JPS5944038B2 - 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 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 of such methods for immobilizing an enzyme, 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 with a diameter of 1 mm to several VTM, such as cellulose, dextran, polysaccharide derivatives such as agarose, polyacrylamide gel, and porous glass. The immobilized enzyme, in which the enzyme is immobilized on such particles, is usually packed into a column, immobilized, and contacted with a substrate solution.
When the substrate has a high molecular weight, it is difficult to diffuse onto the surface of the immobilized enzyme, resulting in a problem that the reaction takes a long time and the reaction yield is low.

The present invention was made to solve the above-mentioned problems, and has a high activity that can move freely in the reaction system like a free enzyme, and therefore, 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 is characterized in that the enzyme is immobilized by ionic bonding on water-dispersed polymer particles having ion exchange groups.Specially, the water-dispersed polymer particles used in the present invention are It is necessary to have an ion exchange group.

Such ion-exchange group-containing -9-free water-dispersed polymer particles are typically obtained by emulsion copolymerization of a monomer having an ion-exchange group, preferably with other monomers. be able to.

Examples of such ion exchange groups include acid groups such as nurphonic acid groups, carboxyl groups, and phosphoric acid groups, and basic groups such as tertiary amine groups and quaternary amine groups.

Specific examples of monomers having such ion-exchange groups include monomers having a nurphonic acid group such as nuthylene nulphonic acid and sulfopropyl methacrylate, and monomers having a carboxyl group such as acrylic acid, methacrylic acid, and itaconic acid. mer, acidhonuphoxyethyl meccrylate,
Monomers having a phosphoric acid group such as 3-chloro-2-acid phosphoxypropyl methacrylate, monomers having a tertiary amine group such as dimethylaminoethyl methacrylate, dimethylaminopropyl methacrylamide,
Monomers having a quaternary amine group such as methacrylamide propyltrimethylammonium chloride and methacryloyloxyethyltriethylammonium chloride can be mentioned.

The monomers to be copolymerized with these monomers having ion exchange groups are not particularly limited as long as they have copolymerizability, but are preferably ethylene, propylene, vinyl chloride, vinyl acetate, vinyl propionate, acrylic esters, Methacrylic acid ester, styrene, methylstyrene, butadiene, isoprene, acrylamide, acrylonitrile,
One or more types of methacrylic acid I-IJ are used.

These copolymerizable monomers are selected so that the resulting copolymer has a glass transition point higher than the temperature at which the enzymatic reaction is carried out.

In the present invention Q, in addition to the monomer having an ion exchange group and the above-mentioned monomer copolymerizable therewith, a polyfunctional polymer for internal crosslinking is further emulsion copolymerized to form a water-dispersed polymer. Preferably, molecular polymer particles are obtained.

Specific examples of such internal crosslinking monomers include ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, dipropylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, triethylene glycol diacrylate, and trimethylolpropane trimethacrylate. Examples include (meth)acrylates of polyhydric alcohols such as methacrylate, trimethylolpropane triacrylate, tetramethylolmethanetetraacrylate, and divinylbenzene.

In the emulsion copolymerization of an internal crosslinking monomer with a monomer having an ion exchange group and a monomer having copolymerizability with this monomer,
It is useful for suppressing the formation of water-soluble polymers and increasing the stability of polymerization.

The water-dispersed polymer particles contain 0.2 to 30% by weight of a monomer having an ion exchange group and 70 to 99% by weight of a copolymerizable monomer.
When an internal crosslinking monomer obtained by emulsion copolymerization of 8% by weight is used in combination, it is preferable to copolymerize in an amount of 200% by weight of the total monomers.

The water-dispersed polymer particles used in the present invention have an average particle diameter of 0.03 to 2μ, preferably 0.07 to 1μ.

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 the amount of immobilized water becomes small and it becomes difficult to disperse in water.

The amount of ion exchange groups contained in the water-dispersed polymer particles is from o, ooi to 5 milliequivalents, preferably from 0.01 to 2 milliequivalents, per gram of the polymer particles.

This is because when the amount of ion exchange groups is too small, the amount of enzyme immobilized is small and the enzymatic reaction cannot be carried out sufficiently, whereas when it is too large, the enzyme tends to be deactivated.

The water-dispersed polymer particles used as a carrier in the present invention can be obtained by emulsion polymerization of the above-mentioned monomers and, if necessary, an internal crosslinking monomer by a conventional method. If an emulsifier is present in the particles, harmful effects such as deactivation of enzymes may occur, so it is preferable not to use an emulsifier during emulsion polymerization.

However, if the emulsifier does not have a harmful effect on the enzyme, it goes without saying that it may be used to stably obtain water-dispersed polymer particles.

Furthermore, in the present invention, a monomer having a functional group is emulsion copolymerized with other copolymerizable monomers and internal crosslinking monomers, if necessary, to form water-dispersed polymer particles. After obtaining, the functional groups can be exchanged with ion exchange groups to obtain water-dispersed polymer particles having ion exchange groups.

Examples of monomers having functional groups that can be converted into ion exchange groups include acrylic esters and methacrylic esters, and polymers containing such monomer components are saponified with acid or alkali. In this way, water-dispersed polymer particles having a carbosacil group as an ion exchange group can be obtained.

Furthermore, when a polymer containing a monomer component having a glycidyl group is reacted with a tertiary amine, a polymer having a quaternary amine group as an ion exchange group can be obtained.

To ionically bond an enzyme to water-dispersed polymer particles, a dispersion of polymer particles and an enzyme solution may be mixed under appropriate conditions such as temperature and pH that do not cause deactivation of the enzyme.

For example, the pH is appropriately determined depending on the isoelectric point of the enzyme, the type of ion exchange group, etc., but is generally preferably 5 to 8.

After ionically bonding the enzyme to the polymer particles in this way,
The immobilized enzyme of the present invention can be obtained by removing 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,
It may also be an extracellular enzyme.

Furthermore, the enzyme does not necessarily need to be highly purified, and delineation fluids 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-phosphogluconate dehydrogenase, apple. Oxidoreductases such as acid dehydrogenase, aspartate acetyltransferase, anupate amine transferase, glycine aminotransferase, glutamate-oxaloacetate amine transferase, glutamate-pyrunate amine transferase, creatine phosphokinase, histamine methyltransferase, pyruvate Kinases, fructokinase, hexokinase, δ-lysine acetyltransnuferase, transferases such as leucine aminopeptidase, asparaginase, acetylcholinesterase, aminoacylase, amylase, arginase, L
-alanine racemase, invertase, urease,
Uricase, urokinase, esterase, β-galactosidase, kallikrein, chymotrypsin, trypsin, thrombin, naringinase, nucleotidase,
Hydrolytic enzymes such as papain, hyauronidase, plasmin, pectinase, hexokinase, pepsin, pectinase, penicillin amitase, phospholipase, honufacuse, lactase, lipase, ribonuclease, rennin, aspartate decarboxylase, aspartase, citrate lyase, glutamate decarboxylase, Lyases such as carboxylase, histidine ammonia-lyase, phenylalanine ammonia-lyase, fumarase, fumarate hydratase, malate synthetase, isomerization such as alanine racemase, glucose isomerase, glucose fonupate isomerase, glutamate racemase, lactate racemase, methionine racemase Examples include enzymes, asparagine synthase, glutathione synthase, pyruvate kinase, and gauze.

As described above, the immobilized enzyme of the present invention is immobilized by ionic bonding on water-dispersed polymer particles having ion exchange groups, and unlike conventional cellulose derivative carrier particles, it is immobilized on water-dispersed polymer particles having ion exchange groups. Since the fermentation enzyme itself can move freely within the reaction system like a free enzyme, the diffusion of the substrate has almost no effect on the reaction. Enzyme reactions can be carried out at high reaction rates.

Furthermore, since the enzyme immobilization conditions are mild, enzyme deactivation during immobilization is less and the activity yield is high.

Furthermore, 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 coagulant, membrane separation using a porous membrane, etc., and can be used repeatedly over a long period of time. be able to.

Example 1 97 g of methyl methacrylate and 3 g of methacryloyloxyethyltrimethylammonium chloride were added to 3 g of distilled water.
00g plus 2-27-azobis-2-amidinopropane dihydrochloride 0. ．． A polymerization initiator aqueous solution prepared by dissolving 1.0 g of water in 10 g of water was added under a nitrogen stream at a temperature of 60°C, and the mixture was heated for 120 r.
Polymerize for 8 hours while stirring at pm, and the average particle size is 0.3μ.
A dispersion of water-dispersed polymer particles was obtained.

This liquid dispersion] 00g was centrifuged and the supernatant was removed.

The precipitated polymer particles were dispersed in distilled water, centrifuged and washed twice.

Next, the precipitated particles were dispersed in 100 ml of a buffer solution (pH 7) prepared from 0.1 M IJ dipotassium hydrogen phosphate and 0.1 M potassium dihydrogen phosphate, rum.

An enzyme solution prepared by dissolving 2 g of urease in 100 ml of the same buffer as above was added to the above dispersion, and after standing at 5°C for 24 hours, centrifugation was performed.

The precipitated polymer particles were washed with a buffer solution to remove unimmobilized urease, and then dispersed again in the buffer solution to obtain immobilized urease according to the present invention.

The amount of urease immobilized in this immobilized enzyme is
The activity yield was 40 m9 per g, and the activity yield measured using 0.03 M urea as a substrate was 95%.

Example 2 Styrene 979 and 3 g of acrylic acid were added to 300 I of distilled water, and an aqueous polymerization initiator solution prepared by dissolving 0.3 g of potassium persulfate in 10 g of water was added at a temperature of 70°C under a nitrogen stream.
React for 8 hours while stirring at 2 rpm, and the average particle size is 0.
．． A dispersion of 4μ polymer particles was obtained.

100 g of this dispersion was centrifuged and the supernatant was removed.

The precipitated particles were washed with a buffer solution prepared from 0.05 MIJ disodium hydrogen phosphate and 0.05 M potassium dihydrogen phosphate (
pH 7) was dispersed in 100 ml.

An enzyme solution in which 2 g of α-chymotrypsin (100 ml) was dissolved in the same buffer as above was added to the above dispersion, and the mixture was heated at 5°C for 24 hours.
After standing for a period of time, it was centrifuged.

The precipitated polymer particles were washed with a buffer solution to remove unfixed α-
Chymotrypsin was removed and the enzyme was dispersed again in a buffer solution to obtain an immobilized enzyme according to the present invention.

The amount of α-chymotrypsin immobilized in this immobilized enzyme was 30 μ per 1 g of polymer particles, and the activity yield measured using 0.05 mM N-acetyl-cymotrysine ethyl ester as a substrate was 90%. .

Example 3 Styrene 709 and 30 g of methidilea acrylate were added to 300 g of distilled water and emulsion copolymerized in the same manner as in Example 2 to obtain a dispersion of polymer particles with an average particle size of 0.35 μm.

Potassium hydroxide was added to this dispersion to adjust the pH to 10 or higher, a saponification reaction was carried out, and then the dispersion was neutralized to pH 4 with hydrochloric acid.

Next, the dispersion was centrifuged, and the precipitated polymer particles were washed with water.

These polymer particles were dispersed in the same buffer as in Example 2 to obtain a dispersion with a fixed content of 30%.

An α-chymotrypsin solution was added to this dispersion in the same manner as in Example 2 to obtain the immobilized enzyme of the present invention.

The immobilized amount of α-chymol IJpusin of this immobilized enzyme was 20 μm per 1 g of polymer particles, and the activity yield was 90 μm using N-acetyl-L-tyrosine ethyl coster as a substrate.
He was in charge.

Claims (1)

[Scope of Claims] 1. An immobilized enzyme characterized in that the enzyme is immobilized on water-dispersed polymer particles having ion exchange groups by ionic bonding. 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. The immobilized enzyme according to claim 1, wherein the ion exchange group is an acid group. 4. The immobilized enzyme according to claim 3, wherein the ion exchange group is a carboxyl group, a sulfonic acid group, or a phosphoric acid group. 5. The immobilized enzyme according to claim 1, wherein the ion exchange group is a primary amine group or a quaternary amine group.