JPS58149680A - Preparation of immobilized enzyme - Google Patents

Abstract

PURPOSE:To obtain a highly active immobilized enzyme having negligibly small diffusion of the substrate to the surface of the immobilized enzyme, by immobilizing the enzyme to a specific water-dispersible polymer particle through covalent bond. CONSTITUTION:Water-dispersible polymer particles having an average particle diameter of 0.03-2mu are prepared by the emulsion copolymerization of (A) 0.2- 10wt% radically polymerizable monomer selected from methyl acrylate, ethyl acrylate and acrylamide, (B) 10-95wt% radically polymerizable monomer copolymerizable with the monomer (A), (C) 1-20wt% polyfunctional monomer for internal crosslinking, e.g. polyacrylate of a polyhydric alcohol, etc., and (D) 1- 60wt% acrylonitrile or methacrylonitrile. The dispersion of the particles is made to react with hydrazine to convert the acrylic acid ester unit or the acrylamide unit contained in the polymer particles to acid hydrazide unit, and an enzyme is bonded to the polymer by covalent bond through the acid hydrazine group.

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 the aqueous solution. However, according to this method, while maintaining the reaction conditions constant,
Do not replenish fresh enzyme or deactivate the enzyme after the reaction (separation of the product and enzyme is very sticky and the enzyme is consumed uneconomically. Because it is official, it is less productive.

In order to solve these problems, it has already been proposed to immobilize an enzyme on a carrier by various methods and to react the immobilized enzyme with a substrate. As one method for immobilizing such an enzyme, a carrier packing method is known in which the enzyme is bonded to a water-insoluble carrier by covalent bonding, ionic bonding, or physical adsorption. However, the carriers conventionally used in this method are usually particles with a diameter of 1 to several dozen, 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. There are problems in that it takes a long time and the reaction yield is low.

The present invention was made in order to solve the above-mentioned problems, and it allows the enzyme to move freely in the same way as a free enzyme by injecting it into the reaction system. The purpose of the present invention is to provide highly active immobilized enzymes that are highly active.

The method for producing an immobilized enzyme according to the present invention includes (a) a small amount of a radically polymerizable monomer selected from methyl acrylate, ethyl acrylate, and acrylamide;
(b) a first radically polymerizable monomer copolymerizable with this monomer, 10 to 95 weight units; (c) a polyfunctional internal crosslinking monomer, 1 to 20 weight units; and (d) reacting hydrazine with a dispersion of water-dispersible polymer particles obtained by emulsion copolymerizing 1 to 60% by weight of acrylonitrile or methacrylonitrile as the second radical copolymerizable monomer. The acrylic acid ester or acrylamide unit of the polymer is converted into an acid hydrazide unit, and the enzyme is covalently bonded to the polymer particles via the acid hydrazide group.

In the present invention, at least one radically polymerizable monomer selected from methyl acrylate, ethyl acrylate, and acryl acrylate is emulsion copolymerized with other monomers described below, and the water-dispersed polymer obtained by The above-mentioned acrylic ester or acrylamide unit possessed by the polymer particles is converted into an acid hydrazide unit by hydrazine.As the monomer to be converted into this strong hydrazide unit, an alkyl ester of acrylic acid having Os or higher, a methacrylic ester, or methacrylamide is used. If used, severe conditions are required for acid hydrazidation, impairing the dispersion stability of the polymer particles, which is undesirable (and also undesirable, since a large amount of thermal energy is required).

The first radically polymerizable monomer is not particularly limited, except for the above-mentioned methyl acrylate, ethyl acrylate, and acrylamide, and acrylonitrile and methacrylonitrile as the second radically polymerizable monomer described below. Various monomers are used, preferably ethylene, propylene, vinyl chloride, vinyl acetate, vinyl propionate, alkyl esters of acrylic acid with Os or higher, methacrylic esters, styrene, methylstyrene, vinyltoluene, butadiene, One or more types of isoprene, methacrylamide, etc. are used. Particularly preferably, alkyl esters of acrylic acid having Os or higher, methacrylic esters, or styrene are used. These radical copolymerizable monomers are selected so that the resulting copolymer has a glass transition point higher than the temperature at which the enzyme reaction is carried out.

In addition, as the radically polymerizable monomer for polyfunctional internal crosslinking, poly(meth)acrylates of polyhydric alcohols are preferable, and specifically, ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and dipropylene glycol dimethacrylate. , 1,3-butylene glycol dimethacrylate, triethylene glycol diacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, tetramethylolmethanetetraacrylate, etc. are used as larel. Divinylbenzene is also used.

Furthermore, in the present invention, it is essential that the water-dispersed polymer particles contain acrylonitrile and/or methacrylonitrile as the second copolymerizable single chain component.

The water-dispersed polymer particles used in the present invention can be obtained by emulsion copolymerizing the above-mentioned monomers in an aqueous medium by a conventional method, but the resulting polymer particles contain an emulsifier. This may cause harmful effects such as deactivation of the enzyme, so it is preferable not to use an emulsifier during emulsion polymerization. This is because, according to the monomer composition of the present invention, stable emulsion copolymerization can be carried out without particularly requiring an emulsifier. However, an emulsifier may be used if necessary, provided that the emulsifier does not have a detrimental effect on the enzyme.

In the present invention, the monomer composition to be emulsion copolymerized is a monomer selected from methyl acrylate, ethyl acrylate, and acrylamide. Monomer with a weight ratio of 1 to 10
980 to 98 weight range is 20 to 900 to 90 weight range monomer 1 to 20 weight range, preferably 2 to 10 weight range, and acrylonitrile or methacrylonitrile 1 to 60 weight range, preferably is 5 to 40 times.

The amount of methyl acrylate, ethyl acrylate and/or acrylamide is related to the amount of enzyme immobilized on the obtained polymer particles; if it is too small, the enzyme cannot be immobilized in a sufficient amount; If it is too high, a large amount of water-soluble components will be produced when the acrylic ester or acrylamide units of the obtained polymer particles are acid-hydrazided with hydrazine, which is not preferable. That is,
When such water-soluble components are generated, some of them are adsorbed to the polymer particles in the water-insoluble polymer particles and remain. is fixed. The immobilized enzyme, on which the 6ζ enzyme is immobilized, is eluted from the water-insoluble water-dispersible polymer particles during the enzymatic reaction.
The activity of the immobilized enzyme itself decreases markedly over time, and since it mixes with the substrate and reaction products, it causes various inconveniences, such as the need to separate them after the reaction.

However, according to the invention, methyl acrylate,
ξ Emulsion copolymerization of at least one monomer selected from ethyl acrylate and acrylamide, an internal crosslinking monomer, and acrylonitrile or methacrylonitrile
From and k, the stability of polymerization is ensured, and! It,
<The formation of water-soluble polymers is inhibited. The reason why such results are obtained is not clear, or the water-soluble low molecular weight polymer mainly composed of methyl acrylate, ethyl acrylate and/or acrylamide produced at the initial stage of polymerization is mixed with acrylonitrile or methacrylonitrile for internal crosslinking. The polymer may effectively copolymerize to make the polymer insoluble in water and stabilize the polymerization. Therefore, when the amount of acrylonitrile or metatallonitrile is too less than the above range,
Alternatively, if the amount is too large, the stability of polymerization will be impaired. Furthermore, if the internal crosslinking monomer is too small, a large amount of water-soluble polymer will be produced as a by-product, while if it is too large, the polymerization will lack stability.

In the present invention, the average particle diameter of the water-dispersed polymer particles is 0.03-21. Preferably it is 0.07 to IP. If the particle size is too small, it will be difficult to recover the immobilized enzyme after dispersing it in water and carrying out the enzymatic reaction. On the other hand, if the particle size is too large, the particle surface area per unit volume will be small, and the enzyme will be difficult to recover. This is not preferable because it reduces the amount of immobilization and makes it difficult to disperse in water.

In the present invention, the aqueous dispersion of water-dispersible polymer particles thus obtained is reacted with hydrazine to convert the methyl acrylate, ethyl acrylate, or acrylamide units contained in the polymer into acid hydrazide units. Conditions for this purpose are selected so that a sufficient amount of hydrazide groups are generated to be used for immobilizing the enzyme without impairing the water dispersibility of the polymer particles. Preferably, hydrazine hydrate with a concentration of 5 to 50 is used, and 1
Allow to react for ~24 hours. It is clear that the higher the reaction temperature and the higher the Jiff of hydrazine hydrate, the more hydrazide groups are produced.

In order to immobilize the enzyme to the polymer particles having hydrazide groups in this way by covalent bonding, it is possible to immobilize the enzyme to the hydrazide groups by a conventionally known method. One method is to react the hydrazide group of the polymer particles with sodium nitrite under mildly acidic conditions to form an acid azide group, and react the amino group of the enzyme with the acid azide group to form a peptide bond. As another method, polymer particles are reacted with succinic anhydride to form succinyl hydrazide groups, and then reacted with an enzyme using water-soluble carbodiimide to form amino groups of the enzyme. It is also possible to form peptide bonds and immobilize the enzyme on polymer particles.After the enzyme is covalently immobilized on the polymer particle in this way, unimmobilized enzyme is removed by centrifugation, membrane separation, etc. Then, the immobilized enzyme according to the present invention is obtained.

The immobilized enzyme according to the invention is used as a dispersion liquid, and the amount of the 6wA fixed fermentation enzyme contacted with the substrate is determined as appropriate depending on the particle size of the immobilized enzyme, the amount of immobilized enzyme, the required reaction rate, the substrate concentration, etc. determined.

The enzyme immobilized in the present invention may be an intracellular enzyme,
It may also be an extracellular enzyme. Furthermore, enzymes do not necessarily need to be highly purified (extracts and partially purified products can also be used.Furthermore, a single enzyme may be immobilized according to the present invention, but multiple enzymes may be immobilized). Examples of enzymes include amino acid oxidase, catalase, xanthine oxidase, glucose oxidase, glucose-6-phosphate dehydrogenase 1. Glutamate dehydrogenase, cytochrome C oxidase, tyrosinase, lactate dehydrogenase, peroxidase, 6 −
Oxidoreductases such as phosphogluconate dehydrogenase, malate dehydrogenase, aspartate acetyltransferase, aspartate aminotransferase, glycine aminotransferase, glutamate-oxaloacetate aminotransferase, glutamate-virupate aminotransferase, creatine phosphokinase, histamine methyltransferase , pyruvate kinase, aractokinase, 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, naringinase, nucleotidase, papain, hyaluronidase, plasmin, pectinase, hexokinase, pepsin, pectinase, penicillin amidase, phospholipase, phosphatase, lactase, lipase, ribonuclease, rennin. Hydrolytic enzymes such as aspartate decarboxylase, aspartase, citrate lyase, glutamate decarboxylase, histidine ammonia lyase, phenylalanine ammonia lyase, fumarase, 7-mer hydratase, lyases such as apple synthetase,
Isomerase enzymes such as asparaginase, glucose isomerase, glucose phos-7ate isomerase, glutamate racemase, lactate racemase, methionine racemase, gauze such as asparagine synthase, glutathione synthase, pyruvate synthase, etc. Ru.

As described above, in the immobilized enzyme of the present invention, the enzyme is covalently immobilized on water-dispersed polymer particles having hydrazide groups, and is different from conventional cellulose derivative carrier particles. Since the immobilized enzyme itself can move freely within the reaction system in the same way as free enzyme, diffusion of the substrate has almost no effect on the reaction. The enzyme reaction can be carried out at a high reaction rate.Moreover, since the enzyme is immobilized on a carrier, after the enzyme reaction, centrifugation, salting out, coagulation precipitation using a flocculant, membrane separation using a porous membrane, etc. It can be easily recovered and used repeatedly over a long period of time.

Furthermore, from the viewpoint of production, the water-dispersible polymer particles used as a carrier in the present invention can be stably emulsion copolymerized without using an emulsifier and without producing undesirable water-soluble polymers. You can get it. Furthermore, by using acrylonitrile or methacrylaterile in combination with an internal crosslinking agent, the obtained water-dispersed polymer particles have high strength and do not cause adhesion between particles.

Example 1 Methyl acrylate) 10y, methyl methacrylate 73g, )lyethylene glycol dimethacrylate?
2 g and acrylonitrile 151 were added to 230 g of distilled water, and an aqueous polymerization initiator solution prepared by dissolving 0.31 potassium persulfate in 10 y of water was added under a nitrogen stream at a humidity of 10°C.
Polymerization was carried out for 8 hours while stirring at 20 m, and the solid content was 30%.
Average particle size 0. An aqueous dispersion of polymer particles of ISp was obtained. The polymerization was very stable, with aggregates of 0.03 arc.

40 d of hydrazine hydrated was added to 10 g of this polymer particle dispersion, and after reacting at a temperature of 50° C. for 6 hours with stirring, the precipitated polymer particles were separated by centrifugation.
The sample was washed with an aqueous solution of M chloride).

The thus obtained polymer particles having acid hydrazide groups were redispersed in 0.3N aqueous hydrochloric acid solution 10011114, cooled to 5°C, and 1M sodium nitrite aqueous solution 50-
was added and reacted at 5° C. for 1 hour to convert the acid hydrazide group to an acid azide group.

The polymer particles precipitated by centrifugation were washed with a 0.1 M aqueous sulfamic acid solution and then with water, and then dispersed in 100 d of water.

An enzyme aqueous solution prepared by dissolving 1.51 g of trypsin in 30 ml of water was added to the above polymer particle dispersion, and reacted at a temperature of 5° C. for 200 hours. After the reaction, the precipitated polymer particles were centrifuged and washed with a buffer solution (pH 7.0) made from 0.1 M dipotassium hydrogen phosphate and 0.1 M potassium dihydrogen phosphate to remove undirected trypsin. was removed and dispersed again in a buffer solution to obtain an immobilized enzyme according to the present invention.

The amount of trypsin identified in this immobilized enzyme is
The activity yield was 201,119 per g, and the activity yield measured using a 1% casein aqueous solution as a substrate was 70%.

Incidentally, the activity yield means the ratio of the actual activity to the theoretical amount of the activity of the immobilized enzyme. Here, the enzyme was reacted with a 1% casein aqueous solution as a substrate at 35°C for 10 minutes, and then high molecular weight proteins were precipitated with 5-stroke tosachloroacetic acid, and the free non-protein decomposed mass was reduced to 280 a
The amount of immobilized enzyme was divided by the amount of free enzyme having the same activity, with the activity of increasing this absorbance by 1.0 per minute as one unit.

After the reaction, the immobilized enzyme was centrifuged, washed with a buffer, and reacted with the substrate again, and it showed almost the same activity as the initial reaction.

Comparative Example 1 Methyl acrylate 10y, methylmethacrylate 51 and acrylonitrile 15y were emulsion copolymerized in the same manner as in Example 12 to obtain a solid content of 30% and an average particle size of 0.15μ.
An aqueous dispersion of polymer particles was obtained. Aggregates were 0.5%.

When trypsin was immobilized on this polymer particle dispersion by the same method as in Example 1, the amount of trypsin immobilized was 15IIISJ per liter of polymer particles, and the activity yield was determined under the same conditions as in Example 1. It was 70%. Further, after this reaction, the precipitated polymer particles were centrifuged and washed with a buffer solution, and then reacted with the substrate again, and the activity was found to have decreased to 70% of the initial activity.

Comparative Example 2 Methyl acrylate) 10f, Methyl methacrylate 8
8f and triethylene glycol dimethacryle) 2y
was subjected to emulsion copolymerization in the same manner as in Example 1 to obtain an aqueous dispersion of polymer particles with a solid content of 29% and an average particle size of 0.25 μm. The polymerization was unstable and the aggregates were outside of 5.

Example 2 10f of acrylamide, 5% of styrene, 18y of methyl methacrylate, 2f of divinylbenzene, and 20p of acrylonitrile were added to 230f of distilled water, and Example 1 and ris
Emulsion copolymerization was carried out using the method described in s. to obtain a dispersion of polymer particles with a solid content of 30 μm and an average particle size of 0.25 μm.

The polymerization was very stable with 0.02% agglomerates.

40 sag of 40% hydrazine hydrate was added to 100 ml of the aqueous dispersion of the polymer particles, and the mixture was reacted with stirring at a temperature of 60° C. for 3 hours to convert the acid amide group of the polymer into an acid hydrazide group. After centrifugation, the precipitated polymer particles were washed with a 0.1M aqueous sodium chloride solution.

The polymer particles were again dispersed in water tooyz, 820 g of anhydrous amber was added, and the pH was adjusted to 4 at room temperature.
After reacting for an hour, the polymer particles were centrifuged and the precipitated polymer particles were washed with water. The thus obtained polymer particles having succinyl hydrazide groups were dispersed in 100 ml of water, and 20 y of l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-meth-p-toluenesulfonic acid was dissolved in 20 ml of water.
A carbodiimide aqueous solution dissolved in 0d was added to the above dispersion, stirred, and the pH was adjusted to S,O.

α-amylase 2,5f was dissolved in 5011 J of water, and p
H5. OK adjusted if? 5. Add to the polymer particle dispersion as an aqueous solution and adjust the pH to 5.6 while stirring.
The α-amylase was immobilized on the polymer particles by reacting at ℃ for 24 hours. After the reaction, the polymer particles precipitated by centrifugation are added to buffer solution KC#c to remove unimmobilized α-amylase1, and then dispersed in the buffer solution again to obtain the immobilized enzyme/number according to the present invention. It was a barrel.

The amount of α-amylarase immobilized in this immobilized enzyme was 307 per polymer particle, and the activity yield was 36. The activity yield is calculated by reacting the immobilized enzyme at 35°C for 10 minutes using a 1% starch aqueous solution as a covering material, and determining the amount of starch decomposed from the iodine starch reaction. The speed (s9/min) was obtained, and the amount of free enzyme having the same activity was divided by the amount of immobilized enzyme.

Patent applicant Nitto Electric Industry Co., Ltd. Agent Patent attorney Itsube Makino

Claims (1)

[Scope of Claims] 111 <aJ A small amount selected from methyl acrylate, ethyl acrylate and acrylamide (both 0.2 to 10% by weight 2° (6) capable of copolymerizing with this monomer) First radically polymerizable monomer 10
-115 weight, (C) polyfunctional internal crosslinking monomer 1-
20% by weight, and (d) acrylonitrile or methacrylonitrile as the second radical copolymerizable monomer.
A dispersion of water-dispersed polymer particles obtained by emulsion copolymerization of 60% by weight is reacted with hydrazine to convert the acrylic ester or acrylamide units of the polymer into acid hydrazide units, and the acid A method for producing an immobilized enzyme, which comprises bonding the enzyme to the polymer particles via a hydrazide group through a covalent bond. (2) Water-dispersed polymer particles are 0.03 to 2μ
2. The method for producing an immobilized enzyme according to claim 1, wherein the immobilized enzyme has an average particle size of . 2. The method for producing an immobilized enzyme according to claim 1, wherein the first monomer is an alkyl ester of acrylic acid having an Os or higher, a methacrylic ester, or styrene. (4) The method for producing an immobilized enzyme according to claim 1, wherein the polyfunctional internal crosslinking monomer is polyacrylate or polyacrylate of a polyhydric alcohol.