JPS6045937B2 - protein adsorbent - Google Patents

Description

[Detailed description of the invention] The present invention relates to a novel protein adsorbent.

The fact that proteins physically adsorb to various substances has been well known.

For example, activated carbon, acid clay, kaolinite, alumina,
Examples include inorganic substances such as silica gel, bentonite, hydroxyapatite, and calcium phosphate, and natural polymers such as starch and gluten. These substances have been put into practical use for purposes such as clarifying liquids containing proteins, removing trace amounts of protein contamination, and purifying and separating proteins, but these substances often have specificity for proteins. There are limitations to solution conditions such as pH, and certain proteins may have chemical effects on coexisting substances.
In reality, the range of application of individual adsorbents is narrow due to reasons such as insufficient mechanical strength. In view of the above, as a result of intensive research, the present inventors have first determined that a monomer containing 2% to 9% by weight of a cyano-containing monomer and 2% to 8% by weight of a crosslinking polymerizable monomer. He invented an excellent protein adsorbent made of a porous copolymer obtained by copolymerizing a polyacrylonitrile-based copolymer with an average pore size of 408 to 9000A, and applied for a patent (patent application). 5
1-128868). After further research, the present inventors discovered that, contrary to the inventors' expectations, even copolymers with a cyano monomer content of less than 20% exhibited significantly high protein adsorption power. The present invention has now been completed.

That is, the present invention provides a compound containing the general formula (
4) (wherein R is hydrogen, alkyl group, etc.) A cyano-containing monomer represented by: and 2% by weight to (a)
A protein adsorbent comprising a porous copolymer obtained by copolymerizing a monomer mixture containing % by weight of a crosslinkable monomer and having an average pore diameter d of 408 to 9000 pores. It is related to.

The protein adsorbent of the present invention has a highly porous structure, as described in detail later, and has the ability to adsorb large amounts of a wide variety of proteins, and is also chemically stable and capable of adsorbing proteins. Since it does not decompose and has high mechanical strength, it is not only easy to operate and can be used on an industrial scale, but also has many advantages such as being easy to manufacture.

Specific examples of the cyano-containing monomer represented by the general formula which is a constitutional unit of the copolymer constituting the protein adsorbent of the present invention include acrylonitrile, methacrylonitrile, α-chloroacrylonitrile, cinnamitrile, etc. be.

The content of these cyano monomers is 1% by weight or more and 2% by weight.
It is preferably 4% by weight or more (a) less than 2% by weight, more preferably 6% by weight or more and less than 2% by weight.
The copolymer of the present invention can contain other monomers copolymerizable with the cyano-containing monomer.

These monomers include hydrocarbon compounds such as styrene, methylstyrene, ethylstyrene, vinylnaphthalene, butadiene, isoprene, hyperylene, etc.; Styrene derivatives such as styrene: Vinyl sulfide derivatives such as methyl vinyl sulfide and phenyl vinyl sulfide: Acrylic acid: Methacrylic acid: Acrylic acid esters such as methyl acrylate and chloromethyl acrylate; cyclohexyl methacrylate;
Methacrylic acid esters such as dimethylaminoethyl methacrylate, glycidyl methacrylate, tetrahydrofurfuryl methacrylate, hydroxyethyl methacrylate, etc. Vinyl ketones such as dimethyl vinyl ketone and ethyl isopropenyl ketone Vinylidene compounds such as vinylidene dichloride and vinylidene bromide: acrylamide, Acrylamide derivatives such as N-butoxymethylacrylamide and N,N-dimethylaminoethyl acrylamide: Fatty acid vinyl derivatives such as vinyl acetate and vinyl phosphoric acid; Thiofatty acid derivatives such as methyl thioacrylate and vinyl thioacetate;
-vinylsuccinimide, N-vinylpyrrolidone, N-
Heterocyclic vinyl compounds such as vinyl phthalimide, N-vinylcarbazole, vinylfuran, vinylimidazole, methylviny, limidazole, vinylprazole, vinyloxazolidone, vinylthiazole, vinylpyridine, methylvinylpyridine, 2,4-dimethyl-vinyltriazine, etc. Although it can be arbitrarily selected from among the above, nonpolar monomers such as hydrocarbon monomers, acrylic esters, and methacrylic esters are preferred. The cross-linked polymerizable monomers serving as constitutional units of the copolymer of the present invention include divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene,
Divinylethylbenzene, trivinylbenzene, divinyldiphenyl, divinyldibenzyl, divinylphenyl ether, divinyldiphenylamine, divinylsulfone, divinylketone, divinylpyridine, divinylquinoline, diallyl phthalate, diallyl maleate, diallyl fumarate, carbonic acid Diallyl, diallyl oxalate, diallyl adipate, diallyl tartrate, diallylamine, triallylamine, triallyl phosphate, triallyl tricarparylate, N,N-ethylene diacrylamide, N,N
- Methylene dimethacrylamide, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, trimethylolpropane trimethacrylate,
Pentaerythritol tetramethacrylate, 1,3-
Butylene glycol diacrylate, trimethylproban triacrylate, pentaerythritol tetra”
Acrylate, triallylisocyanurate, 1,3,
arbitrarily selected from 5-triacryloylhexahydro-1,3,5-triazine, diarylmelamine, etc.
Nonpolar crosslinking monomers such as hydrocarbon crosslinking agents such as divinylbenzene and fatty acid ester crosslinking agents such as ethylene glycol dimethacrylate are preferred.

The content of the crosslinking polymerizable monomer is 2% to 8% by weight, preferably 5% to 7% by weight, and more preferably 8% to 6% by weight. cross-linked polymerizable mono)
If the content of polymer is too low, the degree of swelling and shrinkage will increase and the mechanical strength will decrease. Furthermore, if the degree of crosslinking increases too much, a decrease in the diffusion rate of the liquid within the copolymer will be observed. The copolymer constituting the protein adsorbent of the present invention has an average pore diameter of 408 to
Although the structure has a large number of pores in the range of 9000A, the average pore diameter is more preferably in the range of 50A to 4000A, and even more preferably in the range of 60A to 2500A.

If the average pore size is too small, proteins will enter the pores and cannot be adsorbed, or the diffusion rate will be significantly reduced, which is disadvantageous. If the pore size is too large, the surface area that contributes to adsorption will be small and mechanical This results in disadvantages such as reduced strength. The pore size distribution is also an important factor regarding protein adsorption ability, and when the average pore size is d, the pore size is 0.5
It is desirable that the volume of pores with a diameter of C1 or more and less than 2d is 60% or less, preferably 50% or less of the total pore volume.

There is no particular limit to the lower limit of this value, but since the largest number of pores are around the average pore diameter, it is necessarily 20% or more, but it is generally 30%. A copolymer containing pores having such a wide pore size distribution has a particularly high protein adsorption capacity, as will be described later. Furthermore, pore size also has an important relationship with protein adsorption capacity.

That is, when the weight fraction of the crosslinking polymerizable monomer to the total monomer is X%, the total pore amount per q of dry polymer is 0.0.
It is preferably 5V'Xml or more and 1.5V'XmL or less, and more preferably 0.15VXTL1 or more and 1.3V'Xm.
It is desirable that it be less than l. If the pore size is too small, it will not be possible to provide sufficient adsorption surface, and if the pore size is too large, it will not only reduce the mechanical strength of the copolymer, but also reduce the amount of adsorption per unit volume of copolymer. lower. Next, the method for measuring porosity characteristics adopted in the present invention will be described.

The average pore size, pore size distribution, and pore specific surface area were measured using a mercury intrusion porosimeter.

In this method, mercury is injected into a porous material, and the amount of pores is determined from the amount of mercury infiltrated.The diameter of the pores and the pressure at which mercury is injected into the pores are inversely proportional. The pore size is measured based on the principle of For more information on this method, see
Seisho fine. particle. Measurement (Fi
neParticleMeasurement) Clyde. Oh. Junior and Jie. M. Daala Abaal (
Clyde, Or'R, JrandJ. M.Dalla
Valle) co-author, Sa. Macmillan. Company. new. York (The Macmillan Company
, New York) 1959. With this method it is possible to measure holes up to 35-40A. In the present invention, a pore is defined as a pore communicating from the surface with a pore diameter of 40A or more, and the pore volume and surface area are also values derived from the pore. Further, the average pore diameter is defined as the value of r at which the value of DV/DlOgr becomes the maximum value. Here r is the pore diameter, ■
is the cumulative pore volume measured with a porosimeter. Another value that is an indicator of porosity is bulk specific gravity. The inventors measured the bulk specific gravity using the following method. That is, first, fill a column with a glass filter with resin, thoroughly drain the water,
Find the volume of the column filled with resin at that time. Thereafter, the sample was sufficiently dried and weighed, and the bulk specific gravity was calculated from both values. Next, a method for producing the adsorbent copolymer of the present invention will be described.

Some of the inventors have already discovered a method for producing highly porous crosslinked polymers, and this technique can also be used to produce the porous copolymers of the present invention (Japanese Patent Application Laid-Open No. 51-1
No. 283(4), Japanese Unexamined Patent Publication No. 51-130475).
One method is to use a mixture of copolymerizable monomers,
A mixture of which 2% by weight or more is a cross-linked polymerizable monomer, which has an affinity for the homopolymer of at least one monomer in the mixture and of at least one other monomer. A copolymer is obtained by copolymerizing in the presence of a single liquid that has no affinity with the homopolymer and dissolves the monomer mixture and does not react with each monomer, and then the copolymer is The other is a mixture of copolymerizable monomers, of which 2% by weight or more is a crosslinking polymerizable monomer. and a liquid that has an affinity for the homopolymer of at least one monomer in the mixture, dissolves the monomer mixture, and does not react with each monomer, and at least one of the monomers in the mixture. A copolymer is formed by copolymerizing the monomers in the presence of a mixed liquid consisting of a liquid that has no affinity with the homopolymer and that dissolves each monomer and does not react with each monomer. This method is characterized in that the mixed liquid and unreacted monomers are then removed from the interior of the copolymer.

To further describe this method in detail for the case where a copolymer is synthesized by copolymerizing two types of monomers A and B, when organic liquids are classified as follows, the following (1) to (4) ) are mixed with the monomer mixture;
A porous structure can be obtained by performing a copolymerization reaction.

That is, (1) at least one type of liquid X (2) a mixture of at least one type of liquid and at least one type of liquid Y (3)
Mixture of at least one type of liquid X and at least one type of liquid Z (4) At least one type of liquid Y and at least one type of liquid
Mixture of Seed Liquids Z Here, the liquids X, Y, and Z each have the following properties.

liquid Z: A liquid that shows no affinity for either Polymer A or Polymer B. Here, if a linear polymer of a certain monomer with an average molecular weight of 10,000 or more dissolves in a liquid by 1% or more, it is A liquid is defined as having an affinity for the polymer.

In the case of a cross-linked polymerizable monomer, a mixture consisting of 5 parts of the monomer, 0.1 part of azobisisobutyronitrile, and 1 (4) parts of a liquid is sealed in a glass tube, and the polymerization reaction to be performed is carried out. Heat at the same temperature and time schedule. If the product is transparent, the polymer of the monomer is defined as having an affinity for the liquid. As described above, porous crosslinked copolymers can be easily synthesized by methods (1) to (4), but in order to obtain the copolymer disclosed in the present invention, Methods (2) to (4) are suitable.

That is, in these methods, a copolymerization reaction is carried out in the coexistence of a mixture of a liquid that has an affinity for the copolymer and a liquid that does not have an affinity for the copolymer, resulting in a copolymer having a wide pore size distribution. This is because a polymer can be obtained. In addition, when two types of liquids having different solubility are used in this way, by changing the mixing ratio, it is possible to continuously change the pore size of the copolymer produced. For effective porous structure design, it is necessary to increase the amount of organic liquid added as the proportion of crosslinking monomer to total monomer increases.
That is, when the weight percent of the total liquid relative to the total monomer is D1, and the weight percent of the crosslinking polymerizable monomer relative to the total monomer is X, Formula 1
01.2J and <Dku1σ. It is preferable to satisfy the formula 101.5V or D<1σ. It is more preferable to satisfy 1VY.

The amount of pores in a porous copolymer is essentially determined by the relative amount of liquid added. Next, liquid names used in more specific cases will be listed.

In the copolymerization of acrylonitrile-divinylbenzene-ethylstyrene, liquid Y includes dimethylformamide, N-methylacetamide, nitromethane, dimethylsulfoxide, benzonitrile, γ-butyrolactone, N,N-dimethylacetamide, acetophenone, etc.
Examples of the liquid X include aromatic hydrocarbons such as toluene, xylene, ethylbenzene, and tetralin, cyclohexanone, anisole, chlorobenzene, dichlorobenzene, methyl benzoate, ethyl benzoate, benzyl alcohol, methylene dichloride, chloroform, and dioxane. Liquid Z includes aliphatic hydrocarbons such as heptane and decalin, n-
Examples include aliphatic alcohols such as butanol, cyclohexanol, isooctyl alcohol, amyl acetate, dibutyl phthalate, dioctyl phthalate, and the like.

In the copolymerization of acrylonitrile-ethylene glycol dimethacrylate, liquid Y is dimethylformamide, dimethyl sulfoxide, γ-butyrolactone, N,
N-dimethylacetamide, etc. can be selected, liquid X can be selected from toluene, methyl ethyl ketone, dioxane, cyclohexanone, methylene chloride, chlorobenzene, etc., and liquid Z can be selected from heptane, octane, n-butanol, isopropanol, etc.

Examples of liquids to be used when synthesizing the copolymer of methacrylonitrile-divinylbenzene-ethylvinylbenzene include liquid Y such as pyridine, nitromethane, benzonitrile, cyclohexanone, methyl ethyl ketone,
γ-butyrolactone, etc., as liquid X, toluene,
Examples of the liquid Z include ethylbenzene, tetralin, butyl acetate, and ethyl propionate. Examples of the liquid Z include heptane, butanol, isooctanol, cyclohexanol, and dioctyl phthalate.

The above is just an example; a wide variety of liquids can be used by examining their solubility. The polymerization method for obtaining the copolymer in the present invention may be either radical polymerization or ionic polymerization, but it is generally recommended that the polymerization be carried out by dissolving a radical initiator in a monomer liquid mixture and heating it. Ru.

Suitable radical initiators are selected from radical initiators that dissolve in the liquid and monomer mixture and undergo decomposition at the reaction temperature, examples include acyl peroxides such as benzoyl peroxide, lauroyl peroxide, etc. , azonitrile such as azobisisobutyronitrile, 2,2″-azobis(2,4-dimethylmaleronitrile), peroxides such as ditertiary butyl peroxide, dicumyl peroxide, methyl ethyl ketone peroxide, cumene hydro There are hydroperoxides such as peroxide and tertiary hydroperoxide.The reaction temperature is 10°C to 200°C, preferably 20°C to 1500°C, and more preferably 30°C to 100°C. When polymerizing in an open system, it is necessary to lower the polymerization temperature because the boiling point of acrylonitrile is low. Therefore, it is desirable to use a low-temperature decomposition type initiator as part or all of the polymerization initiator. For example, 2, 2 ″-Azobis (2
, 4-dimethylvaleronitrile), 2,2″-azobis(4-methoxy2,4-dimethylvaleronitrile),
Tertiary butyl pervalerate, diisobutyl peroxocarbonate, and the like are suitable. One of the preferred polymerization methods for synthesizing the copolymer of the present invention is suspension polymerization in water, and in this case, granular resin can be easily obtained.

Acrylonitrile exhibits some solubility in water, but its solubility in water decreases significantly when monomers are added to water-insoluble liquids. However, when polymerizing by adding a water-soluble organic liquid, the suspension polymerization method cannot be used, and instead a method is used in which a lump is obtained by solution polymerization and then ground to an appropriate particle size. It should be done.

Suspending agents used in the present invention include natural polymeric substances such as starch, gum tragacanth, and gelatin; processed natural polymeric substances such as hydroxyethylcellulose, methylcellulose, and carboxymethylcellulose; polyacrylic acid; polyvinylpyrrolidone, polyvinyl alcohol, and partially water-soluble synthetic polymer substances such as polyvinyl acetate, and inorganic substances such as barium sulfate, talc, hydroxyapatite, bentonite, silicic anhydride, and calcium carbonate.

Furthermore, when using acrylonitrile, it is widely recommended to add an inorganic salt such as sodium chloride or calcium chloride to the suspension in order to suppress some of its water solubility.

The above is a typical production method, and it has become clear that the cyano-containing porous crosslinked copolymer obtained by such a method has extremely excellent properties as a protein adsorbent.
The explanation will be given below. 1. The adsorbent of the present invention has a highly porous structure.

The performance of an adsorbent is determined primarily by its adsorption rate and adsorption capacity. For this reason, many common adsorbents have a porous structure, but one of the features of this adsorbent is that its porous structure can be adjusted arbitrarily. That is, the average pore diameter 4
It can take any value in the range of 0A to 9000A, and the pore size can be easily adjusted by the method already disclosed by the inventor. Furthermore, it is desirable that the pore size distribution be wide to some extent. That is, the presence of large pores significantly increases the diffusion rate of proteins in the copolymer, and the presence of a large amount of small pores increases the internal surface area of the copolymer. As a result, the product of the present invention showed high adsorption rate and adsorption capacity. When targeting a specific protein, there will be an optimal porous structure. Although it is not easy to simply describe guidelines for structural design, the first thing to consider is the size of the adsorbed protein. The size of general proteins is in the range of several 10A to several 100A, but in order for the protein to diffuse and be adsorbed within the copolymer, it is necessary to form pores that are at least the size of the protein. 2. The copolymer of the present invention has the ability to adsorb large amounts of various proteins.

The characteristics mentioned in 1 are related to the physical structure, but in order to function as an adsorbent, it is necessary that some kind of interaction exists between the main body and the adsorbed material.

Comparative Example 3 shows the protein adsorption ability of the divinylbenzene-ethylstyrene copolymer, which is not necessarily a high value. On the other hand, it is clear from Example 3 that the copolymer produced by adding 1% by weight of acrylonitrile exhibits several times the adsorption capacity. Considering that the pore structures of both copolymers are almost the same, such a difference in adsorption ability can be considered to be mainly due to the fact that the copolymers contain a small amount of cyano groups. However, the protein adsorption mechanism is slightly different between the porous copolymer of the present invention, which has a low cyano group content, and the porous copolymer of the prior application, which has a high cyano group content. I think so. That is, in the adsorbent of the present invention, hydrophobic bonds play a major role in the adsorption of proteins, and therefore, the adsorbent of the present invention is also effective in adsorbing proteins with a certain degree of hydrophobicity. When the hydrophobic bonding force is strong, the adsorbed protein is difficult to dissolve in water, resulting in a favorable result in that the detachment of the protein is suppressed. The hydrophobicity of proteins is determined by R.E. Feeney (R.E.
．． Feeney) et al., Biohimica. Engineering. Biophysica. Acta (BlOehjmicaetBlOphy
sicaActa) 19, pp. 256-264, 196, it can be measured by the adsorption amount of n-alkanes. 3. The copolymer of the present invention is characterized by high physical strength.

Conventionally, polymers made from starch, cellulose, dextran, etc. as starting materials have been widely used as carriers for protein separation and purification and immobilization. Typical examples are Cephadex, a cross-linked form of dextran, and Cepharose, a cross-linked agarose, but carriers obtained from these natural products have a high degree of swelling and poor mechanical strength, so they cannot be used on a large scale. It becomes difficult to do so. In contrast, the product of the present invention has high mechanical strength unique to three-dimensional crosslinking, and is advantageous when packed in a column for chromatography. 4. Regarding the shape of the adsorbent, it is well known that a spherical adsorbent is the most preferable in terms of adsorption performance, strength, and resistance when the solution is passed through. Polymers can be easily produced into spherical shapes by suspension polymerization in water.

5 One of the preferable conditions for adsorbent resins is that they are inert and can be used in a wide pH range. Polyacrylonitrile, polydivinylbenzene, etc. satisfy this condition, so they are particularly reactive. The copolymer of the present invention satisfies this condition unless a monomer with high properties is added.

While activated alumina and silica gel, which are general-purpose inorganic adsorbents, sometimes decompose the adsorbed substances, the copolymer of the present invention has the advantage that it can be used on a wide range of substrates under a wide range of conditions. are doing. 5 Furthermore, the copolymer of the present invention is
It has great practical characteristics in that it can be used repeatedly, maintains its performance for a long time, and has little deterioration.

As mentioned above, this copolymer has various characteristics that are desirable as a protein adsorbent, and one of the reasons for this is the fact that the crosslinking monomer is included in the structural unit. be. Cyano-containing porous compositions can be obtained, for example, by adding a solution of a polymer containing polyacrylonitrile to a non-solvent and causing reprecipitation, or by depositing polyacrylonitrile on the surface of a porous material by some method. In comparison, the porous structure of this copolymer, which has a three-dimensional crosslinked structure, is easy to design, and the porous structure is less likely to change due to liquid flow or external pressure, and exhibits stable adsorption performance over a long period of time. In addition, the strength of the adsorbent is high, and the polymer constituting the adsorbent does not dissolve in the liquid used. Taking advantage of the various features already described, the protein adsorbent of the present invention can be used for various purposes as described below.

(1) Protein purification and separation.

Since this adsorbent has extremely excellent selective adsorption properties for proteins, it is capable of effectively and selectively adsorbing proteins contained in aqueous media. , vitamins, pigments, inorganic salts, metal compounds,
Proteins coexisting with water-soluble substances such as surfactants can be separated.

Adsorption of a protein from a protein solvent using the adsorbent of the present invention can be carried out at a pH of 1 to 11, but the pH suitable for adsorption is selected depending on the type of protein.

It can also be applied to organic solutions, such as zein in a 70% alcohol solution. Temperatures used during adsorption are 2°C to 50°C, but temperatures around room temperature, especially 2°C, are used.
0°C to 40°C is preferred.

The adsorption operation is carried out by adding the adsorbent of the present invention to a protein solution, and if necessary, stirring or shaking can be performed to facilitate adsorption and shorten the adsorption time. Alternatively, adsorption can be carried out by packing an adsorbent on a column or filter and flowing a protein solution through it. An appropriate adsorption time can be selected depending on the state, concentration, adsorption method, temperature, etc. of the protein solution.

In recent years, industrial application technology for proteins has been studied in many fields as an important new technology, and the development of such an innovative adsorption method from all functional aspects will make an extremely important contribution to the industrial field. Its application range is extremely wide.

In addition, adsorption chromatography, which is currently a representative example of a laboratory-scale separation and purification method for natural organic compounds, is used to
Differences in adsorption power between adsorbents and solvent molecules are utilized, and currently used adsorbents include inorganic substances such as activated carbon, alumina, silica, calcium phosphate, and magnesium silicate.

Compared to these inorganic adsorbents, this adsorbent can be obtained in a good spherical shape, has greater mechanical strength, and can be used repeatedly, making it suitable for large-scale adsorption chromatography. It is often encountered in inorganic adsorbents that this is also possible. Since it does not have the property of decomposing the substance to be separated, it can be used as a carrier for adsorption chromatography because it has the advantage of being able to handle particularly unstable proteins. or,
By utilizing the relationship between the pore size and the amount of protein adsorbed as described above, more selective protein separation becomes possible. Furthermore, on the other hand, the protein adsorbed on the adsorbent of the present invention can be extracted from an aqueous solution adjusted to be acidic or basic, various salt solutions, warm water, or water containing an organic solvent miscible with water such as methanol, ethanol, acetone, etc. Efficient elution is possible with an appropriately selected aqueous solvent, and elution methods may be appropriately combined depending on the type of protein to be eluted and the intended use.

(2) Removal of protein The quality of the product may be significantly lowered due to the contamination of trace amounts of protein; one example of this is the turbidity of sake caused by protein.

By using the adsorbent of the present invention, proteins contained in sake can be removed very efficiently and reliably. Moreover, the product quality can be dramatically improved without any negative effect on the quality of sake. Furthermore, compared to conventional methods such as coagulation-sedimentation using persimmon persimmons, ultracentrifugation, and separation using enzymes, the adsorption and post-treatment operability is much superior, and there is no protein contamination and it is economical. Similarly, this method can be used for brewing beer, wine products and other sake processes. Furthermore, the method for adsorption and removal of proteins can also be applied as a method for removing proteins from high protein content wastewater (eg food processing wastewater).

In this case, the treatment can be carried out in a much shorter time and in a smaller space than with conventional biological methods such as activated sludge method. (3)
Carriers such as immobilized enzymes There are many pairs of substances that exhibit specific interactions in living organisms.

Typical examples include enzymes and substrates, enzymes and inhibitors, and antigens and antibodies. A complex is formed by these combinations. In some cases, this complex is very stable, and in other cases, it exists only as an intermediate and undergoes immediate reaction and decomposition. Enzyme-substrate complexes correspond to the latter case. Many of these compounds are proteins, and by adsorbing and immobilizing them on the adsorbent according to the present invention, they can be used for various purposes. Enzymes immobilized on water-insoluble carriers are well known as immobilized enzymes.

Unlike the chemical bonding method, the conventional physical adsorption method for producing immobilized enzymes has the major advantages of almost no denaturation of the enzyme and the possibility of continuous use while replenishing the enzyme. Its adsorption power is weak and the amount of adsorption is small, so its practical application has been delayed. On the other hand, the amount of protein adsorbed by the adsorbent of the present invention has been shown to be 100 m9 or more per y of dry matter, and once adsorbed enzymes, etc. are hardly eluted by distilled water, deionized water, or tap water. Therefore, it is also possible to use it for enzyme reactions in a state in which enzymes are adsorbed.

Also, affinity. Chromatography is a method based on the principle of immobilizing physiologically active substances on a carrier and separating other physiologically active substances by utilizing the interaction with the carrier, and has attracted much attention in recent years. It is also possible to use the adsorbents of the invention as carriers for this purpose.

Proteins adsorbed and immobilized on adsorbents include enzymes, antigens,
Typical examples include antibodies. Note that when adsorbing these proteins, using a bifunctional reaction reagent such as glutaraldehyde, which is known as a protein cross-linking agent, not only does not interfere with the method of the present invention, but also allows the adsorbent, protein, and It can further strengthen the bonds between proteins and expand its range of application. EXAMPLES Next, the present invention will be explained in detail with reference to Examples, but the scope of the present invention is not limited thereby.

Example 1 Freshly distilled acrylonitrile 10y1 divinylbenzene (purity 56%
, containing 44% vinyl ethylbenzene as impurities,
(hereinafter referred to as 56% divinylbenzene) 90y1 Acetophenone 50y1 Decalin 150y1 Azobisisobutyronitrile 1y12,2''-Azobis(2,4-dimethylvaleronitrile) 1y is added to make a homogeneous solution.

In addition, partially saponified polyvinyl alcohol (viscosity 23Cp)
S saponification degree 88%) 6.25y1 sodium chloride 12.
Add 1270q of distilled water in which 5 Da was dissolved and boil to 300r'P.
At 45°C for 1 hour with stirring at a rotation speed of
It was heated at 0°C for 2 hours, at 60°C for 2 hours, and further at 70°C for 4 hours. During the reaction, the reaction mixture was sampled at regular intervals and the benzene extract was analyzed by gas chromatography to quantify the remaining monomer and measure the polymerization rate. Under the above conditions, the polymerization rate was over 97%. This was confirmed. Note that since acrylonitrile has a low boiling point, it is necessary to increase the efficiency of the reflux condenser by, for example, circulating cold water inside it. The produced copolymer had a good spherical shape and had a diameter in the range of 80 to 150 μm. After performing wet classification using a sieve, unreacted monomers, liquid, etc. were removed with methanol. A portion of the sample was taken and dried under reduced pressure at 60° C. for one hour to prepare a sample for a porosimeter. The remaining copolymer was washed repeatedly with water. The bulk specific gravity of this material was measured to be 0.22, average pore diameter 1400 A, and pore volume 188 mt/y, and the pore volume between 700 A and 2800 A is 0.77
It was m1/g.

Hereinafter, this will be referred to as R-1. Example 2 A granular copolymer was obtained in the same manner as in Example 1, except that 250 g of toluene was added instead of the mixed liquid of acetophenone and decalin.

Its particle size range is 70-180μ, bulk specific gravity 0.25. Average pore diameter 600A1 pore volume 1.52m1/Yl3OO8 to 1200A pores based pore volume is 0.78m1/y1
The surface area was determined to be 180j1'/f.

This product is hereinafter referred to as R-2. Comparative Example 1 Acrylonitrile 20y156 was added to the same equipment as Example 1.
% divinylbenzene 180f1 azo-bisisobutyronitrile 2y12,'2! 2 V of '-azobis(2,4-dimethylvaleronitrile) was added, and 1000 y of pure water in which 5 y of the same partially saponified polyvinyl alcohol as in Example 1 and 15 y of sodium chloride were dissolved was added to give 350 r'P.
After polymerization was carried out under the same temperature schedule as in Example 1 while stirring at a rotational speed of m, the same post-treatment was carried out.

The polymerization rate was 98%, the bulk specific gravity was 0.59, and no pores were observed using a porosimeter. Hereinafter, this copolymer will be referred to as C-1.

Comparative Example 2 56% divinylbenzene was added to 100% of the same equipment as in Example 1.
y1 tetralin 80V1 cyclohexanol 120 da,
A homogeneous solution consisting of 1y of benzoyl peroxide and 9y of methylcellulose (2% aqueous solution with a viscosity of 100CpS)
Add 1500y of pure water dissolved in
Stirring is continued at 0°C for 3 hours and then at 80°C for 4 hours.

The particle size was 60-200μ, and the bulk specific gravity was 0.23.
Measured with a porosimeter, the average pore diameter is 1300A1
Pore amount 1.63m1/y1 pore diameter from 650A to 2600
The amount of pores up to A was 0.53 m1/y. Moreover, the surface area was 240 d/y. This copolymer is referred to as C-2 below.
It is abbreviated as

Example 3 10 ml of solution containing 30 mg of each of the proteins listed in Table 1
0.5y of wet copolymer (R-1) was added to each,
After shaking at 30°C for 3 hours, the precipitate was removed by centrifugation, and the protein concentration in the supernatant was determined by the method of Lowry et al.
hem. R-
The difference from the value of the system without adding 1 was taken as the adsorption amount.

The results are shown in Table 1. The amount of protein adsorption is
The weight of protein adsorbed per y of dry copolymer is shown. Comparative Example 3 The protein adsorption amount of copolymers C-1 and C-2 was measured in the same manner as in Example 3.

The proteins used were serum albumin, α-chymotrypsin, and lipase. The results are shown in Table 2. Example 4 0.5 da of wet copolymer (R-2) was added to 10 ml of a solution containing 30 mg of each of the enzymes shown in Table 2, and 3
After shaking at 0°C for 4 hours, the copolymer was filtered through a glass filter, the titer of the enzyme adsorbed to the copolymer was measured, and the amount of enzyme adsorbed was determined by comparing it with the titer of the adsorbent. The results are shown in Table 3.

The activity of urease was measured by D. Day. Pansleek (
D. D. ■The method of Anslyke et al. (J. BiOl.
Chem, 15, p. 623, 1944); You. Bergmeyer (H.U. Berg
rTlayer) method (BlOchem.Z.327
vol., p. 255, p. 195), α-chymotrypsin was prepared by G. Double. G.W.Schwert
t) et al.'s method (BlOchim.BiOphysjca
Acta. , Vol. 16, p. 570, Cape 195), and pepsin is available from El. M. The method of L. M. Baker et al. (J. BiOl. Chem. Vol. 2ll, p. 701, 1
95 haiku).

Example 5 Hydroxyapatite 10y1 500ml of pure water was placed in a 1e separable flask equipped with a reflux condenser, stirring bar, and thermometer.
Add y and heat to 50°C.

A mixed solution of methacrylonitrile 8y, 156% divinylbenzene 18y, 24g of styrene, 90y of toluene, 60y of dioctyl phthalate, 0.5y of lauroyl peroxide was added thereto at 50°C for 2 hours, then at 65°C for 2 hours, and then at 65°C for 2 hours.
Continue stirring at 0°C for 4 hours. The resulting copolymer has a diameter of 1
The pore structure was measured using a porosimeter and the results were as follows. Average pore diameter 250
0A1 pore volume 243m1/f1 pore diameter 1250A~500
The pore volume derived from 0 persons was 1.09 m1/QO, and the bulk specific gravity was 0.19 m1/q. This copolymer is R-3
It is called.

Example 6 In the same manner as in Example 3, the protein adsorption amount of copolymer R-3 was measured.

The results are shown in Table 4. Example 7 Acrylonitrile 3f1 Ethylene glycol dimethacrylate 15f1 Dimethylformamide 50y1 Chlorobenzene 22y1 Tertiary butyl pervalerate 0.
After adding 0.1y of 1J1 benzoyl peroxide to make a homogeneous solution, the tip of the glass container was sealed and heated at 40°C for 2 hours at 60°C.
℃ for 4 hours) and 80℃ for 4 hours.

After cooling, the glass container was crushed to take out the copolymer, the copolymer was crushed in a mortar, thoroughly washed with acetone and water, and wet classified using 80 mesh and 150 mesh sieves. When the pore structure was measured using a porosimeter according to a conventional method, the average pore diameter was 200A, and the pore volume was 200A.
The pore volume resulting from the pore diameters of 100 A to 400 A was 0.79 m1/d. Also, the bulk specific gravity is 0.
It was measured as 17. Hereinafter, this copolymer will be abbreviated as R-4.
Example 8 The amount of protein adsorption of R-4 was measured in the same manner as in Example 3.

γ-globulin, α-chymotrypsin, cytochrome C
The adsorption amounts are 40mg, 100mg, and 120m, respectively.
It was hot at g. Example 9 5y wet copolymer (R-1) was added to 80 ml of a solution containing 300 mg of trypsin, and the mixture was shaken at 30°C for 3 hours.

Claims (1)

[Scope of Claims] 1 1% by weight or more and less than 20% by weight of general formula (A)
) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (where R is hydrogen, alkyl group, etc.) A monomer mixture containing the expressed cyano-containing monomer and 2% to 80% by weight of a crosslinking polymerizable monomer A protein adsorbent comprising a porous copolymer obtained by copolymerizing a porous copolymer having an average pore diameter (d) of 40 Å to 9000 Å. 2. The protein adsorbent according to claim 1, wherein the content of the cyano monomer is 4% by weight or more and less than 20% by weight. 3. The protein adsorbent according to claim 2, wherein the content of the cyano monomer is 6% by weight or more and less than 20% by weight. 4. The protein adsorbent according to claim 1, wherein the cyano-containing monomer is acrylonitrile. 5. The protein adsorbent according to claim 1, wherein the crosslinking polymerizable monomer is divinylbenzene. 6 The cross-linked polymerizable monomer has the general formula (B) (B) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (here R
The protein adsorbent according to claim 1, wherein _1 and R_2 are monomers represented by hydrogen or a methyl group, and n is an integer from 1 to 30. 7. The protein adsorbent according to claim 1, wherein the weight fraction of the crosslinking polymerizable monomer is 5% to 70%. 8. The protein adsorbent according to claim 1, which has an average pore diameter (d) of 50 Å to 4000 Å. 9. The adsorbent according to claim 1, wherein the volume of pores having a pore diameter of 0.5 d or more and less than 2 d is 60% or less of the total pore volume. 10 When the weight fraction of the crosslinking polymerizable monomer to the total monomer is X%, the total pore amount per 1 g of dry polymer is 0.0
The protein adsorbent according to claim 1, which has a volume of 5√(X)ml or more and 1.5√(X)ml or less. 11 The total pore amount per 1 g of dry copolymer is 0.15√(
The protein adsorbent according to claim 10, wherein the protein adsorbent has an amount of 1.3√(X)ml or more and 1.3√(X)ml or less. 12 Protein adsorbent per gram of dry copolymer is 20m
The protein adsorbent according to claim 1, wherein the protein adsorbent has a molecular weight of at least 100 g. 13 Protein adsorption amount per 1g of dry copolymer is 40m
13. The protein adsorbent according to claim 12, which has a molecular weight of at least g. 14. The protein adsorbent according to claim 1, 12 or 13, which is a protein having a molecular weight between 1,000 and 1,000,000. 15. The protein adsorbent according to claim 14, wherein the protein is human serum albumin. 16. The protein adsorbent according to claim 14, wherein the protein is α-chymotrypsin. 17. The protein adsorbent according to claim 14, wherein the protein is lipase. 18. The protein adsorbent according to claim 1, wherein the protein is an enzyme. 19. The protein adsorbent according to claim 18, wherein the enzyme is trypsin. 20 Claim 1 in which the enzyme is chymotrypsin
The protein adsorbent according to item 8. 21. The protein adsorbent according to claim 1, wherein the protein is an antigen. 22. The protein adsorbent according to claim 1, wherein the protein is an antibody. 23 Claim 1 in which the porous copolymer is spherical
Protein adsorbent described in section. 24 1% by weight or more and less than 20% by weight of general formula (A) (A) ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (where R is hydrogen, alkyl group, etc.) Cyano-containing monomer and 2% by weight A monomer mixture containing from 80% by weight of a crosslinkable monomer to a homopolymer of at least one monomer in the monomer mixture and at least one other monomer is used. at least one liquid a that has no affinity for the homopolymer of the monomers, or at least one liquid b that has an affinity for the homopolymer of all the monomers in the monomer mixture. or a mixture consisting of liquid a and at least one liquid c that has no affinity for the homopolymer of all the monomers in the monomer mixture, and which dissolves the monomer mixture. And when X is the weight percent of the cross-linking polymerizable monomer based on the total monomers of the liquid or liquid mixture that does not react with each monomer, 0.
A resin is formed by copolymerization in the presence of 15√(X) times to 1.5√(X) times the weight, and then the liquid and unreacted monomer are removed from the interior of the resin. A method for producing a protein adsorbent made of a porous copolymer.