JPS5948488A - Immobilized coenzyme and its preparation - Google Patents

Abstract

PURPOSE:To obtain an immobilized coenzyme freely movable, easily separable and recoverable, by linking a coenzyme by covalent bond through the functional group of a shell part to particles of a water-dispersible high polymer consisting of a core part of a polymer of the first monomer composition and the shell part of a polymer of a functional group-containing monomer composition. CONSTITUTION:A coenzyme is linked by covalent bond through a functional group of particles to the particles of a water-dispersible high polymer consisting of (A) 50-98wt% core part of polymer of the first ethylenic monomer composition, and (B) 2-50wt% shell part of polymer of the second monomer composition containing an ethylenic monomer having a functional group, to give the desired immobilized coenzyme. The component A preferably consists of 1- 80wt% acrylonitrile, etc., 0-20wt% crosslinking agent and 20-99wt% acrylic acid ester, and the component B preferably consists of 1-70wt% functional group-containing monomer, 1-80wt% acrylonitrile, 0-20wt% crosslinking agent, acrylic acid ester, etc.

Description

[Detailed description of the invention] The present invention relates to an immobilized coenzyme and a method for producing the same.

Enzyme reactions are partially carried out industrially in the manufacturing process of pharmaceuticals, foods, etc., but conventionally, enzymes are dissolved in an aqueous solution of a substrate, and the reaction is carried out in this aqueous solution. However, with this method, it is very difficult to maintain constant reaction conditions, replenish fresh enzyme, and separate the product and enzyme without deactivating the enzyme after the reaction. is difficult and enzymes are consumed uneconomically. Moreover,
Productivity is poor because the reaction is a batch process. 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.

On the other hand, some enzymes require coenzymes to exhibit their catalytic activity. In such a case,
How do you need immobilized enzymes? An extremely important issue is how to bind the iti enzyme. As is well known, a coenzyme combines with an apoenzyme to form a holoenzyme and performs an enzymatic reaction, but the bond between an enzyme and a coenzyme is generally reversible and relatively weak, so This is because the enzyme 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 cellulose, dextran, These are particles of polysaccharide derivatives such as agarose, polyacrylamide gel, porous glass, etc. with a diameter of 1 to 30 cm, and enzymes and coenzymes are bonded to the carrier through a fixed length structure, a so-called spacer group. Even if we increase the degree of freedom by
Immobilized enzyme or immobilization? 1i If the enzymes themselves are all fixed in the reaction system, their range of free movement is extremely limited, so there are very few opportunities for the enzymes and coenzymes to come into contact with each other, and the desired enzymatic reaction will not take place. It's difficult.

The present invention has been made to solve the above-mentioned problems, and it can move freely in the reaction system like free coenzymes, can be easily separated and recovered from the reaction system, and furthermore, free coenzymes can be easily separated and recovered from the reaction system. Particularly, the present invention is characterized by providing an immobilized coenzyme suitable for use in combination with an enzyme or a conventional form of immobilized enzyme, and a method for producing the same. In polymer particles? An object of the present invention is to provide an immobilized coenzyme that immobilizes a high loading amount of ili enzyme and maintains high coenzyme activity over a long period of time, and a method for producing the same.

The immobilized coenzyme according to the present invention has a polymer core of 50 to 98% by weight consisting of a polymer of a first ethylenic monomer composition and a second ethylenic monomer comprising a functional group-containing ethylenic monomer. A coenzyme is fixed by covalent bond via the above functional group to water-dispersible polymer particles composed of a polymer shell portion of 2 to 50% by weight of a polymer of a monomer composition. According to the present invention, such an immobilized enzyme is characterized in that it is
After emulsion copolymerizing the first ethylenic monomer silk composition in an aqueous medium to form a polymer core made of water-dispersible polymer particles, the polymer core is Emulsion copolymerization of a second tR-mer composition containing an ethylenic monomer having a functional group in the presence of the water-dispersible polymer having a polymer shell on the polymer core is obtained. It is produced by forming particles and then covalently immobilizing a coenzyme to the polymer particles via the above-mentioned functional groups.

In the present invention, first, the first monomer composition is emulsion copolymerized in an aqueous medium to form a polymer core made of water-dispersible polymer particles. The polymer core obtained here must not melt and soften at the temperature at which the enzymatic reaction is carried out in the presence of the immobilized coenzyme according to the invention, generally from 5 to 90°C; The m-mer composition is selected such that the glass transition temperature of the polymer particles is at least 0°C, preferably 5°C or higher.

Therefore, in the present invention, as the first monomer composition,
Styrene, methylstyrene, vinyltoluene, vinyl chloride, acrylic ester, methacrylate/L'S X 71
．． In particular, one or more of alkyl esters having 1 to 1 carbon atoms, acryl [1 nitrile, methacrylonitrile, vinyl acetate, vinyl propionate, butadiene, isoprene, acrylamide, methacrylamide], etc. are used.

As is well known, even if an intermediate polymer forms a homopolymer with a glass transition point of 0°C or lower, by copolymerizing it with other monomers, the glass transition of the copolymer produced by iQ can be improved. Therefore, there is no problem in using ethyl acrylate, butyl acrylate, etc., which give the following fB autopolymer, as a monomer component. For example, the glass transition point of the homopolymer of ethyl acrylate is -22°C, but by copolymerizing methyl methacrylate and ethyl acrylate, whose homopolymer glass transition point is 105°C, fI
A copolymer having a glass transition temperature of about 30°C can then be obtained.

In the present invention, the first monomer composition is 1 to 80% by weight.
It is preferred that the composition contains acrylonitrile and/or methacrylonitrile. This is because the presence of this monomer ensures polymerization stability without using an emulsifier, and also improves the dispersibility of the resulting water-dispersible polymer particles.

In particular, the preferred first monomer composition in the present invention is:
fat acrylonitrile and/or methacriconitrile 1-80% by weight, preferably 5-75% by weight, particularly preferably 10-60% by weight, (bl polyfunctional internal crosslinking monomer O-20% by weight, Preferably 0.1-15% by weight
, particularly preferably 0.5 to 10% by weight, and at least one CI'i selected from (cl acrylic acid alkyl ester, methacrylic acid alkyl ester and styrene)
20 to 99 weight M%, preferably 25 to 95 weight M%,
Particularly preferably 40 to 90% by weight. By using such a small polymer composition, polymerization can be carried out stably and non-densely even in the absence of an emulsifier, and the production of undesirable by-products such as water-soluble polymers can be suppressed. Incidentally, the polyfunctional internal crosslinking monomer is useful for increasing the stability of polymerization, as will be described later, and specific examples thereof will be described later.

The emulsion copolymerization of the first monomer composition is carried out in an aqueous medium, and examples of polymerization initiators such as 2,2'-azobisisobutylamidium hydrochloride, 2'2'-azobis Cyanovaleric acid, persulfate, etc. are used. The water-dispersible ini-molecule polymer particles obtained by this process constitute the core of the water-dispersible polymer particles used as a carrier in the present invention, and usually have an average particle size of 0.03 to 1.5.
It is in the range of μ.

In the present invention, it is preferable to carry out emulsion copolymerization without using an emulsifier, and as described above, copolymerization can be carried out without using G or a double emulsifier. If an emulsifier is mixed into the resulting water-dispersible polymer particles, what happens when coenzymes are immobilized? This is because the ili enzyme may be deactivated, and the immobilized coenzyme tends to be easily detached, resulting in harmful effects. However, it is of course possible to carry out emulsion copolymerization in the presence of an emulsifier if the emulsifier does not have such a harmful effect.

Next, in the present invention, a second monomer composition containing a monomer having a functional group is emulsion copolymerized in an aqueous medium in the presence of the polymer particles forming the polymer core described above. and the above 1st
A polymer shell is formed on a polymer core consisting of a monomer composition of. Here, the functional group means an organic group through which a coenzyme can be immobilized by a covalent bond.

As mentioned above, it is preferable not to use an emulsifier in this second stage emulsion copolymerization. In addition, in the present invention, after copolymerization, a chemical reaction is performed. The case where a monomer having a side chain that can be converted into a difunctional group is used as a component of a monomer composition is also included in the case where a monomer having a functional group is copolymerized.

Examples of the functional group include a carboxyl group, a primary amine group, a hydrazide group, and a glycidyl group. Specific examples of monomers having such functional groups include, for example, 01 phosphor having a carboxyl group such as acrylic acid, methacrylic acid, and itaconic acid; Hydroxyl-containing monomers such as methacrylate-1 and glycidyl methacrylate-I.
Examples include tp and M forms having a glycidyl group such as.

In addition, a monomer having an amide group such as acrylamide,
and a second monomer containing a methyl ester group such as methyl acrylate or methyl methacrylate, respectively.
IL copolymerization is carried out to form a copolymer having these monomer compositions as a polymer shell on the polymer core, and then By decomposing the ami-1-group and allowing hydrazine to act on the methyl ester group, a polymer shell made of a copolymer having an amide group or hydrazide group as a functional group can be formed. In addition, the second monomer composition contains a monomer having an ester group such as an acrylic ester, and this is copolymerized in the presence of the polymer core, and then the ester group is hydrolyzed. By doing so, it is also possible to form a polymer shell made of a copolymer having a carboxyl group as a functional group.

In the second monomer composition, monomers with functional groups are often water-soluble, and therefore, their medium emulsion polymerization may result in undesirable water-soluble polymers. Therefore, in the present invention, as the second monomer composition, together with 1 to 70% by weight of the f monomer having the above-mentioned functional group, 1 to 80% by weight of acrylonitrile and/or I-zyl methacrylate, 0 to 2% commercial amount of a polyfunctional internal crosslinking monomer and 20 to 98% of an intermediate copolymerizable with these
It is preferable to emulsion copolymerize the monomer composition containing % by weight. Also here, it is necessary that the polymer shell formed does not melt at the temperature at which the enzymatic reaction is carried out,
Therefore, the monomer composition should be selected such that the glass transition temperature of the polymer shell formed is at least O'C8, preferably 5°C or higher. Methyl styrene, vinyl 1-luene, vinyl chloride, acrylic ester, methacrylic ester, especially alkyl ester having 1 to 4 carbon atoms, acrylonitrile, methacryl tJ-yl-lyl, hinyl rozoate, vinyl propionate, butadiene, isoprene, acrylic Ami 1
For q41, alkyl esters of acrylic acid and methacrylic acid having 1 to 4 carbon atoms and styrene are preferably used.

Therefore, the second monomer composition particularly preferred in the present invention is (a) Il′1. Weight: 1 to 70 weight%, preferably 5 to 65 weight%, Ushiko (!7 or 10 to 60 weight%), (t+) acrylic 1- tolyl/or methacriconitrile 1 to 80 weight%, Preferably 5~
75% by weight, particularly preferably 10-60% by weight, fcl
Polyfunctional internal crosslinking monomer O~20%, preferably 0.1~15%, particularly preferred L <6; 1: o,
5-107m view%, and (d+acrylic acid ester,
At least one monomer selected from methacrylic acid ester and styrene 20-98% by weight, preferably 25-98%
It consists of 95% by weight, particularly preferably 40 to 80% by weight.

By emulsion copolymerizing such a monomer composition, the formation of a homopolymer made of a monomer having a functional group is suppressed, and the polymerization is particularly stable, and the resulting water-dispersible product is Molecular polymer particles also have excellent molecular stability, and furthermore, when an enzyme is immobilized thereon, the m
This is because the separation can also be suppressed.

As the internal crosslinking polyfunctional monomer, poly(meth)acrylate of polyhydric alcohol is preferable, and specifically, ethylene glycol dimethacrylate-1, diethlene glycol dimethacrylate, triethylene glycol Dimethacrylate, dipropylene glycol dimethacrylate-1-11,3-butylene glycol dimethacrylate-1-11-lyethylene glycol diacrylate-1,
Trimethylolpropane trimethacrylate, pantriacryrytone tetraacrylate 1, etc. are used. Cyhinylbenzene is also used respectfully.

In general, monomers with functional groups are hydrophilic, so if they are emulsion polymerized alone, the polymerization will be unstable, and there will be many undesirable by-products such as water-soluble polymers, resulting in poor dispersibility. It is difficult to borrow polymer particles.

In particular, when a water-soluble polymer is produced, a portion of it is adsorbed and remains on the water-insoluble polymer particles, and when the enzyme is solidified using this as a carrier, the enzyme is also immobilized on this water-soluble polymer. be done. With such an immobilized enzyme in which the enzyme is immobilized on a carrier containing a water-soluble polymer, the enzyme is eluted into the substrate solution together with the water-soluble polymer during the enzymatic reaction, and the enzyme activity decreases over time. In addition, the eluted enzyme will be mixed with the substrate and reaction products, resulting in various inconveniences such as the need to separate them after the reaction.

However, according to the invention, monomers having functional groups, acrylonitrile and/or methacrylonitrile, and preferably polyfunctional internal crosslinking monomers are combined in the presence of a pre-prepared polymeric core. Emulsion copolymerization ensures polymerization stability even in the absence of an emulsifier, suppresses the formation of undesirable water-soluble polymers, and allows these monomer compositions to be incorporated into the polymer core. can be stably copolymerized on the surface of the polymer, thus making it possible to obtain water-dispersible polymer particles having a high density of functional groups on the surface.

The reason why such a result is obtained is not necessarily clear, but during emulsion copolymerization of the second monomer composition, the polymer core made of the first monomer composition is already used as the polymerization site. is given, and acrylonitrile and/or methacrylonitrile and a polyfunctional internal crosslinking unit are added to a water-soluble low molecular weight polymer mainly composed of a monomer having a functional group generated in the initial stage of polymerization. In addition to effectively copolymerizing with the polymer particles and making them insoluble in water, the particles have a good affinity with the core polymer particles and are smoothly formed on the surface of the core polymer particles, thus stabilizing the polymerization. . Therefore, in the present invention,
As described above, the second monomer composition desirably contains acrylateryl and/or methacrylonitrile and a polyfunctional internal crosslinking monomer.

In addition, by emulsifying and copolymerizing a second monomer composition such as those described in "2" above the polymer core to form a polymer shell, functional groups that are not effective in immobilizing coenzymes can be added. can be formed at high density on the surface of water-dispersible polymer particles,
Thus, according to the present invention, coenzymes can be immobilized at a high loading amount.

Furthermore, in the present invention, in the water-dispersible polymer particles obtained as described above, the polymer core portion is 50 to 50%.
It is necessary to sequentially emulsion copolymerize the first and second monomer compositions so that the polymer shell content is 98% by weight and the polymer shell is 2 to 50% by weight.

When the acid moiety is less than 2% by weight in the polymer particle, the amount of copolymer functional groups that the polymer particle has on its surface as a polymer shell decreases, and the amount of enzyme that can be immobilized is limited. This is because an immobilized coenzyme having a high coenzyme activity is obtained, resulting in an Iff. On the other hand, when the amount exceeds 50% by weight, the functional groups are hydrophilic, so that the secondary
Even if the monomer composition is copolymerized, in addition to forming an acid moiety, a water-soluble polymer is formed due to the formation of the second monomer composition, causing the above-mentioned disadvantages, and furthermore, This is because the stability of polymerization and the dispersibility of polymer particles may also be impaired.

The water-dispersed polymer particles used in the present invention have an average particle diameter of 0.05μ to 2μ, preferably 0.1μ to 2μ.
It is 1μ. If the particle size is too small, it will be difficult to recover the immobilized coenzyme using the coenzyme as a carrier 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 amount of This is not preferable because the particle surface area becomes small, the amount of coenzyme immobilized decreases, and dispersion in water becomes difficult.

The method of covalently immobilizing the coenzyme to the functional group of the water-dispersible polymer particles as described above is not particularly limited, and conventionally known methods may be employed as appropriate. Ru. As such a method, depending on the type of functional group,
For example, diazo method, carbodiimide method, cyanogen bromide method,
Examples include, but are not limited to, the azide method. For example, when immobilizing nicotinamide-adenine dinucleotide (N/VD), ■-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride and 1-cyclohexyl-3-
(2-morpholinoethyl)carbodiimi F-meth-p-
A water-soluble carbodiimide such as 1-toluenesulfonate may be used to react with a coenzyme under normal conditions. For example, an NAD solution is added to an aqueous solution of carbodiimide that has 5 to 50 times the carboxyl group, the temperature is about 5°C, the pH is 4,
5 to 6.0 and allow the polymer particles and NAD to react overnight.

Alternatively, it is also possible to react the carboxyl groups on the surface of the polymer particles with N-hydroxysuccinimide in the presence of carbodiimide, and then react the amino groups of NAD.

Still another method is to introduce an amino group into the polymer particle by reacting the carboxyl group of the polymer particle with a carbodiimide and a diamine, and then reacting the amino group with NAD by various conventionally known methods. Let it happen. For example, a crosslinking reagent such as glutaraldehyde can be used to attach the amino groups of the coenzyme to the polymer particles.

That is, the dialdehyde is reacted in excess with the amino groups of the polymer particles, and the 7-mino group of the coenzyme is reacted with the other Mliit aldehyde group of the dialdehyde bonded to the polymer particles through one aldehyde group. be. 1. Another method is diazo coupling. For example, p
- react and couple the nitrobenzaldehyde, then reduce the nitro group to an amino group by conventional methods, e.g. with sodium borohydride and sodium didionite, convert this amine group into a diazonium group with sodium nitrite, and of? i Diazo coupling is performed with the amino group of the enzyme.

After covalently bonding the coenzyme to the polymer particles as described above, if the reaction reagent used and the unimmobilized 6-geon are removed by appropriate means such as centrifugation or membrane fraction A11, the present invention can be achieved. An immobilized coenzyme of the invention is obtained.

The immobilized coenzyme according to the invention is used as an aqueous dispersion 11 and contacted with the apoenzyme and the substrate. The apoenzyme may be a M-free enzyme or an immobilized enzyme. For example, when using a conventional immobilized enzyme in which the 7-boenzyme 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 the substrate is packed in a column. pass. After the reaction is completed, the immobilized coenzyme is separated by membrane separation or other appropriate means.

Is it immobilized in the present invention? iti enzymes are not particularly limited, but specific examples include nicotinamide-adenine dinucleotide phosphate (NAD), nicotinamide-adenine dinucleotide phosphate (NΔDP), flavin-adenine dinucleotide (FAI)), pyridoxal phosphate, Enzyme A, coenzyme Q, dianocobalamin, hyotine, adenosine triphosphate (ATP), adenosine sibosphate (ΔDP), adenosine monophosphate-1-' (AMP), and the like can be mentioned.

In the present invention, it is advantageous to interpose a spacer group in order to increase the degree of freedom in binding the coenzyme to the water-dispersible polymer particles.

The spacer group may be bonded to the polymer particle in advance and then bonded to the coenzyme, or the spacer group may be bonded to the coenzyme in advance and then bonded to the polymer particle. Alternatively, spacer groups may be bonded to the coenzyme and the polymer particles in advance, respectively, and the coenzyme and the polymer particles may be bonded to each other using these spacer groups.

As the spacer/group, diamines such as hexamethylene diamine, F decamethylene diamine, xylylene diamine, aminoalkyl carboxylic acids such as 6-aminocaproic acid, etc. are preferably used, or glycylglycylglycine, etc. Also preferably used. However, it is not limited to these examples.

More specifically, in the present invention, it is preferable to bond a spacer group to the coenzyme in advance, and then bond this to the polymer particles. Specific examples of coenzymes to which a spacer group has been bound in advance include, for example, N6-[(6-aminohexyl)carbamoylmethyl]-NAl), N6-sufucyl-NΔ-N6, -(4- Examples include (3-hydroquinbutyl)]-NΔD, N6-[(6-aminohexyl)carba, seylmethyl]-N A I) P, and the like. 1 In particular, in the present invention, polymer particles having a carboxyl group are used, to which a diamine type spacer group as described above is bonded, and furthermore, to this, a polymer particle having a carbon number of 2 in an aliphatic carboxylic acid residue is bonded. ~11 straight-chain aminoalkylcarphonic acids are reacted with water-soluble carbodiimide, and thus polymer particles having a carboxyl group at the terminal i'l1il are preliminarily released into the i(I, on the one hand, and the ?ii enzyme is For example, the thus obtained Δ"T"
It is desirable to bond and immobilize P, NAD, NΔDP, etc. to the polymer particles using water-soluble carbodiimide.

As described above, in the immobilized coenzyme according to the present invention, water-dispersible polymer particles as a water-insoluble carrier form a polymer shell having functional groups at high density on a polymer core. has been done. Therefore, unlike when conventional cellulose derivative particles are used as a carrier, coenzymes are immobilized in high quality and quantity, and the immobilized coenzymes themselves can move freely within the reaction system like free coenzymes. Because it can move, it has a very high chance of contact with the enzyme, ensuring a smooth enzymatic reaction.

Moreover, since the immobilized coenzyme according to the present invention is covalently immobilized on water-insoluble polymer particles, it is not easily detached from the carrier, and furthermore, after the enzymatic reaction, centrifugation,
Salting out, flocculation using a flocculant, and components using a porous membrane!
1S11, etc., and is thus high over the period of time. It can be used repeatedly while retaining ili enzyme activity.

Furthermore, the immobilized sleeve fi? of the present invention? From the viewpoint of production, in general, a monomer composition containing a monomer having a functional group is emulsion copolymerized to form water-dispersible polymer particles having the functional group at high density on the surface. However, according to the present invention, a polymer core is formed in advance, and 11! Since the monomer composition containing the polymer is emulsion copolymerized, polymer particles can be obtained by stable copolymerization, and the resulting water molecules lJ<
Unlike the case where the second monomer composition is emulsion copolymerized, the polymer particles have functional groups at a dense density on the surface, and as a result, the water obtained by the method of the present invention has functional groups on the surface. It is possible to immobilize a coenzyme in a high loading amount on the dispersible polymer particles.

In particular, according to the present invention, the monomer composition suppresses the formation of undesirable water-soluble polymers without using any emulsifiers, and stably maintains moisture content and properties! According to a preferred monomer composition that can obtain '1133 child polymer particles and further uses (meth)acrylom 1-lyl and an internal crosslinking monomer in combination in the second intermediate composition, The water-dispersible polymer particles obtained have high strength and do not stick to each other.

Examples of the present invention are listed below, but the present invention is not limited to these Examples.

Example 1 80 g of methyl methacrylate and 15 g of acrylonitrile
was added to 421 g of distilled water, and 0.3 g of 2,2'-azobis-2-amidinopropane dihydrochloride was added to 10 g of water.
The dissolved aqueous polymerization initiator solution was added under a nitrogen stream at a temperature of 60°C, and polymerized for 4 hours while stirring at 1.20 rpm to obtain water-dispersible polymer particles as a polymer core. . Next, 0.03 g of the same polymerization initiator as above was added to this.
Add a polymerization initiator aqueous solution dissolved in 10 ml of water, and then add 5.5 g of methyl methacrylate and 5 g of acrylonitrile.
, a mixture of 3 g of methacrylic acid and 10.5 g of I-lyethylene glycol dimethacrylate was added over a period of 1 hour;
By reacting for 3 hours, a polymer shell having a carboxyl group as a functional group was formed on the core. The water a liquid of the polymer particles thus obtained had a solid content of 20% and an average particle size of the polymer particles of 0.45 μm.

The polymerization was very stable, with 0.08% agglomerates.

When this dispersion was centrifuged and the carboxyl groups in the liquid were quantified, only 0.9% of the charged amount of carboxyl groups was determined, indicating that the formation of water-soluble polymerization was small.

Next, the above polymer particles were dispersed in distilled water to a concentration of 20 wt.
Add an aqueous solution of 2.5 g of -cyclohexyl-3-(2-mol heptatrinoethyl)carbodiimitome+--p-+-luenesulponate dissolved in 30 ml of water to the above dispersion, and add ■)) 1. It was adjusted to 5.8 with hydrochloric acid. After this,
N-((6-aminohexyl)carbamoylmethyl coenzyme aqueous solution was added to the dispersion liquid, and at a temperature of 4°C,
6. Slowly for 24 hours while adjusting pl+ to 5.8 with hydrochloric acid. The coenzyme was covalently immobilized on the polymer particles by stirring. Next, the supernatant was discarded after 81 t of centrifugation, and the precipitated polymer particles were washed with water to obtain an immobilized NAD derivative according to the present invention. 2% of the above NAD derivative remained in the supernatant, and it was determined that 98% of the NAD derivative used was immobilized in ml FuN.

When this immobilized coenzyme was measured using glyceraldehyde bosphate dehydrogenase, the activity yield was 67% based on free N-((6-aminohexyl)carbamoylmethyl)-NAD. Ta.

In addition, activity yield is the principle of activity of immobilized coenzyme + %
I Defined as the ratio of actual activity to Ffl.

Reference Example This reference example shows that when the monomer composition is copolymerized stepwise in the same manner as in Example 1, the first and second t't
The amount of carboxyl groups in the supernatant of the aggregate and dispersion after polymerization when copolymerized with various >-m compositions is shown. The results are shown in the table.

Furthermore, when the monomer composition of Experiment No. 4 was copolymerized all at once, the aggregates were 7.3%, the carboxyl group content was 6%, and the polymer particles in the dispersion had carboxyl groups per 1 g. It had 0.08 mmol of groups. However, the polymer particles obtained by stepwise copolymerization had 0.35 mmol of carboxyl groups per gram.

Example 2 The carboxyl group-containing polymer particles obtained in Example 1 were dissolved in 50 ml of distilled water to give a solid content of 20% by weight.
&sa・1. Next, the same force as in Example ■F2.5
Add an aqueous solution' in which g is dissolved in 30 ml of water, and dilute the solution with dilute hydrochloric acid.
l+ was adjusted to 5.8. Furthermore, 1 ml of m-xylylenediamine was dissolved in 10 ml of water, and p+1 was diluted to 5 with dilute hydrochloric acid.
．． An aqueous solution adjusted to a temperature of 8 was added, and the mixture was slowly stirred at a temperature of 4°C for 24 hours while maintaining pl+ at 5.8.

Next, the mixture was centrifuged, and the precipitated polymer particles were washed repeatedly with distilled water, thereby obtaining polymer particles having amino groups.

This polymer particle was again dispersed in distilled water so that the solid content was 20%, and 2 g of N-succinyl-NADo was added to 1 ml of water.
Add 0 ml of cofermented green aqueous solution to the above dispersion, and while adjusting the PL+ to 5.8 with dilute hydrochloric acid, add 1-cyclobenyl-1-cyl-3-(2-mole noethyl)carbodiimide-1- Metho-p-toluenesulpo-1-
An aqueous solution of 2,58 dissolved in 30 ml of water was added, and ρ11
The mixture was stirred for 24 hours at a temperature of 4°C while adjusting the temperature to 5.8°C.

Thereafter, at 11 minutes, the liquid was centrifuged, and the precipitated polymer particles were washed repeatedly in the same manner as in Section 2 to obtain an immobilized NAD derivative according to the present invention. It was confirmed that 92% of the NAD derivative used was immobilized.

When the same reaction as in Example 1 was carried out using this immobilized coenzyme, the activity yield was 88% based on N6-scudinyl-NAD.

Example 3 The polymer particles having amine groups obtained in Example 2 were
Similarly, 6-amitzhexicnic acid was reacted to increase the number of carbon atoms in the spacer group. N-(2-aminoethyl)-N8 was bound to this polymer particle in the same manner as in Example 1 to obtain an immobilized coenzyme according to the present invention. The active yield of this is N6-(2-aminoethyl)-NAD group!
1, it was 81%.

Furthermore, it was confirmed that the coenzyme activity was maintained over a long period of time through the following series of reactions.

Using a membrane module equipped with a polyamide hollow fiber membrane (NTU-8010 manufactured by Nitto Electric Industry Co., Ltd.) with a molecular weight cut-off of 1oooo, 25 ml of the immobilized NAD dispersion obtained in step 2 was placed inside the membrane.
and a phosphate buffer (0.1 M, pH 7, (1)) containing 1 kg each of alcohol dehydrogenase and Lafti 1-dehydrogenase is circulated;
On the other hand, in the space outside the membrane, 0.5M ethanol, 0.05
Phosphate buffer (50 mM, pH 7,0) containing M pyruhic acid and 1 m M J archetype geltadeone was passed through as substrate/8 solution and reacted continuously at a temperature of 25°C. The resulting reaction solution was heated at 100°C for 10 minutes to decompose unreacted pyruvic acid and inactivate the enzyme and coenzyme, and then the amount of lactic acid produced was determined by lactate dehydrogenase. When the relative activity of the immobilized NAD derivative was examined over time, it was found that it retained 92% even after two weeks.

Claims (10)

[Claims] (1) A polymer core of 50 to 98 M% consisting of a polymer of the first ethylenic monomer composition and a second monomer composition containing an ethylenic monomer having a functional group. A coenzyme is covalently immobilized via the above-mentioned functional group to water-dispersible polymer particles composed of a polymer shell portion of 2 to 50% by weight. Immobilized coenzyme. (2) The first monomer composition contains (al acrylonitrile and/or methacrylonitrile 1 to 80% by weight, (b) polyfunctional internal crosslinking monomer 0 to 20% by weight, and (Cl acrylic acid The immobilized coenzyme according to claim 1, characterized in that the immobilized coenzyme comprises 20 to 99% by weight of at least one monomer selected from 9 alkyl ester, methacrylic acid alkyl ester, and styrene. (3) The second monomer composition includes (1 to 70% market weight of a monomer having an al functional group, 1 to 80% market weight of monomer having an al functional group, (1 to 80% market weight of a monomer having a bl acrylonitrile and/or methacrylonitrile, and 0 to 20% by market weight of a functional internal crosslinking monomer, and 20 to 9% of at least one small amount selected from (dl acrylic acid alkyl ester, methacrylic acid alkyl ester, and styrene)
8% by weight of the immobilized coenzyme according to claim 1. (4) The functional group in the second ethylenic monomer composition is at least one selected from a primary amino group, a hydroxyl group, a glycidyl group, and a carboxyl group. The immobilized coenzyme according to item 1. (5) The immobilized enzyme according to claim 1, wherein the water-dispersible polymer particles have an average particle diameter of 0.05 to 2μ. (6) After emulsion copolymerizing the first ethylenic monomer composition in an aqueous medium to form a polymer core made of water-dispersible polymer particles, the polymer core is A water-dispersible polymer having a polymer shell on the polymer core by emulsion copolymerizing a second monomer composition containing an ethylenic monomer having a functional group in the presence of forming polymer particles;
A method for producing an immobilized coenzyme, characterized in that the coenzyme is then covalently immobilized on the polymer particles via the above functional group. (7) The first monomer composition contains (Al acrylonitrile and/or methacriconitrile 1 to 80% by weight, (b) polyfunctional internal crosslinking monomer 0 to 20% by weight, and (Cl acrylic acid The method for producing an immobilized coenzyme according to claim 6, characterized in that it consists of 20 to 99% by weight of one monomer selected from alkyl esters, alkyl methacrylates, and styrene. . (8) The second monomer composition includes (1 to 70% by weight monomers having an al functional group, (b) 1 to 80% by weight acrylonitrile and/or methacrylonitrile), (c+ polyfunctional internal 0 to 20% by weight of a crosslinking monomer, and 20 to 20% by weight of at least one monomer selected from (d+alkyl acrylate, alkyl methacrylate, and styrene)
98% by weight of the immobilized coenzyme according to claim 6. (9) Claim 6, characterized in that the functional group in the second ethylenic monomer composition is at least one selected from a primary amino group, a hydroxyl group, a glycidyl group, and a carboxyl group. A method for producing the described immobilized coenzyme. (10) The water-dispersible polymer particles are 0. ．． 05-2/
7. The method for producing an immobilized coenzyme according to claim 6, wherein the immobilized coenzyme has an average particle size of 7.