JPH0314432B2 - - 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. It is difficult 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.

In particular, when an immobilized enzyme in which an enzyme is immobilized on a water-insoluble carrier by ionic bonding is used in an aqueous solution with high ionic strength, the enzyme is easily desorbed.
It has the disadvantage that enzyme activity quickly decreases;
According to the covalent bonding method, there is little risk of enzyme desorption, but the reaction during immobilization is complicated, and the enzyme is often deactivated during the immobilization process.
Furthermore, the cost is also high.

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. The purpose of the present invention is to provide an active immobilized enzyme and a method for producing the same, in particular, the enzyme can be stably immobilized on a carrier while suppressing desorption using an ionic bonding method, and therefore,
An object of the present invention is to provide an immobilized enzyme whose enzyme activity is maintained at a high level over a long period of time, and a method for producing the same.

The immobilized enzyme according to the present invention has an enzyme immobilized by ionic bonding on water-dispersed polymer particles having ion exchange groups, and a polymer consisting of a polyamine and a Schiff base of dialdehyde is deposited thereon. According to the present invention, such an immobilized enzyme is characterized by immobilizing the enzyme on water-dispersible polymer particles having ion exchange groups by ionic bonding, and then dispersing the polymer particles in water. It is obtained by reacting a polyamine and a dialdehyde in a liquid to deposit a layer of a polymer comprising the Schiff base of the polyamine and dialdehyde on the surface of the polymer particles.

The water-dispersed polymer particles used in the present invention have an average particle diameter of 0.03 to 2μ, preferably
It is 0.07μ to 1μ. If the particle size is too small, it will be difficult to recover the immobilized enzyme using this carrier as a carrier after dispersing it in water and performing an enzyme reaction.
If the particle size is too large, the particle surface area per unit volume becomes small, the amount of enzyme immobilized decreases, and it becomes difficult to disperse in water, which is not preferable.

Furthermore, the water-dispersed polymer particles used in the present invention must have an ion exchange group, and such a polymer must contain a monomer having an ion exchange group and a monomer copolymerizable therewith. (hereinafter sometimes referred to as a copolymerizable monomer) by emulsion copolymerization according to a conventional method, with or without using an emulsifier.

Examples of the ion exchange group of the monomer having an ion exchange group 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. can be mentioned.
Specific examples of monomers having such polar groups include monomers having sulfonic acid groups such as styrene sulfonic acid and sulfopropyl methacrylate;
Monomers having a carboxyl group such as acrylic acid, methacrylic acid, and itaconic acid; monomers having a phosphoric acid group such as acid phosphoxyethyl methacrylate and 3-chloro-2-acid phosphoxyethyl methacrylate; Monomers having a tertiary amino group such as dimethylaminoethyl methacrylate and dimethylaminopropylmethacrylamide, quaternary monomers such as methacrylamidepropyltrimethylammonium chloride, and methacryloyloxyethyltriethylammonium chloride.
Monomers having a grade amino group can be mentioned.

The monomer to be copolymerized with the monomer having an ion exchange group as described above has copolymerizability, and the resulting copolymer has a glass transition point higher than the temperature at which the enzyme reaction is performed. Various materials can be used without particular limitation as long as they have, but preferably ethylene, propylene, vinyl chloride, vinyl acetate, vinyl propionate, acrylic ester, methacrylic ester, styrene, methylstyrene, One or more of vinyltoluene, butadiene, isoprene, acrylamide, methacrylamide, acrylonitrile, methacrylonitrile, etc. are used.

Furthermore, in the present invention, in addition to the monomer having an ion exchange group and the copolymerizable monomer with the monomer, it is preferable to emulsion copolymerize a polyfunctional monomer for internal crosslinking. Specific examples of such internal crosslinking polyfunctional monomers include ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, dipropylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, and triethylene glycol dimethacrylate. (Meth)acrylates of polyhydric alcohols such as glycol diacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, tetramethylolmethanetetraacrylate, etc. are preferably used. Divinylbenzene is also preferably used.

The use of a polyfunctional monomer for internal crosslinking suppresses the formation of undesirable water-soluble polymers in emulsion copolymerization of a monomer having an ion exchange group and the copolymerizable monomer, and also prevents the polymerization. Helps increase stability.

In the present invention, 0.2 to 30% by weight of a monomer having an ion exchange group and 70 to 99.8% of the copolymerizable monomer
It is preferable to obtain water-dispersed polymer particles by emulsion copolymerization with It is best to use up to 20% by weight. This is because if too large a quantity is used, the stability of polymerization may be impaired.

In the present invention, if an emulsifier is mixed in the resulting water-dispersed high molecular weight diluent, there is a risk that the enzyme will be deactivated during enzyme immobilization. It is preferable not to use an emulsifier during emulsion polymerization, but if the emulsifier does not have a harmful effect on the enzyme used, it is of course possible to use an emulsifier as necessary.

Furthermore, in the present invention, when obtaining water-dispersed polymer particles having ion exchange groups, as described above, the monomer having ion exchange groups is co-emulsified with the copolymerizable monomer in advance. However, if necessary, a monomer having a functional group that can be converted into an ion exchange group after polymerization is emulsion copolymerized with a copolymerizable monomer to form water-dispersible polymer particles. After that, the functional groups of this polymer may be converted into ion exchange groups by a chemical reaction. Examples of monomers having functional groups that can be converted into ion exchange groups include acrylic esters and methacrylic esters, and polymer particles containing such monomer components are treated with acid or alkali. In this way, water-dispersed polymer particles having carboxyl groups as ion exchange groups can be obtained. Further, when polymer particles containing a monomer component having a glycidyl group are reacted with a tertiary amine, polymer particles having a quaternary amino group can be obtained.

In the present invention, in order to ionically bond the enzyme to the water-dispersed polymer particles having the above-mentioned ion exchange group, the dispersion of the polymer particles is mixed with the polymer particle dispersion under appropriate conditions such as temperature and pH that do not cause deactivation of the enzyme. What is necessary is just to mix it with an enzyme solution. For example, PH is appropriately determined depending on the isoelectric point of the enzyme and the type of ion exchange group, but is generally preferably 5 to 8. Thereafter, if necessary, unimmobilized enzymes are removed by appropriate means such as centrifugation or membrane separation, thus obtaining water-dispersed polymer particles with enzymes immobilized on their surfaces by ionic bonds. .

After the enzyme is ionically bonded to the polymer particles in this way, polyamine and dialdehyde are reacted in an aqueous dispersion containing the polymer particles, thereby bonding the enzyme to the surface of the polymer particles on which the enzyme is immobilized. An immobilized enzyme can be obtained in which a polymer of Schiff's base consisting of a polyamine and a dialdehyde is deposited.

The polyamine used in the present invention means an amine compound or polymer having two or more amino groups in the molecule that can react with an aldehyde group,
Preferably, aromatic, aliphatic or alicyclic diamines or triamines are used, and specifically, phenylene diamine, xylylene diamine, phenylene triamine, triaminotoluene, ethylene diamine, hexadimethylene amine, etc. are used. . Moreover, polyethyleneimine can also be used. Next, as for the dialdehyde, aromatic or aliphatic dialdehydes are preferably used, and specifically, glutaraldehyde, succinic dialdehyde, glyoxal, maleic dialdehyde, terephthalic dialdehyde, etc. are used.

In the reaction of depositing a Schiff base polymer on the surface of water-dispersed polymer particles on which an enzyme is immobilized, the equivalent ratio of polyamine and dialdehyde is
10:1 to 1:10, preferably 5:1 to 1:
It is 5. If either one of them is used in excess beyond this range, the amount of Schiff base polymer formed will be too small, and therefore it will be difficult to stabilize the enzyme, which is not preferred.

The above reaction can be carried out, for example, by adding aqueous solutions of polyamine and dialdehyde to a dispersion of water-dispersed polymer particles having an immobilized enzyme at a temperature and pH that does not cause deactivation of the enzyme, and stirring the mixture. However, the order in which the respective aqueous solutions of polyamine and dialdehyde are added is not particularly limited. The reaction usually proceeds smoothly when carried out under acidic conditions using hydrochloric acid or the like. Further, the reaction is usually carried out at room temperature, but in cases where there is a risk that the enzyme may be deactivated, it is carried out at a low temperature. The time required for the reaction is usually several hours to 10 minutes for the reaction at room temperature.
It takes about an hour. Next, the immobilized enzyme of the present invention can be obtained by removing unreacted polyamine and dialdehyde by appropriate means such as centrifugation or membrane separation.

In the present invention, although the enzyme is immobilized on water-dispersed polymer particles by ionic bonding by the method described above, it is not always clear why it is much more difficult to desorb compared to conventional enzymes immobilized by ionic bonding. However, the polyamine and dialdehyde react to form a Schiff base, which becomes water-insoluble as the molecular weight increases, deposits on the surface of the dispersed polymer particles, and becomes immobilized on the surface. This is probably because the enzyme coats the enzyme, and a part of the enzyme also reacts with the aldehyde group of the dialdehyde through its amino group, resulting in crosslinking of the enzyme. However, the present invention is not limited in any way by theory.

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 oxtase,
Glucose oxidase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, cytochrome C oxidase, tyrosinase, lactate dehydrogenase, peroxidase,
6-phosphogluconate dehydrogenase, oxidoreductases such as malate dehydrogenase, aspartate acetyltransferase, aspartate aminotransferase, glycine aminotransferase, glutamate-oxaloacetate aminotransferase, glutamate-
Transferases such as pyruvate aminotransferase, creatine phosphokinase, histamine methyltransferase, pyruvate kinase, fructokinase, hexokinase, delta-lysine acetyltransferase, leucine aminopeptidase, asparaginase, acetylcholinesterase, aminoacylase , amylase, arginase, L-arginine deiminase, invertase, urease, uricase,
Urokinase, Etherase, β -Galact Sidase, Caricrain, Kimotolypsin, Trypsin, Trompsin, Ning Bin, Ningginase, Nurin Ginase, Papain, Papine, Pepinz, Pectinase, Pectinase, Hesperinase, Penzirinase, Pennicillinz, Pennicilliper, Hosfoerase Turase, lactase, lipase, ribonucleaese, renin Hydrolases such as aspartate decarboxylase, aspartase, citrate lyase, glutamate decarboxylase, histidine ammonia lyase, phenylalanine ammonia lyase, fumarase, fumarate hydratase, malate synthetase, alanine racemase, Examples include isomerases such as glucose isomerase, glucose phosphate isomerase, glutamate racemase, lactate racemase, and methionine racemase, and ligases such as asparagine synthase, glutathione synthase, and pyruvate synthase.

As described above, the immobilized enzyme according to the present invention has an immobilized enzyme immobilized on water-dispersed polymer particles by ionic bonding, but unlike the immobilized enzyme by the conventional ionic bonding method, the enzyme is immobilized by ionic bonding. It does not desorb easily even in strong aqueous solutions,
Enzyme activity is maintained over a long period of time. Furthermore, the immobilized enzyme of the present invention is different from conventional carriers using cellulose derivative particles, etc., because the immobilized enzyme itself can move freely within the reaction system like a free enzyme. Therefore, even in the case of a high molecular weight substrate, the enzymatic reaction can be carried out at the same high reaction rate as that of the free enzyme.

In addition, since the operation is extremely simple and the enzyme immobilization conditions are gentle, there is little deactivation of the enzyme during immobilization, and it is possible to obtain an immobilized enzyme with a high activity yield. Furthermore, 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 used repeatedly over a long period of time.

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

Example 1 Methacryloyloxyethyltrimethylammonium chloride 3g, methyl methacrylate 80
g, triethylene glycol dimethacrylate 2
g and 15 g of acrylonitrile were added to 230 g of distilled water, and an aqueous polymerization initiator solution prepared by dissolving 0.3 g of 2,2'-azobis-2-amidinopropane dihydrochloride in 10 g of water was added at a temperature of 60°C under a nitrogen stream, and the mixture was heated at 120 rpm. The mixture was polymerized for 8 hours with stirring to obtain an aqueous dispersion of polymer particles with a solid content of 30% and an average particle size of 0.3 μm.

Next, an enzyme aqueous solution in which 2 g of urease was dissolved in 100 ml of a buffer (PH7) prepared from 0.1 M dipotassium hydrogen phosphate and 0.1 M potassium dihydrogen phosphate was added to 100 ml of the above polymer particle dispersion, and the mixture was heated at 5°C. After standing for 24 hours, it was centrifuged, the precipitated polymer particles were washed with a buffer solution to remove unfixed urease, and dispersed again in 300 ml of distilled water, thus
Water-dispersed polymer particles with urease immobilized by ionic bonding were obtained.

Next, 24 ml of a 1% m-xylylenediamine aqueous solution whose pH was adjusted to 6 with hydrochloric acid was added to this dispersion and stirred, and then 30 ml of a 1% glutaraldehyde aqueous solution was added and stirred at 5°C for 5 hours. and reacted. After the reaction is completed, the precipitated polymer particles are centrifuged and washed with water to remove unreacted xylylene diamine and glutaraldehyde, and then dispersed again in a buffer solution to obtain the immobilized enzyme according to the present invention. Ta.

The amount of urease immobilized in this immobilized enzyme was 40 mg per gram of polymer particles, and the activity yield, defined as the ratio of the actual activity to the theoretical amount of activity of the immobilized enzyme, was 60%. Ta. The activity yield is calculated using 0.03M urea aqueous solution as the substrate and 10% at 35℃.
The activity was determined by reacting the immobilized enzyme for minutes, determining the amount of ammonia produced (μmol/min) by hydrochloric acid titration, and dividing the amount of free enzyme with the same activity by the amount of immobilized enzyme. .

Next, this immobilized enzyme and the immobilized enzyme as a comparative example in which the enzyme was immobilized on the previously obtained water-dispersed polymer particles by ionic bonding were used to immobilize the enzyme in an aqueous solution with high ionic strength. We investigated how the removability differs.

That is, urease was immobilized on the previously obtained water-dispersed polymer particles by ionic bonding to obtain an immobilized enzyme. The activity yield of this comparative immobilized enzyme was 75% by the method described above.

After washing the immobilized enzyme according to the present invention and the immobilized enzyme as the comparative example above with 1M sodium chloride, their activities were measured, and it was found that the immobilized enzyme of the present invention retained 97% of its initial activity. However, the activity of the comparative immobilized enzyme decreased to 50% of its initial activity. Therefore, it is clear that the immobilized enzyme according to the present invention is difficult to desorb even in an aqueous solution with high ionic strength.

Furthermore, after measuring the activity of the immobilized enzyme of the present invention as described above, washing with a buffer solution,
The procedure of measuring the activity again was repeated five times, but the activity remained 96% of the original activity, and the activity remained high even after repeated use. In contrast, in the case of the comparative immobilized enzyme, the activity decreased to 25% of the initial activity.

Example 2 A polymerization initiator aqueous solution prepared by adding 3 g of acrylic acid, 50 g of styrene, 25 g of methyl methacrylate, 2 g of divinylbenzene, and 20 g of acrylonitrile to 230 g of distilled water, and dissolving 0.3 g of potassium persulfate in 10 g of water was heated at a temperature of 70°C under a nitrogen stream. The mixture was added to the bottom and polymerized for 8 hours while stirring at 120 rpm to obtain an aqueous dispersion of polymer particles with a solid content of 30% and an average particle size of 0.2 μm.

An enzyme aqueous solution prepared by dissolving 2 g of α-chymotrypsin in 100 ml of a buffer solution (PH7) prepared from 0.05 M sodium dihydrogen phosphate and 0.05 M dipotassium hydrogen phosphate was added to the above polymer particle dispersion, and the mixture was left at 5°C for 24 hours. After that, the mixture was centrifuged, and the precipitated polymer particles were washed with a buffer solution to remove unfixed α-chymotrypsin, and then dispersed again in 300 ml of distilled water.

Next, 36 ml of a 1% aqueous hexamethylene diamine solution whose pH was adjusted to 6 with hydrochloric acid was added and stirred, and then 30 ml of a 1% aqueous glutaraldehyde solution was added and reacted by stirring at 5° C. for 5 hours. After the reaction is completed, the precipitated polymer particles are centrifuged and washed with the same buffer solution as above to remove unreacted hexamethylene diamine and glutaraldehyde, and then dispersed again in the buffer solution for immobilization according to the present invention. obtained the enzyme.

The amount of α-chymotrypsin immobilized in this immobilized enzyme was 32 mg per gram of polymer particles, and the activity yield was 40%. The activity yield was 60%.
The activity yield was determined from the carboxyl group production rate (μmol/min) by alkaline titration by reacting the enzyme at 30°C using 0.05mM N-acetyl-L-tyrosine ethyl ester as a substrate.

Claims (1)

[Claims] 1. An enzyme is immobilized by ionic bonds on water-dispersed polymer particles having ion exchange groups, and a polymer consisting of a polyamine and a Schiff base of dialdehyde is deposited thereon. An immobilized enzyme characterized by: 2. The immobilized enzyme according to claim 1, wherein the water-dispersed polymer particles have an average particle size of 0.03 to 2μ. 3. After immobilizing the enzyme on water-dispersed polymer particles having ion exchange groups through ionic bonding, a polyamine and dialdehyde are reacted in an aqueous dispersion of the polymer particles to form the polymer particles. 1. A method for producing an immobilized enzyme, which comprises depositing a polymer consisting of the above polyamine and a Schiff base of dialdehyde on the surface of the immobilized enzyme. 5. The method for producing an immobilized enzyme according to claim 4, wherein the water-dispersed polymer particles have an average particle size of 0.03 to 2μ.