JPH06238141A - Filter membrane - Google Patents

Abstract

PURPOSE:To easily separate and refine the component produced by microbes by grafting polymers having trapping groups having affinity with the objective component on the surface of the filter membrane and inner surface of narrow pores of the membrane having the membrane base consisting of an inorg. material membrane having narrow pores. CONSTITUTION:The membrane base material of this filter membrane consists of an inorg. membrane having narrow pores. Polymers carried with trapping groups having affinity with the objective component are grafted on the surface of the membrane and on the inner surface of narrow pores. The narrow pores formed on the membrane base has the average pore diameter between >=5nm and <=50mum, or fractional mol.wt. between >=1X10<3> and <=1X10<8>. A soln. containing the objective component is filtered with the filtering membrane to specifically trap the objective component on the trapping groups. Then a soln. of high salt concn. on soln. having isoelectric point pH of urea or grafted polymer is filtered with the membrane on which the objective component is trapped by the trapping groups. Thus, the component trapped by the trapping groups is desorbed and recovered in the transmitting liquid.

Description

Detailed Description of the Invention

[Background of the Invention]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a filtration membrane used in an ultrafiltration method or a microfiltration method and a method for separating a specific component by the filtration membrane.

[0002]

2. Description of the Related Art In recent years, a separation technique using a porous membrane has been remarkably developed. For example, a technique for separating a solvent and ions by a reverse osmosis method, a technique for separating a solvent and a solute by an ultrafiltration method,
Furthermore, it has been put to practical use as a technique for separating particle components in a solvent by a microfiltration method.

Membrane materials conventionally used in such separation techniques include organic materials such as cellulose acetate, polyacrylonitrile, polyamide, polysulfone, and polyethylene, and aluminum oxide, zirconium oxide, silicon oxide, and the like. Inorganic materials are known.

A film made of an organic material (organic film) is formed by an evaporation method, a coacervation method, a stretching method or the like, while a film made of an inorganic material (an inorganic film) is formed by sintering inorganic fine particles.
The film is formed by a method utilizing the phase separation phenomenon.

These membranes are used as membranes for so-called bioindustry for solid-liquid separation of substance production and separation of polymer components. For example, in the case of enzyme production by fermentation of bacterial cells, the target enzyme must be isolated from a fermentation liquid in which particle components such as bacterial cell residue are dispersed. Usually, for polymer components such as enzymes, first perform solid-liquid separation operation to remove hybrid components such as bacterial cells and bacterial cell residues, and then perform a multi-step purification operation to further isolate and concentrate the target polymer. Was going on. Filtration and centrifugal separation have been adopted as solid-liquid separation operations, and adsorption chromatography and affinity chromatography have been adopted as purification operations. Although filtration and centrifugation with a filtration membrane have low separation accuracy, they have the feature that they can be easily operated even in a system with a very high degree of hybridization, while chromatography has high separation accuracy but is affected by hybridization components. Since it has the characteristic of being easy, the target component can be isolated by combining both.

However, the operation of combining these operations in multiple steps is complicated and increases the cost for separation and purification.

[Outline of the Invention]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a filtration membrane capable of separating and purifying a specific component, for example, a component produced by a microorganism, by a simple operation.

Another object of the present invention is to provide a simple method for separating a specific component using the filtration membrane.

[0009]

The filtration membrane according to the present invention comprises:
A filtration membrane using a membrane made of an inorganic material having pores as a membrane substrate, in which a polymer carrying a capture group having an affinity for a specific component is grafted on the surface and the inner surface of the pores. The one.

The first method for separating a target component from a solution containing the target component according to the present invention is the above-mentioned filtration membrane, which comprises a high-capacity group carrying a capture group having an affinity with the target component to be separated. Prepare a filtration membrane with molecules grafted,
The solution containing the target component is filtered through the filtration membrane, whereby the target group is specifically captured by the capturing group, and the high concentration salt solution, urea or graft is captured by the filter membrane capturing the target component in the capturing group. A solution of the polymer thus obtained having an isoelectric point pH is filtered, whereby the components trapped by the trapping groups are eliminated and recovered in the permeate.

Further, the second method for separating the target component from the solution containing the target component according to the present invention is a method for separating a component having a different sign charge from a solution containing a component having a different sign charge. A filter membrane prepared by grafting a polymer carrying an ion dissociative group as a trapping group on the filter membrane, and filtering a solution containing a component having a charge of a different sign with the filter membrane. , Thereby causing a component having a charge having a sign opposite to that of the ion dissociative group to be trapped by the trapping group, while collecting a component having a charge having the same sign as the charge of the ion dissociating group in the permeate, A high-salt-concentration solution is filtered through the filtration membrane that has captured a component having a charge having a sign opposite to that of the base charge, whereby the component captured by the filtration membrane is desorbed and recovered in the permeate. And what That.

[Detailed Description of the Invention] The filtration membrane according to the present invention uses a membrane made of an inorganic material as a membrane substrate. This membrane substrate has pores set between the size of the component that is desired to pass through the membrane and the size of the component that is not desired to pass through. For example, the size of the microorganism is about 1/10 μm to 10 μm, and the size of the crushed material particles in the solution obtained by crushing the culture of the microorganism is 10 nm or more. On the other hand, a polymer component produced by a microorganism generally has a size of about several to several tens of nm and a molecular weight of about 10 ³ to 10 ⁵ . Therefore, the desired polymer component is separated from the culture of the microorganism or its crushed product by filtration using a membrane in which the average pore size or the molecular weight cut-off is set according to the polymer component produced by the microorganism. can do. That is, in order to separate a target polymer component from such a culture of microorganisms or a crushed product thereof, pores having an average pore diameter of 5 nm to 50 μm, preferably 50 nm to 5 μm.
The following pores, or the molecular weight cutoff is 1 × 10 ³ or more 1 ×
A film having pores represented by 10 ⁸ or less, preferably 1 × 10 ⁴ or more and 1 × 10 ⁸ or less may be used.

The membrane substrate is preferably made of an inorganic material from the viewpoint of its strength. Specific examples of preferable inorganic materials include aluminum, zirconium, iron, silicon, tin, titanium, nickel, cobalt, carbon, aluminum oxide, zirconium oxide, silicon oxide, tin oxide, titanium oxide, silicon carbide, aluminum carbide, silicon nitride. , Aluminum nitride, magnesium oxide, nickel oxide, and composite oxides thereof. In particular, aluminum oxide, zirconium oxide, silicon oxide, tin oxide, titanium oxide and their composite oxides, or carbon, silicon carbide and aluminum nitride are preferable.

Further, the film base material is not limited to one made of an inorganic material, but may be made of an inorganic material as a base and formed as a composite with an organic material. For example, there is one in which a thin film made of an organic material is formed on the surface of a filtration film made of an inorganic material. Specifically, a composite film formed by applying a cellulose acetate solution to the surface of an aluminum oxide film, immersing it in water and causing a JP cervation reaction in the cellulose acetate solution can be mentioned.

Such a membrane substrate can be manufactured by a known method or a method similar thereto while adjusting the pores to be set in a predetermined range. For example,
The inorganic material particles are molded with a binder and then fired to obtain porosity, which can be used as a membrane substrate. In this case, the pore diameter or the molecular weight cutoff can be determined by appropriately selecting the size of the particles of the inorganic material that is the starting material. Specific production can be carried out according to the methods described in, for example, JP-A-59-48646, JP-A-3-143535, and JP-A-4-100505.

The filtration membrane according to the present invention comprises the above-mentioned membrane surface of the membrane substrate and the inner surface of the pores of the membrane, which are grafted with a polymer carrying a capturing group having an affinity for a specific component.

The capturing group having an affinity with a specific component is
It means a group having a structure or property capable of specifically capturing a component to be separated by the filtration membrane according to the present invention, such as a protein molecule, by adsorption or the like. Specific examples of such a capturing group include, for example,
Quaternary ammonium, quaternary phosphonium, an ionically dissociable group selected from amino groups, etc., which is dissociated to have a positive charge
Biochemical specific binding to specific molecules such as ionic dissociative groups selected from sulfonic acid group, phenyl sulfonic acid group, carboxyl group, hydroxyl group, phosphoric acid group, which take a negative charge by dissociation, and monoclonal antibodies And a functional group that is hydrophobically bonded and is selected from trimethylsilane, octadecyldimethylsilane, and the like.

Specific examples of the polymer into which such a trapping group is introduced and which is grafted on the surface of the membrane substrate include, for example,
Styrene, methyl methacrylate, ethyl methacrylate,
Examples thereof include polymers or copolymers of monomers such as acrylic acid, vinyl acetate, vinyl alcohol, vinyl chloride, butadiene, cellulose, acrylonitrile, acryloamide, and methacrylonitrile. That is, the polymer to be grafted may be a homopolymer or a copolymer. When it is a copolymer, it may be a block polymer or a random polymer thereof. The polymer to be grafted may be linear or branched.

Here, the bond between the capturing group and the polymer may be at the end of the polymer chain or in the chain.

Specific examples of the polymer into which a preferable capturing group is introduced include sulfonated styrene in which a phenyl group is sulfonated, and one in which an epoxy group is introduced using glycidyl methacrylate and a hydrophobic amino acid is coupled. To be

These polymers are reacted with the functional groups present on the surface of the membrane material to graft polymer chains on the surface of the filtration membrane. The polymer to be grafted may be already grafted as a polymer when reacted with a functional group present on the surface of the membrane material, or may be brought into contact with a functional group on the surface of the membrane material as a monomer to simultaneously graft. The monomer may be polymerized by a polymerization reaction. Further, the capturing group may be previously supported on the polymer chain and grafted, or after the polymer chain is grafted, it may be introduced and supported on the polymer chain.

In these specific reactions, the initiator, its reaction conditions, etc. can be appropriately determined in consideration of the types of functional groups on the monomer and the film surface, the thickness of the graft film to be formed, and the like. . A specific reaction is, for example, S. Y
amashita et al. Appl. Polymer
Sci. , 17, 2935 (1973) and the like.

The graft reaction may be carried out by utilizing the functional group originally possessed by the membrane surface, but the functional group on the membrane surface is replaced with another functional group to modify the membrane surface. Good. As such a modification method, a silane coupling agent such as trimethylchlorosilane or octadecyldimethylchlorosilane is used to provide an alkylsilane group on the surface, a method for providing a phenyl group using dimethylphenylchlorosilane, and γ-aminopropyltrisilane. Examples thereof include a method of providing an amino group using ethoxysilane.

The reaction between the polymer chain and the capturing group may be appropriately determined in consideration of the types of both. For example, “Immobilized Enzyme” edited by Ichiro Chibata, Kodansha Scientific (19
It can be carried out according to the method described in 75).

The thickness of the polymer to be grafted is preferably 500 μm or less, more preferably 1 nm or more and 100 μm or less, and most preferably 20 on the surface of the membrane substrate.
It is not less than nm and not more than 100 μm. The graft membrane is also formed on the inner surface of the pores of the membrane, and its thickness is 1/2 of the pore diameter.
The thickness of the graft film is preferably in the range of 1/3 to 2/3 of the pore diameter.

The filtration membrane according to the present invention can be used as a membrane for so-called bio-industrial substance production solid-liquid separation and polymer component separation. For example, in the case of enzyme production by bacterial cell fermentation, the target enzyme can be efficiently separated and purified from particle components such as bacterial cell residue by a simple operation. Separation of the target component by the filtration membrane of the present invention is performed as follows.

First, a filtration membrane according to the present invention is prepared in which a polymer carrying a capturing group having an affinity with the target component to be separated is grafted. The pores of this filtration membrane must be set to have an average pore size or a molecular weight cutoff capable of filtering the target component. Next, the solution containing the target component is filtered through the filtration membrane according to the present invention. By this filtering operation, the target component is specifically captured by the capturing group when passing through the filtration membrane.

According to a more preferred embodiment of the present invention, the filtration membrane is subsequently subjected to back pressure washing. Interestingly, this washing operation removes components other than the target component from the filtration membrane. The reason is estimated as follows. By the filtration operation, the particle components larger than the pores are captured by the polymer chains on the membrane surface, and the particle components capable of penetrating the pores are captured by the polymer chains existing on the inner surface of the pores. However, the components other than these target components are only physically trapped in the polymer chain, and since this comb-shaped graft polymer has a certain degree of flexibility, it can be washed by back pressure washing. That is, when the permeated liquid is pumped from the permeate side of the filtration membrane, it is easily desorbed by the pressure. On the other hand, the target component is not desorbed from the capturing group at a pressure of about backwashing, and is kept captured by the capturing group as it is.

The fact that components other than the target component can be removed from the filtration membrane by back pressure washing means that the filtration membrane of the present invention also has a high wash recovery property. That is, the filtration membrane according to the present invention, even if the particle component clogs the pores of the membrane, by performing back pressure washing,
It has the advantage that its membrane performance can be easily recovered. Further, since the membrane material of the filtration membrane according to the present invention is made of an inorganic material, it has excellent mechanical strength. Therefore,
For back pressure cleaning, higher pressure, eg 500 KPa
With the above pressure, the solution can be pumped from the permeation side. This is also advantageous in that more efficient back pressure cleaning can be performed.

Subsequently, the target component is trapped in the trapping group, and the solution having a high salt concentration, urea or the grafted polymer having an isoelectric point pH is filtered using a filtration membrane optionally washed by the above washing operation. To do. As a result, the target component trapped by the trapping group can be eliminated and collected as a permeate. Not being bound by the theory below,
The reason why the target component trapped in the trapping group can be recovered by filtering the solution having such a high salt concentration, urea or the isoelectric point pH of the grafted polymer is considered as follows. . That is, when the graft polymer chains in the membrane and the membrane pores come into contact with a high salt concentration solution, urea or a solution of the grafted polymer at the isoelectric point pH, electrostatic repulsion between the polymer chains is generated. It undergoes a major morphological change that is accompanied by a contraction of the polymer chains. As a result, it is considered that the target component captured by the capturing group is eliminated.

A specific example of the high salt concentration solution is an aqueous solution of a salt comprising a combination of a cation selected from lithium, sodium, potassium, ammonium and guanidine and an anion selected from chlorine, sulfuric acid and tannic acid. However, more preferable examples include sodium chloride solution, ammonium sulfate solution, guanidine hydrochloride, tannate and the like. The concentration is preferably 10 mmol / l or more, more preferably 100 m to 10 mol / l. The composition of the solution of the grafted polymer at the isoelectric point pH is not particularly limited, but it is preferable that it does not affect the membrane material and the graft polymer.

Further, according to the filtration membrane of the present invention, it is possible to efficiently separate the components having the different sign charges from the solution containing the components having the different sign charges. For example, each protein can be separated and purified from a solution containing both a protein having a positive charge and a protein having a negative charge.

First, a filtration membrane according to the present invention, on which a polymer carrying an ion dissociative group as a trapping group is grafted, is prepared. The pores of this filtration membrane must be set to have an average pore size or a molecular weight cutoff capable of filtering the component to be separated and purified. Next, a solution containing components having charges of opposite signs is filtered through this filter membrane. By this filtration operation, a component having a charge having a sign different from that of the ion-dissociative group as the trapping group is electrostatically adsorbed to the trapping group.
On the other hand, the component having the same sign as the ion-dissociable group is separated from the particle component that cannot pass through the pores and passes through the pores of the filtration membrane in the permeated liquid in the same manner as in a normal filtration separation operation. Will be collected.

According to a more preferred embodiment of the present invention, the filtration membrane is subsequently subjected to back pressure washing. As in the case of the separation and purification operation described above, the components other than the target component captured by the capturing group are removed from the filtration membrane by this back pressure washing.

The high-salt-concentration solution is filtered using a filtration membrane that has captured a component having a charge having a sign different from that of the ion dissociative group and that has been washed by the above washing operation. It is considered that this reduces the electrostatic interaction between the trapped substance and the trapping group, hydrophobic binding, affinity such as specific binding, and as a result, the target component captured by the trapping group is released. To be

[0036]

The present invention will be explained in more detail by the following examples, but the present invention is not limited to these examples.

Example 1 Production of filtration membrane Inner diameter 7 mm, outer diameter 10 mm, length 200 mm, pore diameter 0.2 μ
m or 0.5 μm tubular internal pressure type alumina ceramic membrane is disclosed in JP-A-3-143535 and JP-A-4-1005.
Prepared as described in No. 05. The film was dehydrated by heating at 500 ° C. and then refluxed in a 5% toluene solution of trimethylchlorosilane for 8 hours to replace the hydroxyl group on the alumina film surface with trimethylsilane. Next, using benzoyl peroxide as a polymerization initiator, styrene was graft-polymerized at 50 ° C. on the methyl group of trimethylsilane on the film surface. This polystyrene was heated to 50 ° C. in trimethyl ether to introduce a chloromethyl group. Further, the chloromethyl group and triethylamine are reacted in DMF at 50 ° C.,
A quaternary ammonium group was introduced.

Example 2 Evaluation of Wash Recovery Property The membrane performance of the filtration membrane produced in Example 1 was evaluated by repeating solid-liquid filtration of the fermentation broth and washing. For comparison, a polystyrene chain was grafted in the same manner as in Example 1 as shown in Table 1 below, but a filtration membrane in which no further group was introduced and a polystyrene chain were further grafted. The membrane performance of the non-performed filtration membrane was similarly evaluated.

As a fermentation broth, a fermentation broth of Bacillus macerans (IFO-3940) which secretes β-galactosidase outside the body was reported by Miyazaki et al. (Agric. Biol. Chem., 5).
2, 625 (1988)).

The filtration operation was performed as follows. That is,
Fermentation liquid with a membrane surface velocity of 1.0 m · s ⁻¹ and filtration pressure of 50 kPa
Under a cross-flow condition of 30 minutes, and then back pressure washing in which the permeated liquid was returned at 200 kPa for 60 seconds was performed to remove the clogging of the membrane. Next, a 1.0 mol / l sodium chloride aqueous solution was filtered through the filter membrane to separate and collect β-galactosidase in the permeate. This filtration-back pressure washing operation-
The cycle of filtering the aqueous sodium chloride solution was repeated 5 times.

The membrane permeation flow rate after repeating 5 times was measured,
The average permeation flow rate of the membrane was corrected by correcting the loss of the permeate due to back pressure washing. Also, the initial membrane permeation velocity (Jφ) of the filtration membrane
And the membrane permeation flow rate (J ₅ ) after repeating the filtration-backpressure washing operation 5 times were compared. Furthermore, the recovery rate of the enzyme was expressed as a percentage by comparing it with the yield when separated and purified by a gel chromatogram. The results are shown in the following table.

[0042]

[Table 1]

[0043]

EFFECTS OF THE INVENTION According to the present invention, by grafting a polymer carrying a trapping group having an affinity with a specific component on the surface of the membrane and the surface of the inside of the pores, it is possible to achieve solid-liquid separation of a substance produced in bioindustry, It can be used as a membrane for separating molecular components, and only the specific component of interest can be recovered by a simple operation.

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Claims (9)

[Claims] 1. A filtration membrane using a membrane made of an inorganic material having pores as a membrane substrate, wherein the surface and the inner surface of the pores are:
A filtration membrane obtained by grafting a polymer carrying a capture group having an affinity for a specific component. 2. The membrane substrate has an average pore diameter of 5 nm or more and 50 μm.
Pore or molecular weight cut off below 1 × 10 ³ or more 1 × 10 ⁸
The filtration membrane according to claim 1, which has pores represented below. 3. The thickness of the grafted polymer is 500 μm
The filtration membrane according to claim 1, wherein: 4. The capturing group is an ion-dissociative group that dissociates to carry a positive or negative charge, a ligand that biochemically specifically binds to a specific molecule, or a functional group that hydrophobically binds to a specific molecule. The filtration membrane according to claim 1. 5. The polymer comprises one or more monomers selected from styrene, methyl methacrylate, ethyl methacrylate, acrylic acid, vinyl acetate, vinyl alcohol, vinyl chloride, butadiene, cellulose, acrylonitrile, acryloamide, and methacrylonitrile. The filtration membrane according to claim 1, which is a polymer or a copolymer. 6. The filtration membrane according to claim 1, wherein the membrane substrate is made of a composite of an inorganic material and an organic material. 7. A method for separating a target component from a solution containing the target component, wherein the filtration membrane according to any one of claims 1 to 6 has affinity with the target component to be separated. A filter membrane on which a polymer carrying a capture group having is grafted is prepared, and a solution containing the target component is filtered through the filter membrane, whereby the target component is specifically captured by the capture group, and the capture group is A high salt concentration solution, a solution of urea or a grafted polymer having an isoelectric point pH is filtered through the filtration membrane having the target component trapped thereinto, and thereby the component trapped in the trapping group is eliminated to remove the permeate. A method characterized by recovering in. 8. A method for separating a component having a different sign charge from a solution containing a component having a different sign charge, the filtration membrane according to any one of claims 1 to 6. , A filter membrane prepared by grafting a polymer carrying an ion dissociative group as a trapping group is prepared, and a solution containing a component having a different sign of charge is filtered through the filter membrane, whereby the charge of the ion dissociative group A component having a charge of a different sign is captured by a capturing group, while a component having a charge of the same sign as the ion dissociative group is recovered in the permeate, and has a charge of a different sign from the charge of the ion dissociating group. A method comprising filtering a high-salt-concentration solution through the filtration membrane that has captured the components, thereby desorbing the components captured by the filtration membrane and collecting them in the permeate. 9. A solution having a high salt concentration is lithium, sodium,
9. An aqueous solution of a salt comprising a combination of a cation selected from potassium, ammonium and guanidine and an anion selected from chlorine, sulfuric acid and tannic acid, the concentration of which is 10 mmol / l or more. The method described.