JPS5924659B2 - protein adsorbent - Google Patents

Description

[Detailed description of the invention] The present invention relates to a new adsorbent for proteins.

Traditionally, inorganic substances that physically adsorb proteins include activated carbon, porous glass, acid clay, kaolinite,
Alumina, silica gel, bentonite, hydroxyapatite, calcium phosphate gel, starch among organic substances,
Gluten and the like are known, and these protein-adsorbing substances are widely used for purposes such as adsorption, separation and recovery of proteins, removal of contaminated proteins, clarification of liquids, and immobilization of proteins.

However, these substances have weak adsorption power as protein immobilization carriers, and proteins tend to separate from them. Furthermore, some adsorbent substances may have a negative effect on proteins, and others may not have sufficient mechanical strength, poor operability, equipment limitations, contamination of fine particles, or poor manufacturing methods. It had various drawbacks such as difficulty. The present inventors have conducted painstaking research to develop a new protein adsorbent with industrial versatility that does not have these drawbacks. We found that a linear molded product of a nitrile group-containing polymer obtained by blend-molding a mixture of hydrophilic polymers and substantially removing the hydrophilic polymer adsorbed a surprisingly large amount of protein. Based on this knowledge, we developed the present invention. I came up with an invention.

The adsorbent of the present invention is produced by blend-molding a mixture of a nitrile group-containing polymer containing 20% by weight or more, preferably 35% by weight or more, and a highly hydrophilic polymer together with a small amount of a plasticizer, and then forming a highly hydrophilic polymer. The polymer can be obtained by substantially washing off the polymer with a suitable solvent. The molded product of the present invention thus obtained is a linear collection of nitrile group-containing polymers, which means that the nitrile group-containing polymers present linearly in the melt-extruded product are The highly hydrophilic polymer filled between these and connecting them is dissolved and removed, resulting in a bare state.
Therefore, in the present invention, a linear molded product of a nitrile group-containing polymer refers to an aggregate of such linear products. Further, in the present invention, "substantially washing and removing" means removing the nitrile group-containing polymer so that a linear form is realized. That is, the present adsorbent adsorbs a large amount of protein with an adsorption force strong enough to be used as an immobilization carrier, and is chemically extremely stable and does not denature the protein. Furthermore, it will not be damaged by adsorption operations or column filling. These features of the present adsorbent, together with the fact that its manufacturing method is extremely simple, allow it to be used in a wide range of applications as a versatile adsorbent. As the highly hydrophilic polymer used in the production of the adsorbent of the present invention, those having a water absorption rate of 50% or more at room temperature can be used, and among them, polyvinyl alcohol and vinyl alcohol copolymers, polyethylene glycol, and polyethylene glycol-containing polymers can be used. Polymers, polyacrylamide and acrylamide copolymers, polyN-vinylpyrrolidone and N-vinylpyrrolidone copolymers, polyacrylic acid and acrylic acid copolymers, polymethacrylic acid and methacrylic acid copolymers, carboxymethyl cellulose, etc. is suitable. Among these, highly hydrophilic polymers having an amide group or carboxyl group in the molecule are particularly preferred for the purpose of the present adsorbent. Furthermore, considering that the highly hydrophilic polymer will be substantially removed later, it is more desirable from a production standpoint that the highly hydrophilic polymer is soluble in water or hot water. The plasticizer used in producing the adsorbent of the present invention includes:
Various highly polar compounds such as dimethylacetamide, dimethylformamide, and water can be used, but water is usually used. Blend molding as used in the present invention means mixing the nitrile group-containing polymer and the hydrophilic polymer in a general mixer for polymer molding, and molding the mixture in a general molding machine.

As a mixer, a general mixer such as a second machine, a Banbury roll, an extruder, etc. can be used. General molding machines such as press machines, extruders, and injection molding machines can be used as molding machines, but extrusion molding, which allows mixing and molding at the same time, is preferred. The temperature during melt extrusion varies depending on the type of highly hydrophilic polymer, the type and amount of plasticizer, etc., but is usually 150 to 200°C.

The nitrile group-containing polymer used in the present invention basically has a nitrile group content of 20% by weight or more,
Preferably, the content is 35% by weight or more, as long as it can maintain a linear shape and is insoluble in the cleaning solvent.

Specifically, acrylonitrile polymers and copolymers, methacrylonitrile polymers and copolymers, cyanide pinylidene polymers or copolymers, cyanoethyl cellulose,
Various vinyl polymers and condensation polymers such as N-cyanoethyl-poly-β-alanine are used. In addition, as a copolymerization component of acrylonitrile and methacrylonitrile, various vinyl monomers that can be copolymerized with these can be used, but comonomers having (N-substituted) amide groups or carboxyl groups in the molecule are especially suitable for this adsorption method. preferred for agent purposes. However, the content of nitrile group is 20% by weight?
If it is less than this, the effects of the present invention cannot be achieved. The nitrile group-containing polymer linear molded product used in the present invention can be used as an adsorbent as it is, but it can also be used in various forms such as paper, nonwoven fabric, thread, granules, and fabric. It can also be processed and used.

So far, commonly available nitrile group-containing polymer linear molded products include bonnel (manufactured by Mitsubishi Rayon Co., Ltd.), engineered kudzulan (manufactured by Nippon Exlan Co., Ltd.), and cashmilon (manufactured by Asahi Kasei Industries, Ltd.) for clothing. There are acrylic fibers for use.

However, these polymers not only have a structure different from that of the adsorbent of the present invention, but also have a significantly low protein adsorption ability. In order to form a complex between the adsorbent of the present invention and a protein, which is made of the molded article produced as described above or its processed product, the temperature is 2°C to 90°C, preferably 5°C to 60°C,
It is sufficient to bring the adsorbent into contact with the protein within a pH range of 1 to 11; outside this range, protein adsorption is extremely slow or hardly occurs.

The contact time varies depending on the type of protein, concentration, temperature, pH, adsorbent and its amount, contact method, etc. The adsorption operation is carried out by adding the adsorbent of the present invention to a protein-containing solution, and the formation of an adsorption complex can be facilitated by shaking or stirring as necessary. The purpose can also be achieved by packing the adsorbent on a column or filter and flowing the protein solution through it. When a protein and an adsorbent are brought into contact to form a complex, depending on the purpose, it is recommended to use a crosslinking agent such as glutaraldehyde, hexamethylene diisocyanate, N,N'-ethylene bismaleimide, etc. This method not only does not impede the effectiveness of the adsorbent in any way, but also forms a stronger composite to obtain a preferred composite. The protein that forms a complex with the adsorbent of the present invention is not particularly limited in its type as long as it has a molecular weight of 1,000 to 1,000,000.
Globulin, hemoglobin, proteases, amylase, glucoamylase, invertase, lactase,
Pectinase, β-galactosidase, cellulase, lipase, esterase, urease, naringinase,
Nucleoside phosphorylase, transphosphorylase, kinase, transaminase, lyase,
Aldolase, carboxylase, decarboxylase, marizquenzyme, tryptophanase, hydratase, especially fumarase, aspartase, cysteine desulfhydrase, albinine deiminase, dehydrogenase, peroxidase, catalase, oxygenase, cytochrome, xanthine oxidase, amino acid oxidase, racemase, xylose isomerase,
thrombin, adenyldeiminase, penicillinase,
Enzyme proteins such as urigase and cholesterol oxidase, proteinaceous hormones such as insulin, gonadotropin, vasopressin, and growth hormone, peptide antibiotics such as bacitracin and colistin, α-phetoprotein, carcinoembryonic antigen, histones, protamine, and prolamin. There are many proteins such as the following.

Among these, α-chymotrypsin, bacitracin, cytochrome C1 ovalbumin, catalase, γ-globulin, hemoglobin, urease, serum albumin, trypsin, etc. provide preferable complexes in terms of adsorption amount, strength, and use. It is known that these proteins are not released by ordinary washing methods such as water or phosphate buffers, and form strong complexes. The complexes consisting of these proteins and the adsorbent of the present invention are novel, and in particular those having a protein amount of 25TI9 or more per adsorbent 19 have not been found in known nitrile group-containing polymers. be. Among them, the amount of protein is 40ml! The above-mentioned enzymes can of course be used for the purpose of adsorption, removal and recovery, and can provide extremely excellent reaction efficiency as immobilized enzymes. Complexes with antibodies or antigens also provide high efficiency as carriers for affinity separations. In recent years, the development of industrial utilization techniques for proteins has progressed significantly, and there is an extremely high demand for techniques for adsorption, recovery, concentration, removal, and immobilization of proteins.

The adsorbent of the present invention is an adsorbent that can be applied to any of these fields. Namely, removal of contaminated proteins from foods and brewed products, removal and concentration of proteins from blood, concentration recovery from enzyme production solutions, recovery of proteins from waste solutions, and production of immobilized proteins such as antigens, antibodies, and enzymes. It also has a surprisingly wide range of applications, such as affinity separation and enzymatic reaction applications. Next, the present invention will be explained in more detail using practical examples.

Examples 1 to 6 Comparative Examples 1 to 6 A 1:1 mixture of an acrylonitrile copolymer consisting of 90% acrylonitrile and 10% methyl acrylate and various hydrophilic polymers listed in Table 1 was added to 15 weight? of water was added thereto and melt extrusion was performed at a temperature of 200°C to obtain a sea-island-shaped and band-shaped molded product containing a hydrophilic polymer as a sea component.

Next, this strip-shaped molded product is 3 to 5 m7! Cut it to the length of
The hydrophilic polymer of the sea component was eluted with hot water at about 80°C to obtain a polyacrylonitrile linear molded product. In order to compare with this molded adsorbent, polyacrylonitrile powder, spongy body, and flaky body were manufactured.

First, add polyacrylonitrile powder to ethyl acetate at 20%
weight? At a concentration of 90% by weight of acrylonitrile? It was obtained by dissolving monomers having a composition of 10% and methyl acrylate, and polymerizing the mixture at 180° C. for about 10 hours in a nitrogen atmosphere using azobisisobutyronitrile as an initiator. The particle size of the powder was approximately 30μ. A spongy body was obtained by dissolving a polyacrylonyl powder obtained in dioxane in dimethylformamide and gelling it while mixing air into the solution as described below. The apparent specific gravity was 0.12. The flakes were obtained by dissolving this polyacrylonitrile powder in dimethylformamide at a concentration of 5%, and pouring this solution into water under high speed stirring. The bulk density of this flaky body is 0.39/CC, and the specific surface area is 45rf
It was I/9. These molded products and granular activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.), each with a dry weight of 0.159, were added to 10 ml of a solution containing 2.8 η/ml of human serum albumin (
PH6. O) and shaken at 30°C for 3 hours. After shaking, a complex of the molded product and human serum albumin was obtained. The protein in the supernatant was extracted using the method of LowryOH et al.
l, J. BlOl. Chem. Volume 193, page 265, 1
951), and the difference from the control without moldings was taken as the adsorption amount. The results are summarized in Table 1. The amount of protein adsorbed was expressed as the weight of protein adsorbed per 100Tn9 of the dried molded product. The bonnell of Comparative Example 1 is manufactured by Mitsubishi Kasei Corporation, and the cashmilon of Comparative Example 2 is manufactured by Asahi Kasei Corporation.

As shown in Table 1, all of the practical examples showed an adsorption amount of 7Tf19 or more, and the adsorbent could be removed immediately after adsorption to obtain a supernatant.

On the other hand, in Comparative Examples Nos. 3 and 5, which adsorbed a relatively large amount of protein, the adsorbent was not completely recovered even after centrifugation at 3000 rpm for 30 minutes, and the supernatant was only obtained after centrifugation at 6000 rpm for 40 minutes. I was able to get the liquid. The adsorption amount of other comparative examples was much smaller than that of the practical example. Practical Example 7 Five types of copolymers shown in Table 2 containing acrylonitrile, methacrylonitrile, and vinylidene cyanide were synthesized by a known radical polymerization method using dioxane as a solvent.

To an equal mixture of these polymers and polyN-vinylpyrrolidone was added 20% water based on the total polymer to give 18
A belt-shaped molded product containing poly-N-vinylpyrrolidone as a sea component was produced by melt extrusion at "C".

Next, this strip-shaped molded body was cut into lengths of about 5 mm and then heated at 70 degrees Celsius.
PolyN-vinylpyrrolidone, a sea component, is removed in hot water from
The desired linear molded product was obtained. Each of these linear molded products was weighed to have a dry weight of 0.159, and 10 ml of a solution (PH
6. O) and shaken at 30°C for 3 hours. After shaking, the complex of the molded product and α-chymotrypsin and the supernatant were separated. The amount of protein adsorption was measured using the same method as shown in Practical Example 1 below, and as shown in Table 2,
It showed an adsorption amount of 7η or more. Examples 12 to 19 0.159 of the molded product shown in Practical Example 4 was added to 10 ml of each protein solution shown in Table 3, and the amount of protein adsorption was measured in the same manner as Practical Example 1.

As a comparative example, cashmilon fiber (manufactured by Asahi Kasei Corporation) was measured in the same manner. The results are summarized in Table 3. As a result, it was found that it has an adsorption capacity three and a half times or more compared to cashmilon fiber.

Furthermore, it exhibited an amazing adsorption capacity of 15η or more for trypsin and hemoglobin. Practical Example 20 The molded product and trypsin complex (immobilized trypsin) obtained in Example 18 was washed three times with 0.01M phosphoric acid buffer.

The relative activity after washing remained unchanged at 80% after the second wash. 10 ml of a solution of benzoyl arginine ethyl ester hydrochloride in 0.05M Tris buffer (349/l, PH8.0) was added to the washed immobilized trypsin, and the mixture was shaken at 25°C to carry out a reaction. After the reaction, the immobilized trypsin was recovered by centrifugation, fresh substrate solution was added, and the reaction was repeated. The reaction solution was prepared by David Schubert (G.
W. Schwert et al. (Biohimi)
BiOhlm.BiOphy
sicaActa) vol. 16, p. 570, 1955).
The reaction was repeated 10 times and the results are shown in the attached drawing. 1
Approximately 40% by the third reaction for the activity of the first reaction? loses its activity, but after that the activity remains almost constant.

That is, about 60% of the initial activity forms a strong complex and can be used repeatedly without loss of activity.

[Brief explanation of the drawing]

The drawing is a graph showing changes in relative activity when a complex of trypsin and a molded article was repeatedly reacted (Example 20).

Claims (1)

[Claims] 1 A line of nitrile group-containing polymer obtained by blend-molding a mixture of a nitrile group-containing polymer containing 20% by weight or more of nitrile groups and a highly hydrophilic polymer, and then substantially removing the highly hydrophilic polymer. A protein adsorbent characterized by comprising a shaped article.