JPS6032497B2 - Protein adsorption material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new adsorbent material for proteins.

Conventional protein adsorption materials include activated carbon, bentonite,
Known materials include alumina, acid clay, calcium phosphate gel, hydroxyapatite, and starch.

These adsorption substances, such as activated carbon and alumina, may denature proteins.

It also had drawbacks such as weak adsorption, easy protein release, insufficient mechanical strength, poor operability, and difficulty in manufacturing and molding. The inventors of the present invention have worked hard to solve these problems and develop an industrial adsorption material with even better operability.As a result, we have developed a material that contains nitrile groups of 2.0% by weight or more and has a bulk density of 0.5% by weight. It was discovered that a nitrile-containing monovinyl polymer powder of 35 ta', preferably 0.28 m/m or less, has a surprisingly high adsorption capacity, and based on this knowledge, the present invention was completed.

In other words, when producing a nitrile-containing monovinyl polymer, a powder with a low bulk density is obtained by polymerizing it under specific conditions, and the powder can be used as an adsorbent with high adsorption capacity, which is excellent both in terms of production and use. Economic efficiency was achieved.

The substance for protein adsorption of the present invention (hereinafter referred to as an adsorbent) is a nitrile-containing monovinyl monomer such as acrylonitrile, methacrylonitrile, vinylidene cyanide, etc. Used as a general term for military monomers.

) alone or by copolymerizing it with other monovinyl comonomers containing a copolymerizable ethylenic double bond. Some powders have a weight of 20 Wt. % or more of nitrile groups, and the bulk density is required to be 0.35 m/kg or less (preferably 0.28 m/kg) or less. The amount of nitrile in the powder is 20Wt. If the bulk density is less than 0.35% or exceeds 0.35 ta', the adsorption capacity will be markedly reduced and the object of the present invention cannot be fully achieved. In order to obtain the adsorbent of the present invention, the monovinyl monomers mentioned above are usually homopolymerized or copolymerized using a radical initiator.

In order to keep the bulk density of the obtained powder below 0.35 m/machine, it is necessary to appropriately select the polymerization solvent, initiator, etc. As a means of obtaining a powder with a low bulk density, solution polymerization or suspension polymerization using an initiator soluble in a solvent in a polymerization solvent that hardly causes swelling of the resulting polymer powder is usually employed. At this time, not only the type of solvent and initiator but also polymerization conditions such as polymerization temperature, monomer concentration, and initiator concentration affect the shape of the powder particles. Acrylonitrile, methacrylonitrile, used in the production of the adsorbent of the present invention,
Specific examples of monovinyl monomers that can be copolymerized with nitrile-containing monovinyl monomers such as vinylidene cyanide include the following. For example, olefins such as ethylene and propylene; vinyl ethers such as ethyl vinyl ether and n-butyl vinyl ether;
Halogenated olefins such as vinyl chloride, vinylidene chloride, and tetrafluoroethylene; methyl (meth)acrylate, butyl (meth)acrylate, 2-hydroxyethyl (
meth) acrylate, polyethylene glycol (meth)
(Meth)acrylates such as acrylates; vinylesters such as pinyl acetate and vinyl benzoate; styrene, Q
- Aromatic vinyl compounds such as methylstyrene; amide vinyl monomers such as (meth)acrylamide, N.N-dimethyl(meth)acrylamide, and N-vinylpyrrolidone.
; maleimide derivatives; carboxyl group-containing monomers such as (meth)acrylic acid and maleic anhydride, and their salts; sulfonic acid group-containing monomers and their salts, such as styrene sulfonic acid and methallyl sulfonic acid: 2-hydroxyethyl (meth) ) Phosphate group-containing monomers such as acrylate phosphate esters and their salts: Basic monomers and their salts such as vinylpyridine, vinylimidazole, N.N-diethylaminoethyl (meth)acrylate, etc., and these comonomers may be used alone Monomers containing sulfonic acid groups, monomers containing carboxyl groups, basic monomers, monomers containing phosphoric acid groups and their salts, amide monomers, and polyoxyalkylene glycol-containing monomers are used in combination. comonomer
as 0.3 to 3.5 Wt. %, the adsorption capacity is further improved. The adsorbent of the present invention can be used as it is, but if necessary, it can also be used after being subjected to various moldings while maintaining its bulky structure.

As such a monovinyl polymer containing a nitrile group, an acrylonitrile synthetic fiber such as Cashmilon (manufactured by Asahi Kasei Corporation) is easily available.

However, such fibers not only have a structure significantly different from that of the adsorbent of the present invention, but also have an extremely low adsorption capacity. In order to form a complex between the powder produced as described above and the protein, the temperature is 0°C to 90qo, preferably 5°C.
The adsorbent and the protein may be brought into contact at a temperature of .degree. C. to 6.degree. C. and a pH of .about.11.

The contact time varies depending on the type of protein, concentration, temperature, bait, amount of adsorbent, and contact method, but it can be accelerated, especially by shaking or rope worship. The adsorption operation can be most easily carried out by adding the present adsorbent to a protein-containing solution. Further, if necessary, the purpose can be achieved by filling the adsorbent in a layered manner and flowing a protein-containing liquid. Since this adsorbent is in the form of a fine powder, it was able to create a good suspension state, and it demonstrated an effect beyond expectations in that it was able to significantly improve the efficiency of contact adsorption with proteins. When a protein and an adsorbent are brought into contact to form a complex, it is recommended to use a binder such as glutaraldehyde, hexamethylene diisocyanate, N・N'-ethylene bismaleinimide, etc. depending on the purpose. 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 has a molecular weight of 100.
The type of protein does not matter as long as it has a molecular weight of 0 to 1 million, but for example, serum proteins such as albumin, globulin, especially antibodies, hemoglobin, proteases, amylase, glucoamylase, invertase, lactase, vectinase, Galactosidase, cellulase, lipase, esterase, urease, naringinase, nucleoside phosphorylase, transphosphorylase, kinase, transaminase, lyase, aldolase, carboxylase, decarboxylase, marizquenzyme, tryptophanase, hydratase, especially fumarase, asbaltase , cysteine desulfhydrase, arginine deiminase, dehydrogenase, peroxidase, catalase, oxygenase, cytochrome, xanthine osidase, amino acid oxidase, racemase, xylose isomerase, thrombin, adenyl deiminase, benicillinase, uricase, cholesterol oxidase, and other enzyme proteins. , protein hormones such as insulin, gonadotropins, banpretusin, and growth hormone;
Bacitracin, colistin, etc., peptide antibiotics, Q-
Many proteins include fetoprotein, carcinoembryonic antigen, histones, protamine, and prolamins.

Among them, Q-chymotrypsin, bacitracin, and Titoku. C, ovalbumin, catalase, globulin hemoglobin, urease, serum albumin, trypsin, etc. provide preferable complexes in terms of their adsorption amount, strength, and uses. It is known that these proteins are not released by ordinary washing solutions such as water or phosphate buffer, and form strong complexes. The complexes consisting of these proteins and the adsorbent of the present invention are novel, and in particular those with a protein content of 40 9 or more per adsorbent have not been found in known nitrile-containing polymers. It is something that These new complexes can be used for adsorption, removal, and recovery purposes as well as
As an immobilized enzyme, excellent reaction efficiency is limited. Complexes with antibodies or antigens are also excellent carriers for affinity separation. 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 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 and recovery from enzyme production solutions, recovery of proteins from waste solutions, production of immobilized proteins such as antigens, antibodies, enzymes, etc. It also has a surprisingly wide range of applications, including affinity separation and enzymatic reaction applications. Next, the present invention will be explained in more detail with reference to Examples.

Examples 1 and 2 Acrylonitrile 95Wt. %, n
-Butyl acrylate 5Wt. Disperse a monomer composition consisting of % in water at a ratio of monomer composition: water = 1:4.5, and m Using three radical initiators, azobisisobutyronitrile (comparative example), '1' and '2
Polymerization was carried out at a temperature of 55°C for females and 65°C for handling to obtain a powder.

The average particle size of these powders was 20 to 30 r. A dry weight of 0.15 kg of each of these powders and powdered activated carbon was added to 1 oz of a solution containing 3.0 ml of human serum albumin (Book 6.0),
After a while, a complex of albumin and powder was obtained.

Proteins in the upper light were extracted using the method of Lowry et al. (Lowhi, 0.
Day. etal. J. Bjol. Chem. Volume 193, 2
65, 1951), and the difference from the control without molded material (powder) was taken as the adsorption amount. The results are summarized in Table 1. The amount of protein adsorbed is the dry molded product (powder) 1
It is expressed as the weight of protein adsorbed per 00 cape. The powder using azobisisobutyronitrile, which has a high bulk density compared to the examples in Table 1, showed only one-third or less of the adsorption amount.

Furthermore, in the case of inorganic adsorbent activity and charcoal, the adsorption amount was even lower. Example 3 For comparison with the adsorbent of the present invention, polyacrylonitrile sponges and flakes were produced as follows.

The sponge state was obtained by dissolving the powder of Comparative Example 1 described above in dimethylformamide and gelling it while mixing air. The apparent specific gravity was 0.16. The flakes were obtained by dissolving the same powder used in the production of the cavernous bodies in dimethylformamide at a concentration of 5% and injecting this solution into water under a high-speed flywheel. The bulk density of this flaky body is 0
．． 36 specific surface area was 36〆/night. A dry weight of 0.15 ml of each of these polymers was added to 10 w' (pH 6.0) of a solution containing 3.1 ml of Q-chymotrypsin per 1 volume, and the mixture was heated at 30 qo for 3 hours, and each sample was added every hour. The amount of adsorption was measured by the method of Example 1.

The results are shown in Table 2. It was shown that the adsorbent of this invention not only has a large adsorption amount but also has excellent adsorption efficiency. Table 2 Example 4 m Acrylonitrile 85Wt. %, methyl methacrylate 15Wt. %■ Methacrylonitrile 90Wt.
%, styrene lOWt. %'3' Acrylonitrile 8
5Wt. %, polyethylene glycol (degree of polymerization 8) monomethacrylate 15Wt. % {4} Acrylonitrile 50Wt. %, tetraethylene glycol diacrylate 5Wt. %, styrene 25
Wt. %, acrylic acid 20Wt% (5) Acrylonitrile 85Wt%. %, N-N-dimethylacrylamide 1
5Wt. % of five kinds of copolymer powders were polymerized in an aqueous solvent using a combination of ammonium persulfate and sodium monobisulfite as a radical polymerization initiator to obtain a powdered adsorbent with a low bulk density.

Complexes of these powdered adsorbents and each protein listed in Table 3 were formed by the method of Example 1, and the amount of protein adsorbed was measured.

3rd, the smallest amount of adsorption is 5 p, and the largest is 13 p.
reached c.

Example 5 ○} Acrylonitrile 30Wt. %, styrene 70W
t. %■ Acrylonitrile 50Wt. %, styrene 5
0Wt. %■ Acrylonitrile 80Wt. %, styrene 20Wt. %■ Vinylidene cyanide 70Wt. %,
Styrene 30Wt. % of four types of copolymer powders were prepared using azobisisobutyronitrile as an initiator.

At this time, as the polymerization solvent {1' monopod, methanol (80
%)-water (20%) mixed solvent was used, and in the case of {4}, acetone was used. Complexes of these powders and tribucin were formed in the same manner as in Example 1, and the amount of adsorbed protein was measured.

The trypsin solution used was 9/9 of 3.3 (pH 6).
．． 2). As shown in Table 4, all of the examples showed adsorption amounts exceeding 9 out of 5. Further, a complex of copolymer (1) of this example with trypsin (immobilized trypsin) was washed three times with 0.01M phosphate buffer. The relative activity after washing was 85%. It was found that there was no decrease in activity from the second time onwards, indicating that a complex was being formed.

Table 4 Washed immobilized trypsin with a solution of penzoyl arginine ethyl ester hydrochloride in 0.09M Tris buffer (
10 w' of (pH 8.0) was added thereto, and the reaction was carried out by shaking at 25 qo.

After the reaction, the immobilized trypsin was collected by centrifugation, a new substrate solution was added, and the reaction was repeated. The reaction solution is di-
According to the method of G. W. Schwert et al. (BiMhim.Bioph侭icaA
(Vol. 16, p. 570, 1955) The results of repeating the reaction seven times are shown in the drawing.

Mutual activity was expressed as a percentage of the first activity. After the second reaction, 70% of the reaction rate of the first reaction was maintained, and there was no decrease in activity, indicating that the complex of the present invention is excellent as an immobilized enzyme.

[Brief explanation of the drawing]

The figure is a graph showing the change in relative activity when the trypsin-adsorbent complex is reacted repeatedly.

Claims (1)

[Scope of Claims] 1. Obtained from only nitrile-containing monovinyl monomers or a mixture of these monomers and at least one monomer selected from the group consisting of acrylic esters, acrylic amide, and styrene; The bulk density of a homopolymer or copolymer containing 20% by weight or more of nitrile groups is 0.35
A substance for protein adsorption, characterized in that it consists of a powder having a particle size of less than g/cm^3. 2. Nitrile containing 20% by weight or more obtained from a nitrile-containing monovinyl monomer alone or a mixture of these monomers and at least one monomer selected from the group consisting of acrylic ester, acrylic amide, and styrene. The bulk density of the homopolymer or copolymer containing the group is 0.35
g/cm^3 or less and a protein-containing solution at a temperature of 0°C to 90°C and a pH of 1 to 1.
1. A method for adsorbing proteins, which comprises contacting in step 1. 3. The method according to claim 2, wherein the temperature is 5°C to 60°C.