JPS6150959B2 - - Google Patents

Description

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

Enzyme reactions are used in the manufacturing process of foods and medicines due to their substrate specificity and high reactivity at room temperature and pressure. Conventionally, enzymes are dissolved in an aqueous solution of the substrate, and the enzyme is reacted in the aqueous solution. The reaction is carried out with
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, various methods have already been proposed to immobilize an enzyme on a water-insoluble carrier and to react the immobilized enzyme with a substrate.

On the other hand, some enzymes may require coenzymes to exhibit their catalytic activity. In such cases, how to bind the necessary coenzyme to the immobilized enzyme is an extremely important issue in order to allow the enzyme reaction to proceed smoothly. As is well known, a coenzyme combines with an apoenzyme to form a holoenzyme and performs an oxygen reaction, but the bond between an enzyme and a coenzyme is generally reversible and relatively weak. This is because the coenzyme is easily separated from the apoenzyme. As with enzymes,
It is already known that coenzymes can be used by immobilizing them on water-insoluble carriers, but the carriers conventionally used for immobilizing enzymes and coenzymes are usually polysaccharides such as cellulose, dextran, and agarose. These are particles of derivatives, polyacrylamide gel, porous glass, etc. with a diameter of 1 mm to several mm. Enzymes and coenzymes are bonded to the carrier via a chain structure of a certain length, a so-called spacer group, and their degree of freedom is increased. Even if the temperature is increased, if both the immobilized enzyme and the immobilized coenzyme itself are immobilized in the reaction system, the free movement range of each is extremely limited, so there is very little opportunity for the enzyme and coenzyme to come into contact with each other. The target enzymatic reaction is difficult to carry out.

The present invention has been made in order to solve the various problems described above, and its general purpose is to provide an improved immobilized coenzyme, and in particular, it aims to provide an improved immobilized coenzyme, and in particular, it aims to eliminate free cooxygen in the reaction system. You can move in the same way,
Another object of the present invention is to provide an immobilized coenzyme that is easy to separate and recover from a reaction system and is suitable for use in combination with free enzymes and conventional forms of immobilized enzymes.

The immobilized coenzyme according to the present invention is characterized in that the coenzyme is immobilized on water-dispersed polymer particles having functional groups by covalent bonds.

Water-dispersed polymer particles are required to have a functional group for covalently bonding a coenzyme, and such polymer particles typically contain a monomer having a functional group, preferably another monomer. It can be obtained by emulsion copolymerization with monomers. Examples of such functional groups include carboxyl groups, hydroxyl groups, amino groups, hydrazide groups, and glycidyl groups. As a monomer having such a functional group,
Specifically, monomers having a carboxyl group such as acrylic acid, methacrylic acid, and itaconic acid; monomers having a hydroxyl group such as hydroxyethyl acrylate and hydroxyethyl methacrylate;
Mention may be made of monomers having glycidyl groups such as glycidyl methacrylate.

In addition, water-dispersed polymer particles having an amino group or a hydrazide group can be prepared by using monomers having an amide group such as acrylamide, methyl acrylate,
Each monomer having a methyl ester group, such as methyl methacrylate, is preferably emulsion copolymerized with other monomers, and the above-mentioned amide in the resulting copolymer is subjected to Hofmann degradation, and the methyl ester group is It can be obtained by reacting with hydrazine. To obtain water-dispersed polymer particles containing a carboxyl group as a functional group by emulsion copolymerizing a monomer having an ester group such as an acrylic ester and then hydrolyzing the ester group. I can do it.

The monomer to be copolymerized with the monomer having these functional groups is not particularly limited as long as it has copolymerizability, but preferably ethylene, propylene, vinyl chloride, vinyl acetate, vinyl propionate, acrylic ester, One or more of methacrylic acid ester, styrene methylstyrene, butadiene, isoprene, acrylamide, acrylonitrile, methacrylonitrile, etc. 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, in addition to the monomer having an ion exchange group and the monomer copolymerizable therewith, an internal crosslinking agent or a polyfunctional polymer is further emulsion copolymerized to form a water-dispersed polymer. It is also possible to obtain coalesced particles. The internal crosslinking agent not only increases the glass transition temperature of the resulting polymer particles, but also reduces the undesirable water-soluble polymer formation during emulsion copolymerization when the functional group of the monomer having a functional group is ionic such as a carboxyl group. suppresses the generation of Specific examples of such internal crosslinking agents include ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, dipropylene glycol dimethacrylate,
1,3-butylene glycol dimethacrylate,
Examples include polyol poly(meth)acrylates such as triethylene glycol diacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, and divinylbenzene.

The water-dispersed polymer particles contain 0.2 to 30% by weight of a monomer having a functional group and 99.8 to 70% by weight of a copolymerizable monomer.
It is obtained by emulsion copolymerization of % by weight. When an internal crosslinking agent is used in combination, it is preferable to copolymerize up to 20% 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 2μ.
It is 1μ. If the particle size is too small, it will be difficult to recover the immobilized coenzyme after dispersing it in water and performing an enzymatic reaction with the enzyme. On the other hand, if the particle size is too large, the particle surface area per unit volume will be small. This is not preferred because the amount of coenzyme immobilized decreases and it becomes difficult to disperse it in water.
The amount of functional groups contained in the water-dispersed polymer particles is 0.001 to 5 milliequivalents, preferably 0.01 to 2 milliequivalents, per gram of the polymer particles. When the amount of functional groups is too small, the amount of coenzyme immobilized is small and the enzymatic reaction is not carried out sufficiently; on the other hand, when it is too large,
This is because there is a tendency for coenzyme deactivation to occur upon immobilization of the coenzyme.

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 agent, in a conventional manner. It is preferable not to use an emulsifier during emulsion polymerization, since the presence of co-enzymes may cause harmful effects such as deactivation of coenzymes. However, if the emulsifier does not have a harmful effect on the coenzyme, it can of course be used to stably obtain water-dispersed polymer particles.

The method for binding the coenzyme to the water-dispersed polymer particles is not particularly limited, and conventionally known methods may be employed as appropriate. This method is as follows. Examples include, but are not limited to, the diazo method, the carbodiimide method, the cyanogen bromide method, and the azide method.

In addition, in the present invention, if necessary, a coenzyme is bound to the polymer particles via a spacer such as hexamethylene diamine, dodecamethylene diamine, or glycylglycylglycine, and the coenzyme to be immobilized is bonded to the carrier. The range of free movement can be expanded.

Furthermore, according to the present invention, the coenzyme can be immobilized in advance on a monomer having a functional group as described above, and the coenzyme can be emulsion copolymerized with other copolymerizable monomers. Water-dispersed polymer particles having immobilized thereon are obtained.

The coenzyme immobilized in the present invention is not particularly limited, but specific examples include nicotinamide-adenine dinucleotide (NDA), nicotinamide-adenine dinucleotide (NDA), and nicotinamide-adenine dinucleotide (NDA).
Examples include adenine dinucleotide phosphate (NADP), flavin-adenine dinucleotide (FAD), pyridoxal phosphate, coenzyme A, and the like.

The immobilized coenzyme of the invention is used as a dispersion and contacted with the apoenzyme and the substrate. The apoenzyme may be a free enzyme or an immobilized enzyme. For example, when using a conventional immobilized enzyme in which the apoenzyme is immobilized on a cellulose derivative or polyacrylamide gel particles, the immobilized enzyme is packed in a column, and the immobilized coenzyme of the present invention containing a substrate is loaded into the column. Let it pass. After the enzymatic reaction is completed, the immobilized coenzyme is separated by membrane separation or other appropriate means.

As described above, the immobilized coenzyme according to the present invention has the following features:
Coenzymes are covalently immobilized on water-dispersed polymer particles having functional groups, and unlike conventional cases where they are immobilized on particulate carriers such as cellulose derivatives or polyacrylamide gels, immobilized coenzymes are Since it can move freely within the reaction system like a free coenzyme, it has a very high chance of coming into contact with the enzyme, ensuring a smooth enzymatic reaction. Moreover, since the coenzyme is covalently immobilized on the water-insoluble polymer particle carrier, it is not easily detached from the carrier. Furthermore, after the enzymatic reaction, it can be easily recovered by centrifugation, salting out, flocculation using a flocculant, membrane separation using a porous membrane, etc., and can be used repeatedly and stably over a long period of time.

Example 1 97 g of methyl methacrylate and 3 g of acrylic acid as a monomer having a functional group were added to 230 g of distilled water, and a polymerization initiator aqueous solution prepared by 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 and reacted for 8 hours while stirring at 120 rpm to obtain a dispersion of polymer particles with an average particle size of 0.25 μm. 50 g of this dispersion was centrifuged to remove the supernatant containing the water-soluble polymer and electrolyte from the initiator, and then the precipitated polymer particles were again dispersed in 50 ml of distilled water.

Dissolve 1.5 g of N-cyclohexyl-N'-[β-(N-methylmorpholino)ethyl]carbodiimide-p-toluenesulfonate in 30 g of water, and add a carbodiimide aqueous solution adjusted to pH 5.0 with hydrochloric acid to the above dispersion. In addition, 0.2 g of NAD was dissolved in 10 g of water, a coenzyme solution adjusted to pH 5.0 with hydrochloric acid was added, and after being left at a temperature of 5°C for 24 hours, unreacted carbodiimide and unfixed NAD were removed. , and the supernatant was removed by centrifugation. The precipitated particles were washed with water to obtain the immobilized coenzyme of the present invention.

When this immobilized coenzyme was measured using glyceraldehyde phosphate dehydrogenase, the activity yield was 35% based on free NAD.

Example 2 95 g of methyl methacrylate and 5 hydroxyethyl methacrylate as a monomer with functional groups
g was added to 230 g of distilled water, and emulsion polymerization was carried out in the same manner as in Example 1 to obtain a dispersion of polymer particles with an average particle size of 0.3 μm. 50 g of this dispersion was centrifuged, and the precipitated particles were again dispersed in 50 ml of distilled water.

A 10N aqueous sodium hydroxide solution was added to this dispersion to adjust the pH to 11-12, and an aqueous cyanogen bromide solution prepared by dissolving 3 g of cyanogen bromide in 50 ml of water was gradually added to the dispersion while stirring. At this time, an aqueous sodium hydroxide solution was added to keep the pH of the dispersion at 11 to 12, and the dispersion was cooled so that the temperature did not exceed 30°C. After the reaction is complete, centrifuge and discard the supernatant.
The reaction product was washed with cold water, centrifuged, and purified by repeating this process, and the purified reaction product was dispersed in 50 ml of 0.1 M sodium bicarbonate aqueous solution to obtain a cyanogen bromide-activated polymer particle dispersion. .

The pH of this dispersion was adjusted using an aqueous sodium hydroxide solution.
10.0, add 2g of 6-aminohexanoic acid to 10% of water.
ml of an aqueous solution was added to adjust the pH to 10.0, and the mixture was reacted at a temperature of 25 to 30°C for 20 hours. After the reaction is completed, centrifuge to remove the supernatant, wash the reaction product with cold water, centrifuge, repeat this operation, and then disperse the precipitated particles again in 50 ml of water to create an aqueous dispersion with spacer groups introduced. type polymer particles were obtained.

NAD was added to the polymer particles in the same manner as in Example 1.
was immobilized to obtain the immobilized coenzyme of the present invention. The activity yield was 43% in the same manner as in Example 1.

Claims (1)

[Scope of Claims] 1. A coenzyme is immobilized to water-insoluble water-dispersible polymer particles having a functional group and having an average particle diameter of 0.03 to 2 μm via the functional group by covalent bonding. Characteristic immobilized coenzymes. 2. The immobilized coenzyme according to claim 1, wherein the functional group is a carboxyl group, a hydroxyl group, an amino group, a hydrazide group, or a glycidyl group.