JPH09169794A - Method for separating proteins - Google Patents

Abstract

(57) [Summary] (Modified) [Problem] Bacteria, viruses and cells in microorganisms and their products during the separation of proteins in the fermented food industry and in the production of cultured biochemical substances, and in the separation of medical products. The present invention provides a method for selectively separating proteins such as bacteria, viruses, cells, etc. according to their types and properties. A method for separating proteins, in which proteins are adsorbed on hydroxyapatite having an aspect ratio of 5 or more and then an aqueous salt solution is circulated through the proteins to selectively separate the proteins.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the separation of microorganisms and their products during the isolation of proteins in the fermented food industry and the production of cultured biochemical substances, or the proteins such as bacteria, viruses and cells in medical biological fluids. Related to methods for separating
The present invention relates to a method for selectively separating proteins such as viruses and cells according to types and properties.

[0002]

BACKGROUND OF THE INVENTION As a method for separating proteins in the fermented food industry, separation of microorganisms and their products during culture production of biochemical substances, separation of proteins such as bacteria, viruses and cells in medical biological fluids. Has traditionally been an inorganic porous material such as silica, activated carbon, diatomaceous earth, cellulose, polyethylene,
There is a method using a nonwoven fabric using an organic fiber such as polyester or a hollow fiber (JP-A-4-156933, JP-A-6-63132) or a metal fibrous substance such as stainless steel, Inconel, and Hastelloy (JP-A-5-130858). . Further, porous hydroxyapatite obtained from vertebrate hard tissue or hydroxyapatite produced by a wet synthesis method is also used as a separating material for proteins such as bacteria.

In addition, the above hydroxyapatite 6
Calcined hydroxyapatite calcined at a temperature of 00 ° C to 1400 ° C is also used as a separating material for proteins. As a method for selectively separating proteins, a biological fluid or the like is cultured in a predetermined medium to form colonies such as bacteria, and only specific bacteria among the formed colonies are cultured in another medium. Bacteria of only specific colonies formed there are cultured in another medium. There is a culture method in which only pure bacteria are cultured and separated by repeating this operation.

[0004]

However, in the conventional method using a separating material for proteins, for example, silica, activated carbon,
Since inorganic substances such as diatomaceous earth are porous, bacteria are incorporated into the pores to separate them from other substances.

Therefore, proteins of a size that can be taken up in the pores can be adsorbed, but other proteins remain discharged as they are in the treatment liquid.

[0006] Further, the adsorption power of these inorganic substances to proteins is merely a physical bond, so that they are not strong and their adsorption capacity is small.

Further, organic polymer fibers such as cellulose, polyethylene and polyester, stainless steel, Inconel,
A filter obtained by processing Hastelloy or the like into a fibrous form uses a mesh of fibrous material as a sieve, and only those physically different in size are selectively filtered. Therefore, it has not been possible to selectively adsorb, separate and sterilize the proteins according to the type and properties of the proteins.

The mechanism for separating proteins by hydroxyapatite is different from the physical separation by the above-mentioned separating material, and since it is adsorption by the electric charge of ions, it has a strong adsorptive separation property for specific proteins. In addition, because of the selectivity due to the three-dimensional structure of the adsorption site on the surface, it is sensitive to the difference in the functional groups on the surface of proteins, and it is expected that the adsorption and separation characteristics will be good depending on the type of protein. .

However, the conventional hydroxyapatite used as a material for separating proteins has been either extracted from natural organisms or synthesized by a wet method. These have poor crystallinity and low adsorptivity, and since they are submicron fine powder, they cannot be molded into a filter themselves, and if they are directly used as a separating material, they have liquid permeability. Could not be secured.

Further, the separation of proteins by the culturing method requires a very long time for separation and requires skill in separation.

[0011]

Therefore, as a result of intensive research to solve the above problems, the present inventor has reached the invention described below.

According to the present invention, there is provided a protein separating material comprising hydroxyapatite having an aspect ratio of 5 or more,
After adsorbing the proteins, a saline solution is circulated through it,
Provided is a method for separating proteins, which is characterized by selectively separating proteins.

The hydroxyapatite preferably has an average c-axis length of 5 μm to 200 μm.

In the present invention, hydroxyapatite whiskers are used as a protein separating material. As the hydroxyapatite used in the present invention, any hydroxyapatite that falls within the above range can be used. Particularly, a hydroxyapatite synthesized by a hydrothermal method can be preferably used.

Hydroxyapatite synthesized by the hydrothermal method has excellent crystallinity and thus excellent liquid permeability, and since the adsorption site on the crystal surface has a well-structured structure, adsorption of proteins is strong, Moreover, the adsorption capacity is large.

Further, the above hydroxyapatite is
Since it is a whisker-like shape in which the A-plane is greatly grown in the c-axis direction, it is possible to adsorb and separate not only the C-plane but also the proteins selectively adsorbed on the A-plane.

Therefore, it is sensitive to the difference in the surface functional groups of the proteins, and the selective separation according to the type of the proteins is possible.

The separating material for proteins used in the present invention is
The reason why proteins can be selectively separated is as follows. The hydroxyapatite, which is the base of the present invention, is a hexagonal columnar crystal, and has A-faces corresponding to the side faces and C-faces corresponding to the bottom face having different properties.

An adsorption site (C site) composed of positively charged calcium ions on the A surface, and an adsorption site (P site) composed of negatively charged phosphate ions on the C surface. Exists. Therefore, acidic proteins having many carboxyl groups and phosphate groups are adsorbed at the C site, and basic proteins having many amino groups are adsorbed at the P site. This action allows selective separation.

The separating material for proteins used in the present invention is
It is hydroxyapatite having an aspect ratio of 5 or more. The aspect ratio is required to be 5 or more in order to effectively adsorb acidic proteins by increasing the area ratio of the A surface. The protein separation material used in the present invention has an average c-axis length of 5 μm to 200 μm.
m hydroxyapatite is preferred. The average c-axis length of 5 μm to 200 μm is the average c
This is because if the axial length is 5 μm or less, liquid permeability cannot be ensured, and if it is 200 μm or more, the stitches become too large and some proteins leak from the stitches during adsorption.

The hydroxyapatite whiskers used in the present invention are used in the form of a layer or in the form of a filter which is preformed. When used in this way, proteins larger than the filtration pore size can be separated as filtration residues regardless of the type of proteins.

Proteins smaller than the filtration pore size are selectively adsorbed to the respective adsorption sites of hydroxyapatite according to the properties of the functional groups of the proteins.

As described above, the hydroxyapatite whiskers used in the present invention can be used alone in the form of a layer or a filter, and
This can be used by holding it on a non-woven fabric, cotton made of natural or synthetic organic fibers, cotton made of inorganic fibers, or a filter cloth or filter paper made of these fibers.

In the present invention, the term “proteins” does not mean only pure proteins, but proteins-based constituents, such as microorganisms such as bacteria and their products, or erythrocytes, etc. It is a concept that also includes components.

When an aqueous salt solution is sequentially passed through the hydroxyapatite filter adsorbing the proteins while increasing the concentration, it is desorbed from the proteins having a weak adsorptivity, so that the proteins are selectively separated and collected. It is possible.

Similar results can be obtained even if the stock solution containing the proteins is a salt solution having a predetermined concentration before the filtration. As the salt used here, it is possible to use as long as it is soluble in water, but potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate, sodium hydrogen phosphate, phosphorus Solutions of phosphates such as sodium dihydrogen acid, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate and the like can be optimally used.

Further, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ammonium chloride,
Salts of potassium hydrogen carbonate, sodium hydrogen carbonate, magnesium hydrogen carbonate, ammonium hydrogen carbonate, potassium carbonate, sodium carbonate, magnesium carbonate, ammonium carbonate and the like can also be used.

The proteins can be selectively collected by the above method by the following mechanism. For example,
When the phosphate solution is passed through hydroxyapatite adsorbing proteins, potassium ions, ammonium ions, etc. are adsorbed at the P site, and phosphate ions are adsorbed at the C site.
At this time, antagonism of adsorption with the already adsorbed proteins occurs, and proteins having a smaller adsorbing power are desorbed from the separation material at a lower phosphate concentration.

By using this method, proteins can be selectively separated in a short time easily.

[0030]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be shown by the following examples.

[0031]

Example 1 25 g lactic acid and 85% H ₃ PO ₄ 6.8
Dissolve 9g in 500ml water and add Ca (OH) ₂
7.37 g was added, and the resulting solution was hydrothermally treated at 165 ° C. for 5 hours to synthesize hydroxyapatite.

As a result of measuring the c-axis length and aspect ratio of this hydroxyapatite with an optical microscope, the average c
The axial length was 21 μm and the aspect ratio was 18.

A cylindrical container having a diameter of 8 mm and a height of 100 mm was filled with 10 g of the above hydroxyapatite to prepare a bacteria separation container.

Staphylococcus aureus (average diameter 0.5 μm) and mycoplasma (average diameter 0.2 μm) were placed in this bacterial separation container.
And influenza virus (average diameter 0.1 μm) were respectively adsorbed.

KH ₂ PO ₄ aqueous solution was added to the hydroxyapatite of the present invention adsorbing this bacterium from 0 mM to 5 mM.
3 ml / mi with a linear gradient concentration of 00 mM
flowed at a flow rate of n. FIG. 1 shows the relationship between the circulation time and the type of bacteria contained in the effluent.

It can be seen that mycoplasma was first isolated, then influenza virus was isolated, and finally Staphylococcus aureus was isolated.

From the above results, it can be seen that according to the present invention, proteins can be selectively separated according to their properties.

[0038]

Example 2 10 g of hydroxyapatite synthesized in Example 1 was filled in a cylindrical container having a diameter of 8 mm and a height of 100 mm to prepare a bacteria separation container.

5 ml of a culture broth of recombinant DNA Escherichia coli which produces a polypeptide was injected into this bacterium separation container.
After leaving the above-mentioned bacteria separation container adsorbing this bacterium for 10 minutes, an aqueous KH ₂ PO ₄ solution was circulated at a flow rate of 3 ml / min with a linear gradient concentration gradient of 0 mM to 500 mM. The concentrations of the eluted E. coli and polypeptide were detected over time with an ultraviolet-visible spectrometer at 280 nm. FIG. 2 shows the relationship between the circulation time and the components contained in the effluent.

First, the polypeptide is eluted, and then E. coli is eluted. Therefore, according to the present invention, the bacteria and their products can be selectively separated from the bacterial culture.

[0041]

Example 3 10 g of hydroxyapatite synthesized in Example 1 was filled in a cylindrical container having a diameter of 8 mm and a height of 100 mm to prepare a bacteria separation container. In this bacteria separation container,
5 ml of recombinant DNA E. coli culture solution producing a restriction enzyme was injected. 1 of the above-mentioned bacteria separation container in which this bacteria is adsorbed
After leaving for 0 minutes, NaCl 10 mM to 150 mM
The solution was allowed to flow for 30 minutes at a flow rate of 3 ml / min with a linear gradient concentration gradient of.

The time-dependent changes in the concentrations of the eluted recombinant DNA Escherichia coli and the restriction enzyme were detected by an ultraviolet-visible spectrometer at 280 nm. FIG. 3 shows the relationship between the circulation time and the components contained in the effluent.

As a result, the restriction enzyme was NaCl 80 mM.
It was found that the recombinant DNA Escherichia coli was eluted with NaCl 115 mM and could be clearly separated.

[0044]

[Example 4] Hydroxyapatite synthesized in Example 1
Filling a cylindrical container with a diameter of 8 mm and a height of 100 mm with 10 g
Filled and made a bacteria separation container. In this bacteria separation container,
Inject 5 ml of recombinant DNA E. coli culture solution that produces glycosides
Entered. The above-mentioned container for separating bacteria which adsorbed this bacterium was used.
After leaving for a minute, (NH _Four )_Two HCO_Three From 500 mM
3 ml / with a linear gradient concentration of 2000 mM
It was circulated for 30 minutes at a flow rate of min. Eluted recombinant D
NA concentration of E. coli and restriction enzyme changes with time of 210 nm
UV-Visible spectrometer. Distribution in Figure 4
The relationship between time and the components contained in the effluent is shown.

As a result, the glycoside is (NH ₄ ) ₂ HCO ₃
At 1800 mM, recombinant DNA Escherichia coli (NH ₄ ) ₂ H
It was found that the product was eluted with CO ₃ 1200 mM and could be clearly separated.

[0046]

INDUSTRIAL APPLICABILITY In the present invention, proteins are adsorbed on a protein separating material composed of hydroxyapatite having an aspect ratio of 5 or more, and a salt solution is circulated through the protein adsorbing material.

Using the method for separating proteins of the present invention,
The adsorbed proteins can be selectively separated and sorted. Therefore, it can be effectively used in industries such as fermentation industries that produce products by microorganisms and industries that separate and produce biological fluids to produce useful substances.

[Brief description of the drawings]

FIG. 1 shows the relationship between the circulation time of the KH ₂ PO ₄ aqueous solution of Example 1 and the type of bacteria contained in the effluent.

FIG. 2 shows the relationship between the circulation time of the KH ₂ PO ₄ aqueous solution of Example 2 and the components contained in the effluent.

FIG. 3 shows the relationship between the circulation time of the NaCl aqueous solution of Example 3 and the components contained in the effluent.

FIG. 4 shows the relationship between the circulation time of the (NH ₄ ) ₂ HCO ₃ aqueous solution of Example 4 and the components contained in the effluent.

Claims (3)

[Claims] 1. A method for separating proteins, which comprises adsorbing proteins to hydroxyapatite having an aspect ratio of 5 or more, and then passing an aqueous salt solution through the adsorbed proteins to selectively separate the proteins. 2. The method for separating proteins according to claim 1, wherein the hydroxyapatite has an average c-axis length of 5 μm to 200 μm and an aspect ratio of 5 or more. 3. The method for separating proteins according to claim 1, wherein the salt solution is a phosphate aqueous solution.