JPH0361421B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an immobilized enzyme and a method for producing the same. Enzyme reactions are partially carried out industrially in the manufacturing process of pharmaceuticals, foods, etc., but conventionally the enzyme is dissolved in an aqueous solution of a substrate and the reaction is carried out in this aqueous solution. However, with this method, it is extremely difficult to maintain constant reaction conditions, replenish fresh enzyme, and separate the product and enzyme without deactivating the enzyme after the reaction. and enzymes are consumed uneconomically. Moreover, since the reaction is a batch process, productivity is poor. In order to solve these problems, it has already been proposed to immobilize an enzyme on a water-insoluble carrier and react the immobilized enzyme with a substrate. A typical example of such an enzyme immobilization method is known as a carrier binding method in which an enzyme is bound to a water-insoluble carrier by covalent bonding, ionic bonding, or physical adsorption. However, the carriers conventionally used in this method are usually cellulose, dextran,
Immobilized enzymes, which are particles of 1 mm to several mm in diameter made of polysaccharide derivatives such as agarose, polyacrylamide gel, porous glass, etc., are usually packed into columns, and the enzymes are immobilized on such particles. Since the substrate is immobilized and brought into contact with the substrate solution, if the substrate has a high molecular weight, it is difficult to diffuse onto the surface of the immobilized enzyme, resulting in a problem that the reaction takes a long time and the reaction yield is low. The present invention was made in order to solve the above-mentioned problems, and is capable of moving freely in the reaction system in the same way as free enzymes, so that diffusion of the substrate to the surface of the immobilized enzyme is hardly a problem. An object of the present invention is to provide a method for producing an active immobilized enzyme.
In particular, the present invention aims to immobilize enzymes at a high loading amount on water-dispersible polymer particles having a high density of ion exchange groups or functional groups, and to immobilize enzymes that maintain high enzyme activity over a long period of time. The purpose of the present invention is to provide a chemical enzyme and a method for producing the same. The immobilized enzyme according to the present invention has a polymer core of 50 to 98% by weight consisting of the polymer of the first ethylenic monomer composition, and an ethylenic monomer having an ion exchange group or a functional group. 2 to 50% by weight of a polymer shell made of a polymer of the second monomer composition containing The immobilized enzyme is characterized in that the enzyme is immobilized, and the immobilized enzyme is obtained by emulsion copolymerizing the first ethylenic monomer composition in an aqueous medium to obtain a water-dispersible polymeric polymer according to the present invention. After forming a polymer core consisting of coalesced particles, a second monomer composition containing an ethylenic monomer having an ion exchange group or a functional group in the presence of this polymer core in an aqueous medium. is emulsion copolymerized to form water-dispersible polymer particles having a polymer shell on the polymer core, and then the polymer particles are treated with the ion exchange group or functional group via the ion exchange group or functional group. It is produced by immobilizing the enzyme. In the present invention, first, the first monomer composition is emulsion copolymerized in an aqueous medium to form a polymer core made of water-dispersible polymer particles. The polymer core obtained here must not melt and soften at the temperature at which the enzymatic reaction is carried out, generally from 5 to 90°C, and therefore the glass transition temperature of the obtained polymer particles must be at least The monomer composition is selected so that the temperature is 0°C, preferably 5°C or higher. Therefore, in the present invention, as the first monomer composition, styrene, methylstyrene, vinyltoluene, vinyl chloride, acrylic ester, methacrylic ester, especially alkyl ester having 1 to 4 carbon atoms, acrylonitrile, methacrylic ester, etc. Nitrile, vinyl acetate, vinyl propionate,
One or more of butadiene, isoprene, acrylamide, methacrylamide, etc. can be used. As is well known, even if a monomer forms a homopolymer with a glass transition point of 0°C or lower, by copolymerizing it with other monomers, the glass transition point of the resulting copolymer will change. Therefore, there is no problem in using ethyl acrylate, butyl acrylate, etc., which provide a homopolymer having a glass transition point of 0° C. or lower, as a monomer component. For example, the glass transition point of a homopolymer of ethyl acrylate is -22°C, but by copolymerizing methyl methacrylate and ethyl acrylate, whose homopolymer glass transition point is 105°C,
Usually, copolymers having a glass transition point of about 30°C can be obtained. In the present invention, the first monomer composition is 1 to 80
Preferably it contains % by weight of acrylonitrile and/or methacrylonitrile. By the presence of this monomer, even without using an emulsifier,
This is because the stability of polymerization is ensured, and the dispersibility of the obtained water-dispersible polymer particles is also improved. In particular, the first monomer composition preferred in the present invention includes (a) 1 to 80% by weight of acrylonitrile and/or methacrylonitrile, preferably 5 to 75% by weight, particularly preferably 10 to 60% by weight, ( b) Polyfunctional internal crosslinking monomer 0-10% by weight, preferably 0.1-10% by weight
15% by weight, particularly preferably 0.5-10% by weight, and
(c) 20 to 99% by weight, preferably 25 to 99% by weight of at least one monomer selected from acrylic acid alkyl ester, methacrylic acid alkyl ester and styrene
It consists of 95% by weight, particularly preferably from 40 to 90% by weight. By using such a monomer composition, polymerization can be carried out very stably even in the absence of an emulsifier, and the production of undesirable by-products such as water-soluble polymers can be suppressed. Incidentally, the polyfunctional internal crosslinking monomer is useful for increasing the stability of polymerization, as will be described later, and specific examples thereof will be described later. The emulsion copolymerization of the first monomer composition described above is carried out in an aqueous medium, and as a polymerization initiator, for example,
2,2′-azobisisobutyramidium hydrochloride,
2,2'-azobiscyanovaleric acid, persulfate, etc. are used. The water-dispersible polymer particles obtained in this way constitute the core of the water-dispersible polymer particles used as a carrier in the present invention, and usually have an average particle size in the range of 0.03 to 1.5μ. be.
In the present invention, it is preferable to carry out emulsion copolymerization without using an emulsifier, and as described above, copolymerization can be carried out without using an emulsifier. If the obtained water-dispersible polymer particles contain an emulsifier, the enzyme may be deactivated during enzyme immobilization, and the immobilized enzyme may be easily detached, causing harmful effects. This is because they tend to have an influence.
However, it is of course possible to carry out emulsion copolymerization in the presence of an emulsifier if the emulsifier does not have such a harmful effect. Next, in the present invention, in the presence of the polymer particles forming the polymer core obtained above in an aqueous medium, a second monomer containing a monomer having an ion exchange group or a functional group is added. The composition is emulsion copolymerized to form a polymer shell on a polymer core made of the first monomer composition. In this case as well, it is preferable not to use an emulsifier, as mentioned above. In the present invention, even when a monomer having a side chain that can be converted into the above-mentioned functional group by a chemical reaction after copolymerization is used as a component of the monomer composition, the monomer having the functional group It shall be included when copolymerizing a polymer. In the present invention, an ion-exchangeable group refers to an organic group through which an enzyme can be immobilized through an ionic bond, and a functional group refers to an organic group through which an enzyme can be immobilized through a covalent bond. Therefore, depending on the organic group, it may be both an ion exchange group and a functional group. Examples of the ion exchange group include acidic groups such as sulfonic acid groups, carboxyl groups, and phosphoric acid groups, and basic groups such as tertiary amino groups and quaternary amino groups. Specific examples of monomers having such ion exchange groups include monomers having sulfonic acid groups such as styrene sulfonic acid and sulfopropyl methacrylate, and monomers having carboxyl groups such as acrylic acid, methacrylic acid, and itaconic acid. Monomers with phosphoric acid groups such as acid phosphoxyethyl methacrylate, 3-chloro-2-acid phosphoxyethyl methacrylate, dimethylaminoethyl methacrylate, dimethylaminopropyl methacrylamide, etc. Examples include monomers having a tertiary amino group, monomers having a quaternary amino group such as methacrylamide propyltrimethylammonium chloride, and methacryloyloxyethyltrimethylammonium chloride. In addition, as a functional group, a carboxyl group, a primary
Examples include a class amino group, a hydrazide group, and a glycidyl group. Specific examples of monomers having such functional groups include monomers having a carboxyl group such as acrylic acid, methacrylic acid, and itaconic acid, and monomers having a hydroxyl group such as hydroxyethyl acrylate and hydroxyethyl methacrylate. , a monomer having a glycidyl group such as glycidyl methacrylate. In addition, monomers having an amide group such as acrylamide and monomers having a methyl ester group such as methyl acrylate and methyl methacrylate can be used in an aqueous medium in the presence of polymer particles forming the polymer core. emulsion copolymerize a second monomer composition containing each of these compounds to form a copolymer having these as a polymer shell on the polymer core, and then By subjecting the amide group in this copolymer to Hoffmann decomposition, and by allowing hydrazine to act on the methyl ester group, a polymer shell consisting of a copolymer having an amide group or hydrazide group as a functional group was obtained. can be formed. Alternatively, the second monomer composition contains a monomer having an ester group such as an acrylic ester, and this is copolymerized in the presence of the polymer core, and then,
By hydrolyzing this ester group, a polymer shell made of a copolymer having a carboxyl group as a functional group can also be formed. In the second monomer composition, monomers with ion-exchangeable groups or functional groups are often water-soluble, and therefore emulsion polymerization alone will result in undesirable water-soluble polymers. Sometimes. Therefore, in the present invention, the second monomer composition contains 1 to 70% by weight of the above monomer having an ion exchange group or functional group, and 1 to 80% by weight of acrylonitrile and/or methacrylonitrile. It is preferable to emulsion copolymerize a monomer composition containing 0 to 20% by weight of a polyfunctional internal crosslinking monomer, and 20 to 98% by weight of a monomer copolymerizable with these monomers. In this case as well, it is necessary that the formed polymer shell does not melt and soften at the temperature at which the enzyme reaction is carried out, and therefore the glass transition temperature of the formed polymer shell should be at least 0°C, preferably The temperature is selected to be 5°C or higher. Examples of the copolymerizable monomer include the above-mentioned styrene, methylstyrene, vinyltoluene, vinyl chloride, acrylic ester, methacrylic ester, especially alkyl ester having 1 to 4 carbon atoms, acrylonitrile, methacrylonitrile, vinyl acetate, One or more of vinyl propionate, butadiene, isoprene, acrylamide, methacrylamide, etc. are preferably used, and in particular, C1-C4 alkyl esters of acrylic acid and methacrylic acid and styrene are preferably used. Therefore, particularly preferred in the present invention, the second monomer composition preferably contains (a) 1 to 70% by weight, preferably 5 to 65% by weight, particularly preferably 5 to 65% by weight of a monomer having an ion-exchangeable group or a functional group. (b) acrylonitrile and/or methacrylonitrile 1 to 80% by weight, preferably 5 to 75% by weight, particularly preferably
10~60% by weight, (c) 0~polyfunctional internal crosslinking monomer
20% by weight, preferably 0.1-15% by weight, particularly preferably 0.5-10% by weight, and (d) 20-98% by weight of at least one monomer selected from acrylic esters, methacrylic esters and styrene; It preferably comprises 25 to 95% by weight, particularly preferably 40 to 80% by weight. By emulsion copolymerizing such a monomer composition, the formation of a homopolymer made of a monomer having an ion exchange group or a functional group is suppressed, and the polymerization is particularly stable, and the resulting product is The water-dispersible polymer particles also have excellent dispersion stability, and furthermore, when an enzyme is immobilized thereon, detachment of the enzyme can be suppressed. The above internal crosslinking polyfunctional monomer is preferably poly(meth)acrylate of polyhydric alcohol, specifically, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate,
Dipropylene glycol dimethacrylate, 1,
3-butylene glycol dimethacrylate, triethylene glycol diacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, tetramethylolmethanetetraacrylate, etc. are used. Divinylbenzene is also preferably used. Generally, monomers having ion exchange groups or functional groups are hydrophilic, so if they are emulsion polymerized alone, the polymerization becomes unstable and there are many side effects such as undesirable water-soluble polymers. It is difficult to obtain polymer particles with good dispersibility. In particular, when a water-soluble polymer is produced, a part of it is adsorbed and remains on the water-insoluble polymer particles.
When an enzyme is immobilized using this as a carrier, the enzyme is also immobilized on this water-soluble polymer. With such an immobilized enzyme in which the enzyme is immobilized on a carrier containing a water-soluble polymer, the enzyme is eluted into the substrate solution together with the water-soluble polymer during the enzymatic reaction, and the enzyme activity decreases over time. In addition, the eluted enzyme will be mixed with the substrate and reaction products, resulting in various inconveniences such as the need to separate them after the reaction. However, according to the present invention, monomers having ion-exchangeable or functional groups are combined with acrylonitrile and/or in the presence of a previously prepared polymer core.
Alternatively, by emulsion copolymerization with methacrylonitrile, preferably with a polyfunctional internal crosslinking monomer, stability of the polymerization is ensured even in the absence of an emulsifier, and undesirable water-soluble polymers are eliminated. The formation is suppressed, and these monomer compositions can be stably copolymerized on the polymer core,
In this way, water-dispersible polymer particles having a high density of ionic groups or functional groups on the surface can be obtained. The reason why such a result is obtained is not necessarily clear, but during emulsion copolymerization of the second monomer composition, the polymer core made of the first monomer composition is already used as a polymerization site. For polyfunctional internal crosslinking with acrylonitrile and/or methacrylonitrile, a water-soluble low molecular weight polymer mainly composed of a monomer having an ionic group or a functional group generated in the initial stage of polymerization. The monomers are effectively copolymerized to make them insoluble in water, and their affinity with the core polymer particles improves, allowing them to be smoothly grafted onto the surface of the core polymer particles, thus stabilizing the polymerization. Therefore, in the present invention, the second monomer composition preferably contains acrylonitrile and/or methacrylonitrile and a polyfunctional internal crosslinking monomer, as described above. In addition, by emulsion copolymerizing the second monomer composition as described above on the polymer core to form a polymer shell, it is possible to form a polymer shell with ion-exchangeable groups or functional groups useful for immobilizing the enzyme. The groups can be formed at high density on the surface of water-dispersible polymer particles, and thus, according to the present invention, enzymes can be immobilized at high loadings. Furthermore, in the present invention, in the water-dispersible polymer particles obtained as described above, the polymer core portion is 50 to 98% by weight, and the polymer shell portion is 2 to 98% by weight.
It is necessary to sequentially emulsion copolymerize the first and second monomer compositions so that the total concentration is 50% by weight. When the shell portion is less than 2% by weight in the polymer particle, the amount of ion exchange groups or functional groups of the copolymer that the polymer particle has as the polymer shell portion on its surface is reduced, and the enzyme that can be immobilized is reduced. This is because the amount of immobilized enzyme is limited, making it difficult to obtain an immobilized enzyme with high enzymatic activity. On the other hand, when it exceeds 50% by weight, the ion-exchangeable groups and functional groups are hydrophilic, so the second monomer composition is Even if copolymerization is carried out, in addition to forming a shell, a water-soluble polymer is produced only from the second monomer composition, causing the above-mentioned disadvantages, and furthermore, the stability of the polymerization is affected. This is because the dispersibility of polymer particles may also be impaired. The water-dispersed polymer particles used in the present invention have an average particle diameter of 0.05μ to 2μ, preferably
It is 0.1μ to 1μ. If the particle size is too small, it will be difficult to recover the immobilized enzyme used as a carrier after dispersing it in water and performing an enzyme reaction. On the other hand, if the particle size is too large, the particle surface area per unit volume will be As the size of the enzyme decreases, the amount of enzyme immobilized decreases.
This is not preferred because it becomes difficult to disperse in water. The method of immobilizing the enzyme to the ion exchange group of the water-dispersible polymer particles as described above through ionic bonding, or the method of immobilizing the enzyme to the functional group via this, is not particularly limited. Known methods are employed as appropriate. For example, in order to immobilize an enzyme on water-dispersible polymer particles having an ion-exchange group through ionic bonding, a dispersion of polymer particles can be prepared under appropriate conditions such as temperature and pH that will not cause deactivation of the enzyme. and the enzyme solution may be mixed. For example, the pH during the reaction is the isoelectric point of the enzyme,
Although it is appropriately selected depending on the type of ion exchange group, etc., 5 to 8 is generally preferable. Thereafter, if necessary, unimmobilized enzymes are removed by appropriate means such as centrifugation or membrane separation, thereby obtaining the immobilized enzyme of the present invention. Furthermore, the method of immobilizing the enzyme on the water-dispersible polymer particles having a functional group is not particularly limited, and any conventionally known method may be employed as appropriate. As such a method, depending on the type of functional group,
Examples include, but are not limited to, the diazo method, the carbodiimide method, the cyanogen bromide method, and the azide method. For example, in order to covalently immobilize an enzyme on water-dispersible polymer particles having hydroxyl groups, the hydroxyl groups are epoxidized with epichlorohydrin, and then the amino groups of the enzyme are reacted with the epoxy groups. Enzymes can be attached to polymer particles by forming amide groups. This epoxidation is carried out under conventionally known conditions, for example, using epichlorohydrin in an amount of 1 to 10 times the equivalent of the hydroxyl groups of the water-dispersible polymer particles, in an alkaline aqueous solution at room temperature. React with. Immobilization of the enzyme onto this epoxy group is also carried out under conventional and common conditions. In addition, one method of immobilizing enzymes on carboxyl groups is to use water-soluble carbodiimide.
The amino group of the enzyme and the carboxyl group on the surface of the water-dispersible polymer particles can be directly bonded by forming an amide bond. Examples of water-soluble carbodiimide include 1-ethyl-3
-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-meth-p-toluenesulfonate, and the like. Enzyme immobilization using water-soluble carbodiimide is carried out under conventionally known conventional conditions. Mix the enzyme, keep the temperature around 5℃, and the pH 4.5~
6.0 and react overnight. In addition, in the present invention, when the enzyme is immobilized by covalent bonds through the functional groups of the polymer particles, if necessary, hexamethylene diamine, dodecamethylene diamine, glycylglycylglycine, etc. are added to the polymer particles. Enzymes can be bound via spacer groups to increase the degree of freedom of immobilized enzymes on polymer particles. After the enzyme is immobilized on the polymer particles as described above, if the reaction reagent used and the unimmobilized enzyme are removed by appropriate means such as centrifugation or membrane separation, the present invention can be achieved. Obtain immobilized enzyme. The immobilized enzyme according to the invention is used as an aqueous dispersion and contacted with the substrate. The amount of immobilized enzyme to be used is appropriately determined depending on the particle size of the immobilized enzyme, the amount of immobilized enzyme, the required reaction rate, substrate concentration, etc. The enzyme immobilized in the present invention may be an intracellular enzyme or an extracellular enzyme. Furthermore, the enzyme does not necessarily have to be highly purified, and extracts and partially purified products can also be used. Furthermore, in accordance with the present invention, a single enzyme may be immobilized, or multiple enzymes may be immobilized simultaneously. In the present invention, the enzyme is not particularly limited, and various enzymes can be used. Specific examples include amino acid oxidase, catalase, xanthine oxidase, glucose oxidase, glucose-6-
oxidoreductases such as phosphate dehydrogenase, glutamate dehydrogenase, cytochrome C oxidase, tyrosinase, lactate dehydrogenase, peroxidase, 6-phosphogluconate dehydrogenase, malate dehydrogenase;
Aspartate acetyltransferase, aspartate aminotransferase, glycine aminotransferase, glutamate-oxaloacetate aminotransferase, glutamate-pyruvate aminotransferase, creatine phosphokinase, histamine methyltransferase, pyruvate Kinases, fructokinase, hexokinase, δ-lysine acetyltransferase, transferases such as leucine aminopeptidase, asparaginase, acetylcholinesterase, aminoacylase, amylase, arginase, L-arginine deiminase, invertase, urease, uricase, urokinase, Esterase, β-galactosidase, kallikrein, chymotrypsin, trypsin, thrombin, oringinase, nucleotidase, papain, hyauronidase, plasmin, pectinase, hesperidinase, pepsin, penicillinase, peniciliamidase, phospholipase, phosphatase, lactase, lipase, ribonuclease, rennin, etc. hydrolase, aspartate decarboxylase,
alpartase, citrate lyase, glutamate decarboxylase, histidine ammonia lyase, phenylalanine ammonia lyase,
Lyases such as fumarase, fumarate hydratase, malate synthetase, isomerases such as alanine racemase, glycose isomerase, glycose phosphate isomerase, glutamate racemase, lactate racemase, methionine racemase, asparagine synthase, glutathione synthase, pyruvate synthase. Examples include ligases such as As described above, in the immobilized enzyme of the present invention, water-dispersible polymer particles as a water-insoluble carrier form a polymer shell having ion exchange groups or functional groups densely formed on a polymer core. A section is formed. Therefore, unlike the case of using conventional cellulose derivative particles as a carrier, the enzyme is immobilized at a high loading amount, and the immobilized enzyme itself can move freely within the reaction system like a free enzyme. The diffusion of the substrate has little effect on the reaction, and therefore, even in the case of a high molecular weight substrate, the enzymatic reaction can be carried out at a high reaction rate similar to that of free enzyme. Moreover, since the immobilized enzyme of the present invention is immobilized on a water-insoluble carrier, it can be easily processed by centrifugation, salting out, coagulation precipitation using a flocculant, membrane separation using a porous membrane, etc. after the enzyme reaction. It can be recovered and thus used repeatedly while retaining high enzymatic activity over a long period of time. In addition, from the viewpoint of manufacturing the immobilized enzyme of the present invention, generally, a monomer composition containing a monomer having an ion exchange group or a functional group is emulsion copolymerized,
Although it is difficult to produce water-dispersible polymer particles having these ion exchange groups and functional groups on the surface at high density, according to the present invention, a polymer core is formed in advance, Since a monomer composition containing a monomer having an ion exchange group or a functional group is emulsion copolymerized thereon, polymer particles can be obtained by stable copolymerization, and the resulting water Unlike the case where only the second monomer composition is emulsion copolymerized, the dispersible polymer particles have these ion exchange groups or functional groups at a high density on the surface, and as a result, the present invention Enzyme can be immobilized at a high loading amount on the water-dispersible polymer particles obtained by the above method. Examples of the present invention are listed below, but the present invention is not limited to these Examples. Example 1 80g of methyl methacrylate and acrylonitrile
Add 15g to 421g of distilled water and add 2,2'-azobis-
A polymerization initiator aqueous solution prepared by dissolving 0.3 g of 2-amidinopropane dihydrochloride in 10 g of water was added under a nitrogen stream at a temperature of 60°C, and polymerized for 4 hours while stirring at 120 rpm to obtain a water dispersion as a polymer core. Polymer particles with high molecular weight were obtained. Next, an aqueous polymerization initiator solution containing 0.03 g of the same polymerization initiator as above dissolved in 10 ml of water was added thereto, and a mixture of 5.5 g of methyl methacrylate, 5 g of acrylonitrile, 3 g of methacrylic acid, and 0.5 g of toluethylene glycol dimethacrylate was added. It took 1 hour to add and react for 3 hours to form a polymer shell having a carboxyl group as a functional group on the core. The aqueous dispersion of polymer particles thus obtained had a solid content of 20% and an average particle size of the polymer particles of 0.45μ. An aqueous dispersion of polymer particles with an average particle size of 0.45μ was obtained. The polymerization was very stable, with 0.08% agglomerates. This dispersion was centrifuged and the carboxyl groups in the supernatant were quantified and found to be 0.9% of the charged amount.
Only the carboxyl groups in the sample were quantified, and the formation of water-soluble polymerization was minimal. Next, the above polymer particles were dispersed in distilled water to a solid content of 20% by weight to obtain 50 ml of a dispersion liquid.
Cyclohexyl-3-(2-morpholinoethyl)
An aqueous solution of 2.5 g of carbodiimide-meth-p-toluenesulfonate dissolved in 30 ml of water was added to the above dispersion, and the pH was adjusted to 6.5 with hydrochloric acid. After this, α
- An enzyme aqueous solution prepared by dissolving 500 mg of amylase in 10 ml of water was added, and while the pH was adjusted to 6.5 at 5° C., the mixture was left to react for 24 hours, thereby immobilizing α-amylase on the polymer particles through covalent bonds. Next, the precipitated polymer particles were centrifuged and treated with 0.1M each of sodium chloride, sodium acetate, and calcium acetate.
The immobilized α-amylase according to the present invention was thus obtained by redispersing it in a buffer solution with pH 7.0 dissolved at 0.02M and 0.001M. In this immobilized enzyme, the amount of α-amylase immobilized is 40 mg per 1 g of polymer particles, and
The activity yield was 45%. Incidentally, the activity yield is defined as the ratio of the actual activity to the theoretical amount of the activity of the immobilized enzyme. Here, the activity of the immobilized enzyme, that is, the starch decomposition rate (mg/ The amount of free enzyme having the same activity was divided by the amount of immobilized enzyme. Reference example This reference example shows the case where the monomer composition was copolymerized stepwise in the same manner as in Example 1, and when the first and second monomer compositions were variously changed and copolymerized. The amounts of carboxyl groups in the aggregates and the supernatant of the dispersion after polymerization are shown. The results are shown in the table. Furthermore, when the monomer composition of Experiment No. 4 was copolymerized all at once, the amount of aggregates was 7.3%, the amount of carboxyl groups was 6%, and the polymer particles in the dispersion contained carboxyl groups per gram. It had 0.08 mmol. However, in the case of stepwise copolymerization, the polymer particles had 0.35 mmol of carboxyl groups per gram. Example 2 The carboxyl group-containing polymer particles obtained in Example 1 were dissolved in 50 ml of a phosphate buffer solution with a solid content of 20 ml.
An enzyme aqueous solution prepared by dissolving 50 mg of α-chymotrypsin in the same buffer as above was added to the above polymer particle dispersion, left at 5° C. for 24 hours, and then centrifuged. The precipitated polymer particles were washed with a buffer solution to remove unimmobilized α-chymotrypsin, and then dispersed in the buffer solution again to obtain covalently immobilized α-chymotrypsin according to the present invention. Immobilization of this immobilized enzyme α-chymotrypsin

【table】

[Table] Represents dimethacrylate.
The amount was 4.0 mg per gram of polymer particles, and the activity yield was 90%. Here, the activity yield is 0.05m
The immobilized enzyme was reacted at 30°C using N-acetyl-L-tyrosine ethyl ester of M as a substrate, and the rate of carboxyl group production (μmol/min) was determined by alkaline titration. Example 3 After forming the same polymer core as in Example 1 in the same manner as in Example 1, an aqueous dispersion containing the same was added with 4 g of dimethylaminoethyl methacrylate, 4 g of acrylonitrile, and acrylic acid as a second monomer composition. A polymer shell was formed in the same manner as in Example 1 except that 5.5 g of ethyl and 0.5 g of triethylene glycol dimethacrylate were added, and the solid content was 20
%, an aqueous dispersion of polymer particles with an average particle size of 0.3 μm was obtained. The polymer particles precipitated by centrifugation were dispersed in 100 ml of 0.1 M phosphate buffer (PH7.0) to a solid content of 10% by weight, and 500 mg of urease was added thereto, followed by stirring at 5°C for 24 hours. , urease was immobilized on polymer particles by ionic bonding. Thereafter, the precipitated polymer particles were centrifuged and washed with the same buffer as above to remove unimmobilized urease, and then dispersed again in the same buffer to obtain immobilized urease according to the present invention. The amount of urease immobilized in this immobilized enzyme was 45 mg per 1 g of polymer particles, and the activity yield was
It was 95%. The activity yield is determined by reacting the immobilized enzyme at 35°C for 10 minutes using a 0.03M urea aqueous solution as a substrate, and measuring the activity by determining the amount of ammonia produced (μmol/min) by hydrochloric acid titration. It was determined by dividing the amount of active free enzyme by the amount of immobilized enzyme.

Claims (1)

[Scope of Claims] 1. Contains a 50 to 98% by weight polymer core made of the polymer of the first ethylenic monomer composition and an ethylenic monomer having an ion exchange group or a functional group. 2-50% by weight of the polymer of the second monomer composition
1. An immobilized enzyme, characterized in that the enzyme is immobilized on water-dispersible polymer particles comprising a polymer shell portion and the above-mentioned ion exchange group or functional group. 2 The first monomer composition contains (a) 1 to 80% by weight of acrylonitrile and/or methacrylonitrile, (b) 0 to 20% by weight of a polyfunctional internal crosslinking monomer, and (c) acrylic acid. The immobilized enzyme according to claim 1, comprising 20 to 99% by weight of at least one monomer selected from alkyl esters, alkyl methacrylates, and styrene. 3 The second monomer composition includes (a) 1 to 70% by weight of a monomer having an ion exchange group or a functional group, (b) 1 to 80% by weight of acrylonitrile and/or methacrylonitrile, (c) 0 to 20% by weight of a polyfunctional internal crosslinking monomer, and (d) 20 to 98% by weight of at least one monomer selected from acrylic acid alkyl ester, methacrylic acid alkyl ester, and styrene. An immobilized enzyme according to claim 1, characterized in: 4. The functional group according to claim 1, wherein the functional group in the second ethylenic monomer composition is at least one selected from a primary amino group, a hydroxyl group, a glycidyl group, and a carboxyl group. Immobilized enzyme. 5. Claim 1, wherein the ion exchange group in the second ethylenic monomer composition is at least one acidic group selected from a carboxyl group, a sulfonic acid group, and a phosphoric acid group. immobilized enzyme. 6. A patent claim characterized in that the ionic group in the second ethylenic monomer composition is at least one basic group selected from a tertiary amino group, a quaternary amino group, and phosphoric acid. The immobilized enzyme according to scope 1. 7. The immobilized enzyme according to claim 1, wherein the water-dispersible polymer particles have an average particle size of 0.05 to 2μ. 8 After emulsion copolymerizing the first ethylenic monomer composition in an aqueous medium to form a polymer core made of water-dispersible polymer particles, the polymer core is copolymerized in an aqueous medium. A second monomer composition containing an ethylenic monomer having an ion-exchangeable group or a functional group in the presence thereof is emulsion copolymerized to form an aqueous dispersion having a polymer shell on the polymer core. 1. A method for producing an immobilized enzyme, which comprises forming polymer particles, and then immobilizing the enzyme on the polymer particles via the ion exchange group or functional group. 9 The first monomer composition contains (a) 1 to 80% by weight of acrylonitrile and/or methacrylonitrile, (b) 0 to 20% by weight of a polyfunctional internal crosslinking monomer, and (c) acrylic acid. 9. The method for producing an immobilized enzyme according to claim 8, comprising 20 to 99% by weight of at least one monomer selected from alkyl esters, alkyl methacrylates, and styrene. 10 The second monomer composition contains (a) 1 to 70% by weight of a monomer having an ion exchange group or a functional group, (b) 1 to 80% by weight of acrylonitrile and/or methacrylonitrile.
(c) 0 to 20 weight % of a polyfunctional internal crosslinking monomer, and (d) 20 to 98 weight % of at least one monomer selected from acrylic acid alkyl ester, methacrylic acid alkyl ester, and styrene. %. 9. The method for producing an immobilized enzyme according to claim 8. 11. The functional group according to claim 8, wherein the functional group in the second ethylenic monomer composition is at least one selected from a primary amino group, a hydroxyl group, a glycidyl group, and a carboxyl group. Method for producing immobilized enzyme. 12 Claim 8, wherein the ion exchange group in the second ethylenic monomer composition is at least one acidic group selected from carboxyl group, sulfonic acid group, and phosphoric acid group. A method for producing an immobilized enzyme. 13 The ionic group in the second ethylenic monomer composition is a tertiary amino group and a quaternary amino group,
9. The method for producing an immobilized enzyme according to claim 8, wherein the basic group is at least one kind selected from phosphoric acid. 14. The method for producing an immobilized enzyme according to claim 8, wherein the water-dispersible polymer particles have an average particle size of 0.05 to 2μ.