JPS6012033B2 - immobilized enzyme - Google Patents

Description

[Detailed description of the invention] The present invention relates to immobilized enzymes.

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, 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 such problems, it has already been proposed to immobilize an enzyme into a single substance by various methods and to react a substrate with this immobilized specific enzyme.

As one of the methods for individualizing enzymes, a carrier binding method is known in which the 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 polysaccharide derivatives such as cellulose, dextran, and agarose, polyacrylamide gel, and porous glass, each having a diameter of one to several sides. An immobilized enzyme, in which the enzyme is immobilized on particles like this, is usually packed into a column, immobilized, and brought into contact with a substrate solution.If the substrate has a high molecular weight, it is difficult to diffuse onto the surface of the immobilized enzyme, which inhibits the reaction. 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 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. The purpose of this invention is to provide an immobilized enzyme.

The immobilized enzyme according to the present invention contains 0.2 to 1% by weight of a radically polymerizable monomer having a carboxyl group in 'a}, and a first radically polymerizable monomer that can be copolymerized with the monomer in {b]. body 1
0 to 95% by weight, 'c-polyfunctional internal crosslinking monomer 1 to
2% by weight of acrylonitrile or methacrylonitrile as the second radical copolymerizable monomer [d]
The enzyme is covalently immobilized on water-dispersible polymer particles obtained by emulsion copolymerization of 6% by weight.

The carboxyl group-containing radically polymerizable monomer used in the present invention preferably has the general formula RICH=CR2C
OO day (However, RI represents hydrogen, a lower alkyl group, preferably a methyl group or a carboxyl group, R2 represents hydrogen or a lower alkyl group, preferably a methyl group, and when RI represents hydrogen or a lower alkyl group, R2 represents a carbo-lower It may also be an alkoxy group.

), and preferred specific examples include acrylic acid, methacrylic acid, itaconic acid, crotonic acid, maleic acid, fumaric acid, monoalkylmaleic acid, monoalkylfumaric acid, monoalkylitaconic acid, etc. However, one or a mixture of two or more of acrylic acid, methacrylic acid and itaconic acid is particularly preferably used.

The first radically polymerizable monomer to be copolymerized with the monomer having a carboxyl group is a monomer having a carboxyl group, except for acrylonitrile and methacrylnitrile, which are the second radically polymerizable monomers described below. There is no particular restriction on the use of ethylene, propylene, vinyl chloride, pinyl acetate, vinyl propionate, acrylic ester, methacrylic ester, styrene, methylstyrene, vinyltoluene, butadiene, isoprene, although it is not particularly limited as long as it has copolymerizability with the polymer. , acrylamide, methacrylamide, etc., or two or more thereof may be used.

In particular, acrylic acid ester, methacrylic acid ester, or styrene is preferably used. These copolymerizable monomers are selected so that the resulting copolymer has a transition point higher than the temperature at which the enzymatic reaction is carried out. Further, as the polyfunctional internal crosslinking radically polymerizable monomer, poly(meth)acrylate of polyhydric alcohol is preferable, and specifically, ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, dipropylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, triethylene glycol diacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, tetramethylate.

methane tetraacrylate, etc. are used. Divinylbenzene is also used. Furthermore, in the present invention, as described above, it is essential that the water-dispersed polymer particles contain acrylonitrile and/or methacrylonitrile as the second copolymerizable monomer 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 using 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 the monomer composition of the present invention allows stable emulsion copolymerization 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 as follows:
0.2-1% by weight of monomers having carboxyl groups, preferably 0.5-8% by weight, 10-95% by weight of first monomers, preferably 20-9% by weight, internal crosslinking agent 1-2
of, preferably 2 to 1, and acrylonitrile or methacrylonitrile 1 to 60, preferably 5 to 4, by weight.

The amount of the monomer having a carboxyl group is also related to the amount of enzyme immobilized on the obtained copolymer particles, and if it is too small, the enzyme cannot be immobilized in a sufficient amount;
If the amount is too large, the enzyme is likely to be deactivated when the enzyme is immobilized on the resulting polymer particles, which is not preferable. Next, in general, when a monomer having a carboxyl group and a monomer copolymerizable therewith are emulsion copolymerized, the former monomer has high hydrophilicity, so that free water soluble material is released in the aqueous medium phase. Polymers are often formed.

When such a water-soluble polymer is formed, a portion of it is adsorbed onto the surface of the water-insoluble polymer particles and remains, and when an enzyme is immobilized using this as a carrier, the enzyme is also immobilized on this water-soluble polymer. be converted into The immobilized enzyme, in which the enzyme is immobilized on a carrier containing a water-soluble polymer, is released from the water-impregnable water-dispersible polymer particles during the enzymatic reaction, and the immobilized enzyme itself In addition, the activity of the compound 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 present invention, by emulsion copolymerizing a monomer having a carboxyl group, an internal crosslinking monomer, and acrylonitrile and/or methacrylonitrile, the stability of the polymerization is ensured, and the undesirable water lagoon is prevented. The formation of chemical polymers is inhibited.

The reason why such results are obtained is not clear, but acrylonitrile or methacrylonitrile and internal crosslinking monomers are effective for water-soluble low-molecular-weight polymers whose main components are monomers with carboxyl groups generated in the initial stage of polymerization. It is likely that the polymer is copolymerized to make it insoluble in water and the polymerization is stabilized. Therefore, if the amount of acrylonitrile or methacrylonitrile is too small or too large from the above range, the stability of polymerization will be impaired. In addition, when the amount of internal crosslinking monomer is too small, the amount of water-soluble polymer by-product increases;
If the amount is too large, the polymerization becomes unstable. In the present invention, the average particle size of the water content type polymer particles is 0.
It is 0.03 to 2 degrees, preferably 0.07 to 1 degrees.

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, making it difficult to collect the enzyme. This is not preferred because the amount of immobilization decreases and it becomes difficult to disperse in water. The enzyme can be covalently bonded to the water content polymer particles without any particular limitation, and any conventionally known method can be used.

For example, as one method, the amino group of the enzyme and the carboxyl group on the surface of the water-dispersible polymer particle can be directly bonded by forming an amide bond using water-soluble carbodiimide. Examples of water-soluble carbodiimides include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)lupodiimidemethop-toluenesulfonic acid, and the like. be able to.
Enzyme immobilization using water-soluble carbodiimide is carried out under conventionally known conditions.
Using carbodiimide with a sonic equivalent of 5q, at a temperature of about 5°C,
It is sufficient to maintain the axis at 4.5 to 6.0 and allow the enzyme to mix and react overnight. The second method is to react the carboxyl groups on the surface of water-dispersed polymer particles in the presence of N-hydroxysuccinimide, and then react with the amino groups of the enzyme to form a covalent bond. can.

Furthermore, as a third method, a diamine is applied to the carboxyl group on the surface of a water-dispersed polymer particle to introduce an amino group onto the surface of the polymer particle, and the enzyme is immobilized by a covalent bond with this amino group. You can also do that. For example, the carbodiimide described above can be used to react the carpoxyl group of the enzyme with the amino group on the surface of the polymer particle, and
A cross-linking reagent such as glutaraldehyde can be used to attach the amino/groups of the enzyme to the polymer particles. When dialdehyde is used as a crosslinking reagent, an excess amount of the dialdehyde is reacted with respect to the amino groups of the polymer particles, and the free aldehyde group of the dialdehyde bound to the polymer particles by one of the dialdehyde groups is reacted with the amino group of the enzyme. React the bonds. Further, as a fourth method, a diazo coupling method can also be used.

For example, p-nitrobenzaldehyde is reacted and bonded to polymer particles into which amine/groups have been introduced, and then the nitro groups are reduced to amide/groups by conventional methods such as sodium borohydride and sodium dithionite. This amino group is converted into a diazonium group using sodium sulfite, and this is diazo-coupled with the amino group of the enzyme.
After covalently bonding the enzyme to the polymer particles as described above, the immobilized enzyme of the present invention can be obtained by removing the used reaction reagent and unimmobilized enzyme by centrifugation, membrane separation, or the like.

The immobilized enzyme of the invention is used as a dispersion and contacted with a 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, although a single enzyme may be immobilized according to the present invention, multiple enzymes may be immobilized. Specific examples of enzymes include amino acid oxidase, catalase, xanthine oxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, cytochrome C oxidase, tyrosinase, lactate dehydrogenase, peroxidase, 6-phosulgluconate dehydrogenase, malate Oxidoreductases such as dehydrogenases, aspartate acetyltransferase, aspartate aminotransferase, glycine aminotransferase, glutamate-oxaloacetate aminotransferase, glutamate-pyruvate aminotransferase, creatine phosphokinase, histamine methylnaransferase, Pyruvate kinase, fructokinase, hexokinase, 6-lysine acetyltransferase, leucine aminobef. Transferases such as titase, asparaginase, acetylcholinesterase, aminoacylase, amylase, arginase, L-
Arginine deiminase, invertase, urease, uricase, urokinase, esterase, 8-galactosidase, kallikrein, chymotrypsin, trypsin, thrombin, naringinase, nucleotidase, /ゞ/fin, hyauronidase, plasmin, vectinase, hesveridinase, pepsin, benicillinase, penicillin Amidase, phospholipase, phosphatase, lactase, IJpase, ribonuclease,
Hydrolytic enzymes such as rennin, aspartate decarboxylase, aspartase, citrate lyase, glutamate decarboxylase, histidine ammonia lyase, phenylalanine ammonia lyase, fumarase, fumarate hydratase, lyases such as malate synthetase, alanine racemase , glucose isomerase, glucose phosphate isomerase, glutamate racemase, lactate racemase, isomerase such as methionine racemase, ligase such as asparagine synthase, glutathione synthase, pyruvate synthase, and the like. As described above, the immobilized iQ enzyme of the present invention has an enzyme immobilized by a covalent bond on water-dispersed polymer particles having carboxyl groups, and is different from conventional single cellulose derivative 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 little effect on the reaction. Enzyme reactions can be carried out at high reaction rates.

Moreover, since the enzyme is immobilized on a carrier, it can be easily recovered after the enzyme reaction by centrifugation, salting out, coagulation precipitation using a flocculant, pus separation using a porous membrane, etc., and can be used repeatedly over a long period of time. be able to. Furthermore, from the viewpoint of production, the water-dispersible polymer particles as a carrier used in the present invention can be produced without using an emulsifier.
In addition, it can be stably obtained by emulsion copolymerization without producing undesirable water-soluble polymers.

Furthermore, by using acrylonitrile or methacrylonitrile in combination with an internal crosslinking monomer, the obtained water-dispersed polymer particles have high strength and do not cause adhesion between the particles. Example 1 A polymerization initiator aqueous solution was prepared by adding 3 parts of acrylic acid, 80 parts of methyl methacrylate, 2 parts of triethylene glycol dimethacrylate, and 15 parts of acrylonitrile to 230 parts of distilled water, and dissolving 0.3 parts of potassium persulfate in 10 parts of water. 70
Polymerization was carried out for 8 hours under a nitrogen stream at a temperature of
An aqueous dispersion of French polymer particles was obtained.

The polymerization was very stable, with 0.02% agglomerates. Further, when the dispersion was centrifuged and the top light was examined, only 5% of the carboxyl groups of the charged amount were determined, and the amount of water-soluble polymer by-product was small. Next, to 100 M of the above dispersion, an aqueous solution of 20 molar of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemeth-p-toluenesulfonic acid dissolved in 200 m of water was added, and while stirring , the pH was adjusted to 5.0.

Dissolve 2.5 μl of a-amylase in 500 μl of water and
Add an enzyme aqueous solution with H adjusted to 5.0 to the above dispersion,
2 at a temperature of 5°C while adjusting the pH to 0.5 under the fly.
The enzyme immobilization reaction was performed at Misaki time. Thereafter, the polymer particles precipitated by centrifugation are washed with a buffer solution to remove unfixed α-amylase, carbodiimide from the reaction, and reaction products, and then dispersed again in the buffer solution to obtain the method according to the present invention. Immobilized α-amylase was obtained.

The amount of α-amylase immobilized in this immobilized enzyme was 40 females per polymer particle per night, and the activity yield measured using a 1% starch aqueous solution as a substrate was 40%.

Incidentally, the activity yield means the ratio of the actual activity to the theoretical amount of the activity of the immobilized enzyme.

Here, the activity of the immobilized enzyme and the rate of starch decomposition (m9/min) were determined by reacting the immobilized starch enzyme at 35oo for 10 minutes using a 1% starch aqueous solution as a substrate, and determining the amount of starch decomposition from the iodine starch reaction. The amount of free enzyme obtained and having the same activity was divided by the amount of immobilized enzyme. Comparative Example 1 3 parts of acrylic acid, 82 parts of methyl methacrylate, and 15 parts of acrylonitrile were emulsion copolymerized 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 0.30 mm. Obtained.

Aggregates were 1%. When this dispersion was centrifuged and the upper light was examined, carboxyl groups were determined to be 40% of the charged amount, and a large amount of water-soluble polymer by-product was observed. Comparative example 2 Acrylic acid 3, Methyl methacrylate 9
5 g of triethylene glycol dimethacrylate and 2 g of triethylene glycol dimethacrylate were emulsion copolymerized in the same manner as in Example 1 to obtain an aqueous dispersion of polymer particles with a solid content of 28% and an average particle size of 0.32 μm.

The polymerization was unstable with 6% agglomerates. Also,
When this dispersion was centrifuged and the top light was examined, carboxyl groups were determined to be 20% of the charged amount. Example 2 [1] of 100 of the polymer particle aqueous dispersion obtained in Example 1
-cyclohexyl-3-(2-morpholinoethyl)rubodiimidemeth-p-toluenesulfonic acid 2M in water
Add an aqueous solution dissolved in 0.00', and while using a pestle,
．． Adjusted to 0.

An aqueous solution of metaxylylene diamine (3.0%) dissolved in 30% of water (pH 5.0) was added to the above dispersion, and the mixture was reacted at room temperature for 2 hours while adjusting the pH to 5.0. . After that, the precipitated polymer particles are washed away with water,
Unreacted carbodimide, metaxylylene diamine, reaction products, etc. were removed to obtain water-dispersed polymer particles having amino groups. The polymer particles were redispersed in 100 g of water, 60 g of a 5% aqueous solution of glutaraldehyde was added, and after reacting at room temperature for 2 hours, the polymer particles were purified by centrifugation, and a water-dispersed polymeric polymer having aldehyde groups was prepared. Coalced particles were obtained.

Next, 100% of a buffer solution (pH 7.0) prepared from 0.1M dipotassium hydrogen phosphate and 0.1M dipotassium phosphate
The above polymer particles were dispersed in the polymer particles, and an enzyme aqueous solution in which urease 30 was dissolved in buffer solution 30 Z was added, and the reaction was carried out at a temperature of 5°C for 2 hours to immobilize urease on the polymer particles. .

After the reaction, the polymer particles precipitated by centrifugation were washed with a buffer solution and redispersed in the buffer solution to obtain the immobilized urease according to the present invention. In this immobilization method, the amount of immobilized enzyme urease is 30 females per polymer particle per night, and the activity yield is 50%.
Met.

The activity yield is 35% using a 0.03M urea aqueous solution as a substrate.
The immobilized enzyme is reacted for 10 minutes at I asked for it. Example 3 To 100% of the aqueous dispersion of the water-dispersed polymer having amino groups obtained in Example 2, 200% of a 1% ethanol solution of p-nitrobesozaldehyde was added, and the mixture was reacted at room temperature for 2 hours. After that, the particles were centrifuged and refined to obtain water-dispersed polymer particles having nitro groups.

The polymer particles were redispersed in 100 g of water, and
．． 100 mL of an aqueous solution containing 1M sodium dithionite and 0.9M sodium bicarbonate was added and reacted at room temperature for 2 hours to reduce the nitro groups of the polymer particles to amino groups.

After centrifugation, the mixture was washed thoroughly to obtain water-dispersed polymer particles having amino groups. The polymer particles were dispersed in 100ml of water and added with 0.0ml of 0.1M sodium nitrite.
Add 50% of aqueous hydrochloric acid solution and react at room temperature for 1 hour.
The amino groups of the polymer particles were changed to diazonium groups.

After centrifugation, the chicks were thoroughly washed to obtain water-dispersed polymer particles having diazonium groups. The polymer particles were redispersed in 100% of water, and an enzyme dispersion containing 30% trypsin and 30% of buffer was added thereto, reacted at a temperature of 5°C for 2 hours, and then centrifuged. The precipitated polymer particles were washed with a buffer solution to remove unfixed trypsin.

This was dispersed again in a buffer solution to obtain immobilized trypsin according to the present invention. The amount of trypsin immobilized in this immobilized enzyme was 9/20 per polymer particle, and the activity yield was 40%.

In addition, after reacting the enzyme with 1% casein aqueous solution as a substrate at 35 qo for 10 minutes, high molecular weight proteins were precipitated with 5% trichloroacetic acid, and the amount of free non-protein scales was determined from the absorbance at 28 m. The activity yield was calculated using the activity that increases the absorbance by 1.0 per minute as one unit. Example 4 230 g of distilled water was added to 5 g of methacrylic acid, 23 g of methyl methacrylate, 40 g of styrene, 2 g of divinylbenzene and 30 g of Ac IJ lonitrile, and polymerized in the same manner as in Example 1, solid content 30%, average. An aqueous dispersion of polymer particles having a particle size of 0.25 mounds was obtained.

The polymerization was very stable, with 0.02% agglomerates. 1-Cyclohexyl-3 to 100% of the above dispersion
-(2-morpholinoethyl)carbodiimidemeth-P
- An aqueous solution of 20 parts of toluenesulfonic acid dissolved in 20 parts of water was added, and the pH was adjusted to 5.0 while stirring.
Dissolve urease 3.0 in 500 ml of water and adjust the pH to 5.
．． The above dispersion was added to an enzyme aqueous solution adjusted to 0, and the enzyme immobilization reaction was carried out for 24 hours while adjusting the pH to 5.0 under a hawk frame. Thereafter, the polymer particles were washed by centrifugation and dispersed in a buffer solution to obtain the immobilized urease according to the present invention.

The amount of urease immobilized by this immobilized enzyme was 43:9 per polymer particle per night, and the activity yield was 30% when measured in the same manner as in Example 1 using 0.09M urea as a substrate. .

Claims (1)

[Scope of Claims] 1 (a) 0.2 to 10% by weight of a radically polymerizable monomer having a carboxyl group, (b) a first radically polymerizable monomer that can be copolymerized with this monomer 10 ~95% by weight, (c
) 1 to 20% by weight of a polyfunctional internal crosslinking monomer, and (d
) Oxygen is immobilized by covalent bonds in water-dispersed polymer particles obtained by emulsion copolymerizing 1 to 60% by weight of acrylonitrile or methacrylonitrile as the second radical copolymerizable monomer. An immobilized enzyme characterized by: 2 The monomer having a carboxyl group has the general formula R^1CH
=CR^2COOH (However, R_1 is hydrogen, a lower alkyl group or a carboxyl group, R^2 is hydrogen or a lower alkyl group, and when R^1 is hydrogen or a lower alkyl group, R^2 is a carbo-lower alkoxy group. The immobilized enzyme according to claim 1, characterized in that it is represented by: 3 0 water-dispersed polymer particles
．． The immobilized enzyme according to claim 1, having an average particle size of 0.3 to 2 μm. 4. The immobilized enzyme according to claim 1, wherein the first monomer is an acrylic ester, a methacrylic ester, or styrene. 5. The immobilized enzyme according to claim 1, wherein the polyfunctional internal crosslinking monomer is polyacrylate or polymethacrylate of a polyhydric alcohol.