JPH08173780A - Higher fatty acid esterified porous membrane - Google Patents

Abstract

PURPOSE: To obtain a higher fatty acid esterified porous membrane capable of separating, refining and insolubilizing efficiently a protein such as lipase or the like by a method wherein a functional group having a hydrocarbon having a specific number of carbons is chemically bonded to a surface of a base membrane having porosity and its hole surface by an ester linkage. CONSTITUTION: The porous membrane is formed as a porous membrane wherein a functional group having a 6 or over C hydrocarbon is chemically bonded by an ester linkage, which has preferably 0.1mm equivalent - 5mm equivalent of the functional group having the 6 or over C hydrocarbon chemically bonded to a side chain by an ester linkage per a membrane 1, and is formed as a hollow filamentous porous film of 0.01-5μm average hole diameter, 20-80% hole ratio, and 10μm-5mm membrane thickness. Such the higher fatty acid esterified porous membrane is manufactured by treating with higher fatty acid halide after introducing a hydroxyl group onto a surface of a base membrane having porocity and its hole surface.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a membrane which can efficiently separate, purify and insolubilize proteins such as lipase.

[0002]

2. Description of the Related Art Conventionally, beads, gels, fibers, non-woven fabrics, cloths and the like have been generally used as substances for separating, purifying and insolubilizing lipases. Due to the above problem, the pressure loss was large, the SV (superficial velocity) could not be increased, and the productivity did not increase.

[0003]

The present invention utilizes a porous membrane in which functional groups having a hydrocarbon having 6 or more carbon atoms are chemically bonded by an ester bond, and thus proteins such as lipase can be efficiently treated. The purpose is to separate, purify and insolubilize.

[0004]

The inventor of the present invention has found that the above-mentioned object can be achieved by the following means, and has completed the present invention. That is, by using a porous membrane in which a functional group having a hydrocarbon having 6 or more carbon atoms is chemically bonded by an ester bond, efficient separation, purification and insolubilization of proteins such as lipase can be achieved. I was able to.

Further, the porous membrane has a functional group having a hydrocarbon having a carbon number of 6 or more chemically bound to its side chain by an ester bond, in an amount of 0.1 to 5 milliequivalents per 1 g of the membrane, Average pore diameter 0.01-5 μm, porosity 20-80
%, It was found that this can be achieved more efficiently in the case of a hollow fiber-like porous membrane having a thickness of 10 μm to 5 mm. Hereinafter, the present invention will be specifically described.

As the material of the substrate film used in the present invention, most of the materials currently on the market can be used. For example, cellulose (di or tri) acetate, polysulfone, polyvinylidene fluoride, polyamide, polyethylene, polypropylene. Polyolefins such as ethylene-4
Examples thereof include a copolymer of a polyolefin and a halogenated olefin such as a fluorinated ethylene copolymer, polyvinylidene fluoride or polysulfone.

The structure of the porous membrane of the present invention using the substrate membrane may be a flat membrane (pleats, spirals), a tube, a hollow fiber or the like, with the hollow fiber being particularly preferred. In order to secure strength in water, the higher fatty acid esterified porous membrane of the present invention is preferably made of polyolefin, a copolymer of polyolefin and a halogenated olefin, polyvinylidene fluoride or polysulfone, and the inner and outer surface portions of the membrane and A porous membrane in which a functional group having a hydrocarbon having 6 or more carbon atoms is chemically bound by an ester bond to at least a part of the surface of the pore is preferable, and the binding of the functional group to the porous membrane may be direct. Alternatively, a polymer containing a functional group may be bound.

More preferably, the material of the membrane of the porous membrane is polyolefin, the membrane structure has a three-dimensional network structure, and the carbon number of 6 is present on at least a part or the whole of the inner and outer surface portions of the membrane and the surface portion of the pores. A hollow fiber-like porous membrane in which the above-mentioned functional group having a hydrocarbon or a polymer having the functional group is chemically bonded is preferable. Examples of the functional group having a hydrocarbon having 6 or more carbon atoms include a saturated or unsaturated fatty acid residue having 6 or more carbon atoms. Further, the functional group may be one kind, or several kinds of functional groups having different carbon numbers may be used. Further, the higher fatty acid may be a branched one, but in consideration of the influence on the enzyme adsorption ability of the product, it is preferable to use a linear one. Typical examples of these higher fatty acids include palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid.

The present invention will be described in more detail below. The functional groups mentioned above are preferably 0.
It is desirable to contain 1 to 5 meq, and more preferably 0.5 to 4 meq. Below this range, the ability of the membrane to adsorb proteins such as lipase will be reduced, and above this range, other properties of the membrane, such as mechanical properties, will be reduced.

The average pore size of the porous membrane is 0.01 μm to 5 μm.
A range of m is preferred. If it is less than this range, the water permeability is not sufficient for practical use, and the time required for concentration becomes enormous, or if it is more than this range, there is a problem in the ion adsorption performance. Although there are many methods for measuring the average pore size, in the present invention, the air permeation flow rates of the dry membrane and the wet membrane when the air pressure is changed, which is usually called the air flow method, described in ASTM F316-70. Comply with the method to measure from.

The porosity of the porous film is preferably in the range of 20% to 80%. Here, the porosity is measured by preliminarily immersing the membrane in a liquid such as water, then drying, and measuring the weight change before and after that. When the porosity is outside the above range, it is not preferable in terms of permeation rate and mechanical properties. The thickness of the porous film is preferably in the range of 10 μm to 5 mm. Below this range, a problem occurs in the mechanical strength of the membrane, and above this range, the water permeability is not practically sufficient.

The pore structure of the porous membrane can be variously formed by a molding method. For example, polysulfone can be formed into a three-dimensional network structure film by employing a so-called wet method in which polysulfone is used as a mixed solvent using a solvent or the like and then discharged from a nozzle in a hollow fiber shape and molded with a coagulant or the like. In the case of polyolefin, it is possible to form a porous film by so-called stretching or so-called etching radiation, which is produced by chemical treatment after electron beam irradiation, but the pore structure can be obtained by stretching or etching radiation. The microphase separation method and the mixed extraction shown in, for example, JP-B-59-37292, JP-B-40-957 and JP-B-47-17460, rather than the finger structure or the through-hole structure. Those having a three-dimensional network structure formed by a method or the like are preferable in terms of practical performance. However, the method is not limited to this. In particular, JP-A-55-
Specific surface area 50 to 500 disclosed in Japanese Patent No. 131028.
m ² / g, 7 to 42% by volume of inorganic fine powder having an average primary particle diameter of 0.005 to 0.5 μ, 30 to 75% by volume of an organic liquid having an SP value of 8.4 to 9.9, and a number average molecular weight of 15 1,000
A three-dimensional network obtained by mixing 10 to 60% by volume of a polyolefin resin having a weight average molecular weight of less than 300,000 and melt-molding the mixture, and then extracting an organic liquid and an inorganic fine powder from the molded product. By using a porous membrane having a structure, it is possible to achieve excellent adsorption performance that cannot be obtained by the conventional techniques.

In order to introduce the above-mentioned functional group into the side chain of the polymer constituting the porous membrane, it may be introduced before molding into a membrane, but the functional group should be as uniform as possible on each surface of the membrane. It is preferable to bond to the surface of the pores of the membrane preferentially, so it is preferable to chemically add-bond to the inner and outer surfaces of the membrane and at least a part of the surface portion of the pores after molding into the membrane. preferable.

The amount of the functional group in the present invention is the amount of the porous membrane 1
It refers to milliequivalents per gram, and here, the film 1 g is a value based on the total weight of the film, and for example, a part of the surface of the film or a part of the inside thereof is taken out. Not that. In order to bond functional groups while maintaining the excellent mechanical properties of the membrane, make the surface of the pores of the membrane as uniform as possible,
Since it is preferable to allow the functional group to exist more preferentially,
Naturally, partial heterogeneity is acceptable. Therefore, the meaning of the film 1g mentioned here indicates a value which is uniformly measured over the entire surface of the film, and does not mean the weight from an extremely microscopic viewpoint.

In the method for producing a higher fatty acid esterified porous membrane of the present application, when the base material film does not have a reaction active point for reacting with the functional group, first, the reaction active point is introduced into the base material film. Then, a method of directly reacting the higher fatty acid halide or covalently bonding the higher fatty acid through at least one coupling agent can be mentioned.

Specifically, after generating radicals by irradiating an electron beam or γ-ray to the base material film, a method of introducing the above functional group by graft polymerization, or after coating the above-mentioned functional group-containing polymer into the membrane pores , A method of immobilizing with an electron beam or γ-ray, or a method of reacting a functional group after introduction of a hydroxyl group by contacting ozone water to a base material film,
Considering the uniformity, a method of introducing a functional group by a graft polymerization reaction after γ-ray irradiation is preferable. The degree of esterification with respect to the reaction active point depends on the type of reaction solvent, the amount of acid halide added,
It can be arbitrarily defined depending on the reaction time, reaction temperature and the like.

The reaction active site of the higher fatty acid halide may be any functional group such as a hydroxyl group or an epoxy group as long as it is a functional group capable of esterifying the higher fatty acid halide. The reaction solvent is a solvent capable of dissolving a higher fatty acid halide such as acetone, dioxane, chlorobenzene, toluene, ethyl acetate, dibutyl ether, etc., and is inert and does not substantially affect the acid esterification reaction. It is desirable to use a solvent. Moreover, it is not necessary to use a solvent by reacting only the higher fatty acid halide.

Since this reaction is an esterification reaction and is usually accompanied by a by-product of hydrochloric acid, in order to supplement this, for example, triethylamine, trimethylamine, pyridine, N-alkylmorpholine, N- It is preferable to add an aliphatic, aromatic or alicyclic tertiary amine such as alkyl piperidine or N-alkylpyrrolidine or a base such as ammonium acetate or potassium 2-ethylhexanoate.

The role of the higher fatty acid esterified porous membrane of the present invention is very important. That is, in the case of using the above-mentioned porous film having a side chain having a functional group bonded thereto, the treatment speed is much higher than that in the case of using the functional group-bonded beads, gel, fiber, nonwoven fabric, cloth or the like. The column can be made fast, the column can be made compact, and the amount of desorption liquid can be dramatically reduced, and it can be completely desorbed. Also, because of the structural advantages,
Colloidal substances can be removed at the same time. These are extremely great advantages in efficiently separating, purifying, concentrating and insolubilizing proteins such as lipase.

Further, in the case of a membrane, it is easy to scale up, and it is sufficient to change the amount of the membrane from a small scale such as a lab scale to an industrial plant scale, and other scale-up factors such as water permeation rate can be set. It can be handled without improvement. The present invention will be described below with reference to examples, but these do not limit the present invention.

[0021]

【Example】

[0022]

Example 1 and Comparative Example 1 Preparation of Palmitic Acid Esterified Porous Membrane 22 parts by weight of finely divided silicic acid (Aerosil R-972; Nippon Aerosil Co., Ltd.), 55 parts by weight of dibutyl phthalate (DBP), polyethylene resin powder [Asahi Kasei SH Co., Ltd.
-800 grade] After premixing 23 parts by weight of the composition, it was extruded into a hollow fiber having an inner diameter of 2 mm and a thickness of 0.5 mm with a 30 mm twin-screw extruder, and then, was extruded into 1,1,1-trichloroethane. It was immersed for 60 minutes to extract DBP. Further, after immersing in a 40% aqueous solution of caustic soda at a temperature of 60 ° C. for about 20 minutes to extract finely divided silicic acid, it was washed with water and dried.

After irradiating the obtained porous film with γ-rays at 100 KGy in an N ₂ atmosphere, vinyl acetate was graft-polymerized in the gas phase. After washing and drying, saponification treatment is performed with 1N NaOH to obtain an average pore diameter of 0.25 μm, a film thickness of 0.5 mm, a porosity of 62%, and a graft ratio of polyvinyl alcohol group of 25.
% Film was obtained. The film was immersed in a palmitic acid chloride solution, reacted at 30 ° C. for 15 hours, then washed and dried. As a result, it was possible to introduce a palmitate group of 2.8 meq / g film. Here, the amount of introduced palmitate ester group is quantified by the gravimetric method, and qualitatively by FT-.
By IR measurement. The definition of the graft ratio of the example membrane was as follows.

[0024] Preparation of palmitate gel 100 g vinyl acetate, triallyl isocyanurate 4
1.4 g, decahydronaphthalene 56.4 g, and 2,
Homogeneous mixture consisting of 3.5 g of 2'-azobisisobutyronitrile, polyvinyl alcohol 1% by weight, sodium dihydrogen phosphate dihydrate 0.05% by weight and sodium dihydrogen phosphate dodecahydrate 80% water containing 1.5% by weight
0 ml and 0 ml were put into a 2 l flask, and after sufficiently stirring, 60 ° C
The suspension polymerization was carried out by heating and stirring for 18 hours at 75 ° C. for 5 hours, and a granular polymer was obtained. After filtering and washing with water, and then extracting with acetone, sedimentation classification was carried out several times in water, and the average particle size (D
W) A 9.2 μm gel was obtained. DW is measured by Coulter Counter ZB type (US Coulter Electronics Co.)
Made in. 50 g of this gel is mixed with sodium hydroxide 5
A saponification reaction was carried out by adding 0.0 g of the solution dissolved in 1000 ml of water into a 2 l flask and stirring at 15 ° C. for 20 hours. After the reaction, the gel was washed with water and classified to have a DW of 9.9μ.
m gel was obtained. The saponification rate was 95%. This gel is immersed in palmitic acid chloride solution and stirred,
After reacting at ℃ for 15 hours, it was washed and dried. As a result, it was possible to introduce 20% of palmitic acid ester groups to hydroxyl groups.

The palmitate esterified porous membrane and palmitate esterified gel synthesized by the above-mentioned production method are made into a module and a column each having a diameter of 10 mm and a length of 100 mm, and the same amount of the lipase-containing liquid is added to the water-permeable liquid. Of S
When V (superficial velocity) was changed and water was permeated, the amount of trapped lipase was measured. The trapped lipase was washed with water, and then the lipase activity in the eluate when eluted with a 0.2% aqueous solution of a surfactant (Triton-100) was measured.
Table 1 shows the results.

When a palmitate esterified porous membrane was used, the palmitate esterified gel was used although the amount of lipase captured by raising the SV (superficial velocity) of the lipase-containing water-permeable liquid hardly changed. In this case, as the SV (superficial velocity) of the lipase-containing water-permeable liquid was increased, the amount of trapped lipase was decreased, and when a gel was used, if the SV was increased, the pressure loss became too large, making water impermeability impossible. .

[0027]

[Table 1]

[0028]

EFFECT OF THE INVENTION By using the higher fatty acid esterified porous membrane of the present invention, the enzyme solution can be treated with a high SV, so that the enzyme such as lipase can be efficiently separated without lowering the capture rate. , Can be purified and insolubilized.

Claims (3)

[Claims] 1. A higher fatty acid esterified porous structure in which a functional group having a hydrocarbon having 6 or more carbon atoms is chemically bonded by an ester bond to the film surface and the pore surface of the porous base material film. Membrane. 2. A functional group having a hydrocarbon having 6 or more carbon atoms is chemically bonded by an ester bond and a hydroxyl group is chemically bonded to the surface of the porous base material film and the surface of the pores. A characteristic higher fatty acid esterified porous membrane. 3. A method for producing a higher fatty acid esterified porous membrane, which comprises introducing a hydroxyl group into the membrane surface and the pore surface of a porous substrate membrane and then treating with a higher fatty acid halide.