JPS5945888A - Preparation of immobilized enzyme - Google Patents

Abstract

PURPOSE:To prepare an immobilized enzyme capable of keeping high enzymatic activity for a long period, by saponifying the particles of a specific water- dispersible polymer, and immobilizing an enzyme to the particle through the formed carboxyl group with covalent bond. CONSTITUTION:Particles of a water-dispersible polymer having an average particle diameter of 0.03-2mu are prepared by the copolymerization of (a) 5- 90wt% of a (meth)acrylic acid ester of formula (R1 is H or methyl; R2 is 1-3C alkyl or benzyl), (b) 0-90wt% of a monomer copolymerizable thereiwth (e.g. styrene), (c) 1-20wt% of a polyfunctional monomer for internal crosslinking (e.g. divinylbenzene) and (d) 1-60wt% of (meth)acrylonitrile. The particle is saponified with a 0.01-5N aqueous solution of an acid or alkali at 20-70 deg.C to form carboxyl group. The carboxyl group is made to react with the amino group of an enzyme (e.g. catalase) in the presence of a water-soluble carbodiimide to immobilize the enzyme to the polymer particle with a covalent bond.

Description

[Detailed description of the invention] The present invention relates to a method for producing an immobilized enzyme.

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. It is difficult and enzymes are consumed uneconomically. Moreover,
Productivity is poor because the reaction is a batch process.

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 particles of 1 mm to several mm in diameter, such as polysaccharide derivatives such as cellulose, dextran, and agarose, polyacrylamide gel, and porous glass. An immobilized enzyme, in which the enzyme is immobilized on particles, is usually packed into a column, immobilized, and brought into contact with a substrate solution, so if the substrate has a high molecular weight, it will diffuse onto the surface of the immobilized enzyme and hatch. There are problems in 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 has a high activity that allows the enzyme to move freely in the reaction system like a free enzyme, 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 immobilized enzyme.

The method for producing an immobilized enzyme according to the present invention includes (5 to 98% by weight of al acrylic ester or methacrylic ester,
(2) 0 to 90% by weight of a first monomer copolymerizable with this monomer, 1 to 20% by weight of a tel polyfunctional internal crosslinking monomer
, and (dl) A monomer composition consisting of 1 to 60% by weight of acrylonitrile or methacriconitrile as the second monomer is emulsion copolymerized, and the resulting water-dispersible polymer particles are saponified to form carboxyl generate a group, then
It is characterized by covalently immobilizing the enzyme via this carboxyl group.

In the present invention, acrylic esters and/or methacrylic esters are hereinafter referred to as (meth)acrylic esters. ) is copolymerized by emulsion, preferably in the absence of an emulsifier, and the ester groups in the side chains of the water-dispersible polymer particles thus obtained are saponified. By doing so, carboxyl groups are generated at a high density on the surface, and the enzyme is immobilized via covalent bonds via these carboxyl groups.

Here, (meth)acrylic acid esters include those represented by the general formula % (where R1 represents hydrogen or a methyl group, and R2 represents an alkyl group having 1 to 3 carbon atoms or a benzyl group). are preferably used, and (meth)acrylic acid esters are particularly preferably used.

The first copolymerized with such (meth)acrylic ester
As monomers, except for acrylonitrile, methacrylate trile, and the above (meth)acrylic acid ester,
There is no particular restriction as long as it is copolymerizable with these and the resulting copolymer has a glass transition point higher than the temperature at which the enzyme reaction is carried out, but preferably,
One or more of ethylene, propylene, vinyl chloride, acrylic ester, methacrylic ester, styrene, methylstyrene, vinyltoluene, butadiene, isoprene, acrylamide, methacrylamide, etc. are used. In particular, styrene is preferably used. however,
This first monomer does not necessarily need to be included in the monomer composition as a monomer component.

In addition, as the first monomer, in order to stabilize the polymer particles during polymerization and to stabilize the polymer particles, a monomer having an ionic group is used in an amount of 5% by weight or less of the total monomer composition. Good too. Examples of such ionic groups include acid 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 ionic 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. A monomer having
Monomers with phosphoric acid groups such as acid phosphoxyethyl methacrylate and 3-chloro-2-acid phosphoxyethyl methacrylate; monomers with tertiary amino groups such as dimethylaminoethyl methacrylate and dimethylaminopropyl methacrylamide. Examples include monomers having a quaternary amino group such as methacrylamide propyltrimethylammonium chloride and methacryloyloxyethyltrimethylammonium chloride.

Moreover, as a polyfunctional monomer for internal crosslinking, 1. Preferably, poly(meth)acrylates of polyhydric alcohols include ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, dipropylene glycol dimethacrylate, and 1,3-butylene glycol dimethacrylate. 1. Triethylene glycol diacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, tetramethylolmethanetetraacrylate, etc. are used. Divinylbenzene is also preferably used.

Furthermore, in the present invention, as a monomer for obtaining water-dispersible polymer particles, the monomer composition may contain acrylonitrile and/or methacrylonitrile as a second monomer component. Required.

In the present invention, water-dispersible polymer particles having an ester group in the side chain can be obtained by emulsifying and copolymerizing each of the above-mentioned monomers in an aqueous medium using a conventional method. However, if emulsifiers are mixed in the resulting polymer particles, harmful effects such as deactivation of enzymes may occur during enzyme immobilization, so emulsifiers are not used when making emulsified copolymers. is preferable. According to the above-mentioned monomer composition according to the present invention, stable copolymerization can be carried out without particularly requiring an emulsifier. You may use it accordingly.

The monomer composition in the present invention includes (meth)acrylic acid ester 5 to 98% by weight, preferably 20 to 93% by weight, a first monomer copolymerizable with this monomer 0 to 90% by weight,
Preferably 0 to 80% by weight, 1 to 20% by weight, preferably 2 to 10% by weight of a polyfunctional internal crosslinking monomer, and a second
191 to 60% by weight, preferably 5 to 40% by weight of acrylonitrile or methacriconitrile as a monomer.

In the monomer composition, the amount of (meth)acrylic acid ester monomer is determined by the amount of carboxyl groups produced by saponifying the water-dispersible polymer particles obtained from this, and therefore by the amount of enzymes that can be immobilized on these carboxyl groups. It is also related to the amount of
If it is too small, the enzyme cannot be immobilized in sufficient quantity, and if it is too large, it will impair the stability of polymerization and
This is not preferred because the stability of the resulting polymer particles is also impaired.

Since the (meth)acrylic acid ester used in the present invention is hydrophobic, it does not produce a water-soluble polymer, and by emulsion copolymerizing this with (meth)acrylonitrile, the polymerization is extremely stable. is carried out, with almost no agglomerates being formed. When the amount of (meth)acrylonitrile is less than the above range or too much, it is not preferable because the stability of polymerization may be impaired. Furthermore, the internal crosslinking monomer contributes to increasing the stability of the polymer particles during saponification, which will be described later, and is preferably used within the above range.

According to the present invention, the water-dispersible polymer particles having ester groups obtained as described above are saponified with an aqueous acid or alkali solution, and the ester groups are converted to carboxyl groups. Examples of acids include hydrochloric acid, sulfuric acid, phosphoric acid, etc., and examples of alkalis include sodium hydroxide,
Potassium hydroxide, ammonia, etc. are used as appropriate. The concentration of these acid or alkaline aqueous solutions is usually
It is 0.01 to 5 normal, preferably 0.05 to 2 normal.

In the present invention, it is sufficient to saponify only the surface of the polymer particles, and the saponification conditions are appropriately selected. Usually, saponification is carried out using the above-mentioned acid or alkali at a temperature of 20 to 70"C for several minutes to several tens of hours.

When the concentration of acid or alkali is too high, the stability of the polymer particles deteriorates, and the polymer particles aggregate or become water-soluble. Moreover, when the concentration is too low, saponification takes an unnecessarily long time, which is not preferable. Too high a temperature is also not preferred because the stability of the polymer particles deteriorates.

The water-dispersible polymer particles obtained in this manner have hydroxyl groups on the surface at a high density, but the particle size hardly changes before and after saponification, and only slightly increases after saponification.

The amount of carboxyl groups contained in the water-dispersible polymer particles is 0.005 to 20 milliequivalents, preferably 0.01 to 5 milliequivalents, per gram of the polymer particles.

This is because if the loading amount is small, the enzymatic reaction will not be carried out sufficiently, while if it is too large, the stability of the water-dispersible polymer particles may be impaired.

The water-dispersed polymer particles used in the present invention have an average particle diameter of 0.03μ to 2μ, preferably 0.07μ.
The thickness is between 1μ and 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 This is not preferable because it becomes small, the amount of enzyme immobilized decreases, and it becomes difficult to disperse in water.

The method of covalently immobilizing the enzyme via the carboxyl group of the water-dispersible polymer particles is not particularly limited, and conventionally known methods may be employed as appropriate.

For example, one method is to convert the carboxyl group and the free amino group of the enzyme into a water-soluble carbodiimide, e.g.
Form a covalent bond by reacting with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-meth-p-)luenesulfonate, etc. can be done. Specifically, for example, an enzyme is added to an aqueous solution of the carbodiimide in an amount equivalent to 5 to 50 times the carboxyl group of the polymer particles, and the temperature is about 5°C, and the pn is 4.5.
What is necessary is just to leave it still or to stir, keeping it at about 6.0.

Another method is to react N-hydroxysuccinimide with the carboxyl group on the surface of the water-dispersible polymer particles in the presence of carbodiimide, and then react with the amino group of the enzyme to form a covalent bond. . Furthermore, as another method, diamine is allowed to act on the carboxyl group on the surface of the water-dispersible polymer particles,
It is also possible to introduce amino groups onto the surface of the polymer particles and to immobilize the enzyme through covalent bonds. For example, the carbodiimide described above can be used to convert the carboxyl group of the enzyme into an amino group of the polymer particle, and a cross-linking reagent such as glutaraldehyde can be used to convert the 7-mino group of the enzyme into the polymer particle. Can be combined.

When dialdehyde is used as a crosslinking reagent, an excess amount is reacted with the amino groups of the polymer particles, and the free aldehyde group of the dialdehyde bound to the polymer particles by one aldehyde group is reacted with the amino group of the enzyme. Combine groups.

Furthermore, as another method, a diazo coupling method can also be used. For example, p-nitrobenzaldehyde is reacted and bonded to a polymer particle into which an amino group has been introduced, and then the nitro group is reduced to an amino group using a conventional method such as sodium borohydride and dithorium dithionite. The amino group is converted into a diazonium group using sodium nitrite, and this is coupled to the amino group of the enzyme.

After covalently bonding the enzyme to 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 immobilization of the present invention can be achieved. Get the 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-phosphate dehydrogenase, glutamate dehydrogenase, cytochrome C oxidase, tyrosinase, lactate dehydrogenase, peroxidase, 6-phosphogluconate dehydrogenase, malate dehydrogenase. Oxidoreductase, aspartate acetyltransferase, aspartate aminotransferase, glycine aminotransferase, glutamate-oxaloacetate aminotransferase, glutamate-virupate aminotransferase, taleatin phosphokinase, histamine methyltransferase, pyruvate kinase, fructokinase, hexokinase, Transferases such as δ-lysine acetyltransferase, leucine aminopeptidase, asparaginase, acetylcholinesterase, aminoacylase, amylase, arginase, L-
Alanine racemase, invertase, urease, uricase, urokinase, esterase, β-galactosidase, kallikrein, chymotrypsin, trypsin, thrombin, naringinase, nucleotidase, papain, hyauronidase, plasmin, pectinase, hexokinase, pepsin, pectinase, penicillin amidase, phospholipase, Hydrolytic enzymes such as phosphatase, lactase, lipase, ribonuclease, rennin, aspartate decarboxylase,
aspartase, citrate lyase, glutamate decarboxylase, histidine ammonia lyase, phenylalanine ammonia lyase, fumarase, fumarate hydratase, malate synthetase, alanine racemase, glucose isomerase,
Examples include isomerases such as glucose phosphate isomerase, glutamate racemase, lactate racemase, and methionine racemase, and gauze such as asparagine synthase, glutathione synthase, and pyruvate synthase.

5. As described above, the immobilized enzyme of the present invention has an enzyme immobilized on water-dispersed polymer particles having a high density of carboxyl groups on the surface through covalent bonds via the carboxyl groups, and is different from conventional methods. Unlike when using cellulose derivative particles as a carrier, the immobilized enzyme itself can move freely within the reaction system like a free enzyme, so the diffusion of the substrate has little effect on the reaction. The enzyme reaction can be carried out with the same high reaction rate as the free enzyme even when the substrate is

Moreover, since the immobilized enzyme according to the present invention is immobilized on a water-insoluble carrier, centrifugation, salting out, and
It can be easily recovered by coagulation precipitation using a flocculant, membrane separation using a porous membrane, etc., and thus can be used repeatedly while maintaining high enzyme activity for a long period of time.

Furthermore, since monomers having carboxyl groups are generally water-soluble, emulsion copolymerization with other monomers is not easy.
Homopolymers are often produced, and even if copolymers are obtained, the density of carboxyl groups on the surface of the polymer particles is low, making it difficult to increase the amount of enzyme loaded in the polymer particles. According to the present invention, the monomer composition suppresses the formation of undesirable water-soluble polymers without using an emulsifier, and stably obtains water-dispersible polymer particles, which can then be quenched. to produce carboxyl groups, it is possible to stably obtain water-dispersible polymer particles having carboxyl groups, and since the density of carboxyl groups on the polymer particles is high, the enzyme can be used at a high loading amount. Can be immobilized. Furthermore, by using (meth)acrylonitrile and an internal crosslinking monomer in combination, the water-dispersible polymer particles obtained have high strength and do not stick to each other.

The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 80 g of methyl acrylate, 2 g of dimethacrylate) and 15 g of acrylonitrile were added to 230 g of distilled water, and 0.3 g of 2,2'-azobis-2-amidinopropane dihydrochloride was dissolved in 10 ml of fc water. Add an aqueous polymerization initiator solution at a temperature of 60°C under a nitrogen stream, and polymerize for 8 hours while stirring at 120 rpm.
An aqueous dispersion of polymer particles having a solid content of approximately 30% and an average particle size of 0.4 microns was obtained. The polymerization was very stable, with 0.104% agglomerates.

Next, 100 ml of a 2N aqueous sodium hydroxide solution was added to the above aqueous dispersion of polymer particles, stirred overnight at room temperature, neutralized by adding a 2N aqueous hydrochloric acid solution, and the polymer particles were centrifuged and washed. .

The amount of carboxyl groups per gram (dry weight) of the polymer particles thus obtained was 3.3 mmol.

Add 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide to 100ml of the aqueous dispersion of the above polymer particles.
40g of metho-p-1-luenesulfonate in 200ml of water
Add an aqueous solution dissolved in 1 and adjust the pH to 5.0 while stirring.
Adjusted to. An aqueous solution prepared by dissolving 2.5 g of α-amylase in 500 ml of water and adjusting the pH to 5.0 was added to the above polymer particle aqueous dispersion, and while stirring and adjusting the pH to 5.0, the solution was heated to 5°C. The mixture was allowed to react for 24 hours.

After that, the polymer particles are centrifuged, and the precipitated particles are washed with a buffer solution to remove unfixed α-amylase, unreacted carbodiimide, and reaction by-products, and then dispersed in the buffer solution again. An immobilized enzyme according to the present invention was obtained.

The amount of α-amylase immobilized in this immobilized enzyme was 70 μ/g of polymer particles, and the activity yield was 40%.

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 immobilized enzyme was heated at 35° using 1% starch aqueous solution as a substrate.
The activity of the immobilized enzyme was determined by reacting for 30 minutes at
That is, the starch decomposition rate (■/min) was obtained, and the amount of free enzyme having the same activity was divided by the amount of enzyme immobilized.

Example 2 9 Aqueous dispersion of polymer particles having hydroxyl groups obtained in Example 1 10
An aqueous solution of 20 g of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-meth-p-toluenesulfonate dissolved in 200 ml of water was added to 0 ml of the solution, and while stirring, pi was adjusted to 5.0. Next, pl+ was prepared by dissolving 3.0 g of m-xylylene diamine in 30 ml of water.
5.0 aqueous solution was added to the above dispersion, and the pH was adjusted to 5 while stirring.
．． While adjusting the temperature to 0, the reaction was allowed to proceed at room temperature for 4 hours. After this,
The polymer particles precipitated by centrifugation were washed with water to remove unreacted carbodiimide, diamine, and reaction by-products, thus obtaining amino-attached water-dispersible polymer particles.

The polymer particles were redispersed in 100 ml of water, 60 ml of a 5% aqueous solution of glutaraldehyde was added, and the mixture was allowed to react at room temperature for 24 hours, followed by purification by centrifugation to obtain water-dispersible polymer particles having aldehyde groups. Obtained.

Next, 0.1 M dipotassium hydrogen phosphate and 0.1 M
The above polymer particles were dispersed in 100 ml of a buffer solution (pl (7°00)) prepared from potassium dihydrogen phosphate, an enzyme aqueous solution containing 3 g of urease dissolved in 30 ml of buffer solution was added, and the mixture was incubated at a temperature of 5° C. for 20 ml. The urease was immobilized on the polymer particles by reacting for a period of time. After the reaction, the precipitated polymer particles were centrifuged and washed with a buffer solution, and then redispersed in the buffer solution to obtain the immobilized enzyme according to the present invention. Ta.

The amount of urease immobilized in this immobilized enzyme was 40 μ/g of polymer particles, and the activity yield was 50%. Activity yield is defined as the ratio of the actual activity to the theoretical amount of activity of the immobilized enzyme, here 0.03
Using an aqueous urea solution of M as a substrate, react the immobilized enzyme at 35°C for 10 minutes, measure the activity by determining the amount of ammonia produced (μmol/min) by hydrochloric acid titration, and determine the free enzyme having the same activity. It was determined by dividing the amount by the amount of enzyme immobilized.

Example 3 200 ml of a 1% ethanol solution of p-nitrobenzaldehyde was added to 100 ml of the water dispersion of the water-dispersible polymer particles having amino groups obtained in Example 2, and the mixture was reacted at room temperature for 2 hours. The mixture was centrifuged and washed to obtain water-dispersible polymer particles having nitro groups.

The polymer particles were redispersed in 100 ml of water, and
．． Add 100 ml of an aqueous solution containing IM sodium dithionite and 0.5 M sodium bicarbonate and mix at room temperature for 2 hours.
The reaction was carried out for a period of time to reduce the nitro groups of the polymer particles to amino groups. After centrifugation, the particles were thoroughly washed to obtain water-dispersible polymer particles having amino groups.

The polymer particles were dispersed in 100ml of water, and 0.0ml of water was added to the polymer particles.
1 M sodium nitrite in 0.5 N hydrochloric acid solution 50
m1 was added and reacted at room temperature for 1 hour to convert the amino groups of the polymer particles into diazonium groups.

Thereafter, the polymer particles were thoroughly washed to obtain water-dispersible polymer particles having diazonium groups.

The polymer particles were redispersed in 100 ml of water, an enzyme solution containing 0.3 g of trypsin dispersed in 30 ml of buffer solution was added, and the mixture was allowed to react at a temperature of 5°C for 24 hours, followed by centrifugation. The combined particles were washed with a buffer solution to remove unfixed trypsin. This was dispersed again in a buffer solution to obtain an immobilized enzyme according to the present invention.

The amount of trypsin immobilized in this immobilized enzyme was 3.5 μ/g of polymer particles, and the activity yield was 40
%Met.

In addition, the enzyme was heated at 35°C using 1% casein aqueous solution as a substrate.
After reacting for 10 minutes with 5% trichloroacetic acid, high molecular weight proteins were precipitated with 5% trichloroacetic acid.
The activity yield was determined by setting the activity increased by 1°0 per minute as 1 unit.

Example 4 30 g of ethyl acrylate 1 40 g of styrene 2 g of divinylbenzene and 30 g of acrylonitrile were mixed with 23 g of distilled water.
In addition to 0g, polymerize in the same manner as in Example 1 to obtain approximately 30% solids.
An aqueous dispersion of polymer particles having an average particle size of 0.25 μm was obtained.

The polymerization was very stable, with 0.02% agglomerates.

The above polymer particles were saponified in exactly the same manner as in Example 1 to obtain water-dispersible polymer particles having carboxyl groups.

Add 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide to 100 ml of this aqueous dispersion of polymer particles.
20g of metho-p-toluenesulfonate in 200ml of water
Add an aqueous solution dissolved in
Adjusted to. An aqueous solution prepared by dissolving 3.0 g of urease in 500 ml of water and adjusting the pH to 5.0 was added to the above polymer particle aqueous dispersion, and the mixture was heated to a temperature of 5''C while stirring and adjusting the PL to 5.0. The mixture was allowed to react for 24 hours.

Thereafter, the polymer particles are centrifuged, and the precipitated particles are washed with a buffer solution to remove unfixed urease, unreacted carbodiimide, and reaction by-products, and then dispersed again in the buffer solution. The immobilized enzyme was obtained by

The amount of urease immobilized in this immobilized enzyme is
The activity yield was 58 μ/g, and the activity yield, determined in the same manner as in Example 2 using 0.03 M urea as a substrate, was 30%.

4 Comparative Example 1 In Example 1, polymerization was carried out in exactly the same manner as in Example 1 except that acrylonitrile was not used, but the entire polymer agglomerated during the polymerization and the polymerization could not be carried out smoothly.

Comparative Example 2 In Example 1, polymerization was carried out in exactly the same manner as in Example 1 except that triethylene dimethacrylate was not used, and 6% of aggregates were formed. After removing aggregates,
When this aqueous dispersion of polymer particles was saponified under the same conditions as in Example 1, the entire dispersion became gel-like, and an aqueous dispersion of polymer particles could not be obtained.

Patent applicant: Nitto Electric Industry Co., Ltd. Agent Patent Attorney Itsu Makino

Claims (1)

Scope of Claims: (11 (81 acrylic ester or methacrylic ester 5 to 98% by weight, 1 to 20% by weight of internal crosslinking monomer, and (d) 1 to 60% of acrylonitrile or methacriconitrile as the second monomer.
Emulsion copolymerization of a monomer composition consisting of A method for producing an immobilized enzyme, the method comprising: (2) The method for producing an immobilized enzyme according to claim 1, wherein the water-dispersed polymer particles have an average particle diameter of 0.03 to 2μ. (3) The method for producing an immobilized enzyme according to claim 1, wherein the polyfunctional internal crosslinking monomer is poly(meth)acrylate of a polyhydric alcohol. (4) The method for producing an immobilized enzyme according to claim 1, wherein the acrylic ester or methacrylic ester is a methyl ester or an ethyl ester.