM was added. Also, H
A sample without DAB was prepared for comparison and control. And
Three sets of test tubes having different concentrations of HDAB were prepared. To each group of test tubes, 1 g / L solution of different proteins (bovine serum albumin (BSA), lysozyme, and ovalbumin (manufactured by Egg)) was added and mixed by stirring to dissolve the powder in the test tube. A 5 mL mixture was prepared. The mixed solution in these test tubes was centrifuged at 3000 rpm for 10 minutes to separate into two layers. The upper layer and the lower layer thus separated were collected using a Pasteur pipette. The amount of the upper layer liquid taken out was 2.0 mL and the amount of the lower layer liquid was 3.0 mL in each test tube.

Then, the separated liquids are treated with a wavelength of 280 nm.
And the protein concentration in each solution was measured. The blank in the spectroscopic measurement was a system containing no protein. Then, the ratio of the protein concentration of the upper layer and the protein concentration of the lower layer (upper layer / lower layer) was obtained and used as the partition coefficient of each protein.

FIG. 2 shows the changes in the partition coefficient of the above three proteins when the concentration of hexadecyltrimethylammonium bromide was changed. From the results of FIG. 2, although the effect varies depending on the type of protein, the presence of the cationic surfactant hexadecyltrimethylammonium bromide in the aqueous two-phase partition system allows the protein to be transferred to the upper layer side. It turns out that it will be possible. By utilizing this action and effect, it is possible to obtain the target protein in a high yield from a mixed system in which impurities (or substances not preferably mixed with the target protein) tend to concentrate on the lower layer side in the mixed solution. Become.

[Example 3] Separation of bacterial cells by aqueous two-phase separation method After culturing a strain of the genus Bacillus, the turbidity of the bacterial cells in the culture was adjusted to about 50 using an ultrafiltration membrane. Six samples of the microbial cell turbidity-adjusted culture solution thus prepared were prepared, and polyethylene glycol (PEG) 4000 and sodium phosphate were added to each of them so as to be 14% by weight, and then hexadecyltrimethylammonium was added. Bromide was added to each of the concentrations shown in Table 2. Then, each sample solution was allowed to stand at 25 ° C. for 5 hours, and the turbidity of each of the separated upper and lower layers was measured. The results are shown in Table 2.

Table 2 ──────────────────────────────────── HDAB concentration (mM) 0 5 10 25 50 75 ──────────────────────────────────── Upper layer turbidity 12.3. 0.4 0 0 0 Lower turbidity 65.4.76.2 78.0 77.6 78.0 80.1 ───────────────────── ─────────────── Cell distribution coefficient 0.188 0.005 −−−−−−−− (about 0) −−−−−−−− ─────────── ──────────────────────────

It can be seen that in the above two-layer partitioning system, the addition of hexadecyltrimethylammonium bromide distributes the cells more to the lower layer side.

Example 4 Improvement of Hue by Aqueous Two-Phase Separation Method A large number of test tubes were prepared, and the above-mentioned culture solution obtained in Example 1 was put into each test tube, and the light liquid side of the aqueous two-layer separation was performed. The phase-forming substance (polyethylene glycol 4000) and the heavy liquid side-phase-forming substance (sodium citrate dihydrate) were added in the form of powders so as to be 9% by weight and 14.5% by weight, respectively. A reactive surfactant (hexadecyltrimethylammonium bromide, HDAB) was added at varying concentrations (the one without addition was for comparison). 15 these test tubes
After allowing to stand at 1 ° C. for 1 hour to separate the cells into the lower layer, the hue of the upper layer was measured by the Klett method. The measurement result is shown in FIG. The smaller the value measured by the Klett method (Klett number), the lower the coloring.

HDA as a cationic surfactant
Instead of B, hexadecyltrimethylammonium chloride (cation-A) and alkyltrimethylammonium chloride (alkyl is an alkyl group having 14 to 18 carbon atoms)
The same measurement was performed using (cation-B). The measurement results are also shown in FIG. From the results shown in FIG. 3, the presence of the cationic surfactant in the aqueous two-phase separation system improves the hue including the recovered enzyme and the like, thereby improving the hue of the separated and recovered enzyme and the like. I understand that it will be done.

[0026]

INDUSTRIAL APPLICABILITY The aqueous two-phase separation method of the present invention is excellent in various efficiencies such as separation efficiency and hue improvement efficiency, and is therefore extremely advantageous for industrial use. Then, for example, it is possible to efficiently separate the bacterial cells from the protein or low-molecular substance produced from the bacterial cells, or to separate two or more kinds of proteins.

[Brief description of drawings]

FIG. 1 is a graph showing the results of Example 1 which is an example of the present invention.

FIG. 2 is a graph showing the results of Example 2 which is an example of the present invention.

FIG. 3 is a graph showing the results of Example 4, which is an example of the present invention.

Claims (3)

[Claims] 1. A cationic interface for carrying out an aqueous two-phase separation method utilizing at least one water-soluble polymer and another substance which is combined with the water-soluble polymer to form a two-phase distribution system. An aqueous two-phase separation method, characterized in that an active agent is allowed to coexist in the two-phase distribution system. 2. A microbial cell and a protein or a low-molecular substance produced from the microbial cell, in the presence of a cationic surfactant, at least one water-soluble polymer and the water-soluble polymer. A method of separation utilizing an aqueous two-phase separation system formed from another substance that is combined to form a two-phase distribution system. 3. Two or more kinds of proteins, and at least one kind of water-soluble polymer in the presence of a cationic surfactant,
A method of separating from each other utilizing an aqueous two-phase separation system formed from another substance that is combined with the water-soluble polymer to form a two-phase distribution system.