JPH0632615B2 - Method for producing granular fixed molded article of enzyme or microbial cell - Google Patents

Description

The present invention relates to a method for immobilizing enzyme or microbial cells, and more particularly to a method for immobilizing enzyme or microbial cells as a granular molded product.

As a method for immobilizing an enzyme or a microorganism, a conventional entrapment method,
Many methods such as a physical adsorption method and a covalent bond method are known. When used in a microbial reaction or an enzymatic reaction, the lump-shaped or sheet-shaped immobilized product obtained by these methods is usually cut into fine pieces or ground and then packed in a column. In that case, however, the immobilized products often come in close contact with each other, resulting in poor efficiency of microbial reaction and enzyme reaction, and often cause channeling phenomenon to block the column.

These drawbacks are that if an enzyme or microbial cell can be immobilized as a granular molded product, it will flow easily and the work of packing into the column will be easy, and the contact area between particles will be small and the efficiency of microbial reaction and enzyme reaction will be improved. can do. As a method for immobilizing an enzyme or microbial cell as a granular molded product, a water-soluble polymer having an ability to gel by contact with a polyvalent metal ion and a hydrophilic photocurable resin of a certain kind as an immobilization carrier is used. A method for producing a particulate immobilization product from an aqueous medium by combination with a saccharide is known. (Japanese Patent Laid-Open Publication No. 59-11182) This method can produce a particulate immobilization product from an aqueous medium very easily without impairing the activity of the enzyme or the microbial cell, but it can be used as a carrier for immobilization with a certain hydrophilic light. A liquid composition prepared by adding a photopolymerization initiator, an enzyme, or microbial cells to a polyvalent metal using a water-soluble polymer polysaccharide having the ability to gel by contact between a curable resin and a polyvalent metal ion. A method in which the composition is gelled into particles by dropping into an aqueous medium containing ions, and then the resulting granular gel is irradiated with an actinic ray in an aqueous medium to cure the photocurable resin in the granular gel. Therefore, there is a drawback that the photocurability of the photocurable resin is lowered when the concentration of dissolved oxygen in the aqueous medium is high. This tendency is remarkable especially when hydroquinone or the like which binds to oxygen and exhibits a polymerization inhibiting action is used as a polymerization inhibitor for the photocurable resin. Insufficient curing of the photocurable resin results in unfavorable results such as leakage from particles of enzyme or bacterial cells that are entrapped and immobilized, or turbidity of an aqueous medium.
In order to eliminate the influence of dissolved oxygen, it is necessary to carry out a troublesome operation of dropping particles into an aqueous medium and irradiating with actinic rays in an inert gas.

The present inventors have conducted intensive studies on a method of overcoming these drawbacks and removing the influence of dissolved oxygen in an aqueous medium and a polymerization inhibitor in a photocurable resin when fixing particles in an aqueous medium. Came to

The features of the present invention are: (a) a hydrophilic photocurable resin having at least two ethylenically unsaturated bonds in one molecule in the production of a granular immobilization molded product of an enzyme or a microbial cell (b) a photopolymerization initiator (C) A water-soluble polymer polysaccharide capable of gelling upon contact with polyvalent metal ions (d) A liquid composition containing an enzyme or a microbial cell is treated with polyvalent metal ions and ascorbic acid or ascorbic acid. It is to drip into an aqueous medium containing sodium to gel the composition into particles, and then irradiate the resulting granular gel with actinic rays to cure the photocurable resin in the granular gel. Ascorbic acid or sodium ascorbate contained in this aqueous medium prevents the production of benzoquinone by the oxidation of dissolved oxygen in the aqueous medium such as hydroquinone contained in the photocurable resin due to its reducing action, and thus photocurability It has a remarkable effect in improving the curability of the resin. As a result, it is possible to remarkably reduce the leakage from the entrapped and immobilized enzyme or bacterial cell particles and the turbidity of the aqueous medium. Even when a polymerization inhibitor such as benzoquinone that exhibits a polymerization inhibitory effect regardless of the presence of dissolved oxygen is used to prevent polymerization of the photocurable resin, benzoquinone is polymerized with ascorbic acid or sodium ascorbate in an aqueous medium. It can be reduced to hydroquinone, which has a small inhibiting action, to improve the photocurability.

Hereinafter, the method of the present invention will be described in more detail.

(A) Hydrophilic photocurable resin In the method of the present invention, one of the immobilization carriers is 1
A hydrophilic photocurable resin having at least two ethylenically unsaturated bonds in the molecule is used. The photocurable resin is generally 300 to 30,000, preferably 500.
To 20,000, and an ionic or nonionic hydrophilic group sufficient to have a number average molecular weight in the range of 20,000 and be sufficiently dispersed in an aqueous medium in which an enzyme or microbial cell is suspended. , For example, hydroxyl group, carboxyl group, phosphate group,
A resin containing a sulfonic acid group, an amino group, an ether bond, etc., which is cured to be a resin substantially insoluble in water when irradiated with an actinic ray having a wavelength in the range of about 250 to about 600 nm is preferably used. To be done. Such a photocurable resin has already been known as a carrier for immobilizing enzymes or microbial cells (for example, Japanese Patent Publication No. 55-40 and Japanese Patent Publication No. 55-2).
Nos. 0676 and the like), and the representative ones include the following: (i) Highly oxidative unsaturated polyesters: for example, maleic anhydride, maleic acid, fumaric acid, itaconic acid, itacone anhydride. At least one unsaturated polycarboxylic acid such as an acid,
Acid value obtained by esterification of polyvalent alcohol with polyvalent carboxylic acid component consisting of at least one saturated polycarboxylic acid such as trimellitic acid, trimellitic anhydride, pyromellitic acid and pyromellitic anhydride Is 40-2
Unsaturated polyester salts within the range of 00; at least one unsaturated polycarboxylic acid such as maleic anhydride, maleic acid, fumaric acid, itaconic acid, and itaconic anhydride;
An acid value obtained by reacting an acid anhydride with the residual hydroxyl group in the esterified product with a polyhydric alcohol component containing at least 5% by weight of a polyhydric alcohol having more than 3 hydroxyl groups in one molecule is 40 to 200. Such as unsaturated polyesters within the range.

(ii) Highly oxidized unsaturated epoxides: polyglycidyl compounds such as n-equivalents of Epicoat 828, Epicoat 1001, Epicoat 1004 and the like manufactured by Shell Chemical Co. and polyvalent carboxylic acids such as maleic acid, adipic acid and trimellitic acid ( n-1) an acid value obtained by adding an acid anhydride to a hydroxyl group remaining in the addition reaction product of an equivalent of the unsaturated carboxyl compound such as (meth) acrylic acid with 2 equivalents is in the range of 40 to 200. Saturated epoxides; a compound obtained by adding an acid anhydride to a hydroxyl group remaining in the addition reaction product of the polyglycidyl compound n equivalents as described above and the polycarboxylic acid (n + 2) equivalents as described above (meth) acryl Unsaturated epoxides having an acid value of 40 to 200 obtained by reacting an unsaturated glycidyl compound such as glycidyl acid.

(iii) Anionic unsaturated acrylic resin: Here, the anionic unsaturated acrylic resin is at least 2 selected from (meth) acrylic acid and (meth) acrylic acid ester.
A resin obtained by introducing a photopolymerizable ethylenically unsaturated group into a copolymer containing a carboxyl group, a phosphoric acid group and / or a sulfonic acid group, which is obtained by copolymerizing a certain (meth) acrylic monomer, and The following formula C + 5P + 10S = A (1) [where C is the concentration of the carboxyl group in the resin (mol / k
g), P is the phosphoric acid group concentration in the resin (mol / kg), and S is the sulfonic acid group concentration in the resin (mol / kg)]. To 5 (mol / kg), and the concentration of the photopolymerizable ethylenically unsaturated group in the resin is 0.1 to 5 (mol / kg). Such a copolymer can be synthesized by a known method, in which case acrylic acid as a comonomer,
When an unsaturated carboxylic acid such as methacrylic acid is used, a copolymer containing a carboxyl group can be obtained, and an unsaturated phosphoric acid ester such as Phosmer M and Phosmer C1 [both manufactured by Oil and Fat Products Co., Ltd.] is used as a comonomer. For example, a copolymer containing a phosphoric acid group is obtained, and as a comonomer, (meth) acrylic acid-2-sulfoethyl, (meth)
If an unsaturated sulfonic acid ester such as 3-sulfopropyl acrylate is used, a copolymer containing a sulfonic acid group can be obtained. In order to introduce a photopolymerizable ethylenically unsaturated group into the copolymer thus obtained, a carboxyl group, a phosphoric acid group or a sulfonic acid group present in the copolymer may be glycidyl (meth) acrylate or the like. This is possible by reacting an unsaturated glycidyl compound.

(iv) Cationic unsaturated acrylic resin: For example, (meth)
A copolymer of a (meth) acrylic acid ester containing an unsaturated amino compound such as 2-diethylaminoethyl acrylate, tert-butylaminoethyl (meth) acrylate, and vinylpyridine in an amount of more than 5% by weight, ( Meta)
Unsaturated acrylic resin obtained by reacting unsaturated glycidyl compound such as glycidyl acrylate; unsaturated acrylic resin obtained by quaternizing with unsaturated amino compound after chloromethylation of polystyrene; polyethyleneimine unsaturated glycidyl compound And the additions, etc.

(v) Polyesters of polyethylene glycol and (meth) acrylic acid: For example, diesters of unsaturated monocarboxylic acids such as (meth) acrylic acid of polyethylene glycol having a molecular weight of 400 to 10,000 and containing less than 30% by weight of propylene oxide groups. A dibasic acid such as n mol of maleic anhydride and a molecular weight of (n + 1) mol of 600 to 1000;
An esterification product of 0 polyethylene glycol and 2 mol of an unsaturated monocarboxylic acid such as (meth) acrylic acid;
An esterified product of n basic acid such as trimellitic acid, n + 2 molar polyethylene glycol having a molecular weight of 600 to 10,000 and 3 molar unsaturated carboxylic acid such as (meth) acrylic acid.

(vi) Polyethylene glycol and (meth) acrylic acid 2-
Urethane Adducts with Hydroxyethyl: For example, n
Moles of diisocyanate such as tolylene diisocyanate, xylylene diisocyanate, hexamethylene diisocyanate and (n-1) moles of molecular weight 800-100
00 polyethylene glycol and 2 moles of (meth)
Urethanes with unsaturated monohydroxy compounds such as 2-hydroxyethyl acrylate; n moles of triisocyanates such as Desmodur L (manufactured by Bayer) and (n
-1) Urethanes with an unsaturated monohydroxy compound such as (n + 2) moles of 2-hydroxyethyl (meth) acrylate having a molecular weight of 800 to 10,000 and a trifunctional group such as 1 mole of trimethylolpropane. Hydroxy compound, 4 mol of diisocyanate, 2 mol of polyethylene glycol having a molecular weight of 400 to 10,000, and 2 mol of (meth) acrylic acid 2-
Urethane products with unsaturated monohydroxy compounds such as hydroxyethyl.

(vii) Unsaturated celluloses: for example, water-soluble cellulose such as cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, hydroxyethyl cellulose and an unsaturated glycidyl compound such as glycidyl (meth) acrylate, or itaconic anhydride, maleic anhydride, etc. Addition reaction product with unsaturated acid anhydride.

(viii) Unsaturated polyamide: For example, an adduct of 1 mol of a diisocyanate such as tolylene diisocyanate or xylylene diisocyanate and 1 mol of an unsaturated hydroxy compound such as 2-hydroxyethyl acrylate into a water-soluble polyamide such as gelatin. An unsaturated polyamide that has been subjected to an addition reaction.

The photo-curable resins as exemplified above can be used alone or in combination of two or more kinds. Among these photocurable resins, those that can be particularly advantageously used in the present invention include the above (v)
Examples thereof include polyesters of polyethylene glycol and (meth) acrylic acid, and urethane-added products of polyethylene glycol of (vi) and 2-hydroxyethyl (meth) acrylate.

(B) Photopolymerization initiator For the purpose of promoting the photopolymerization reaction of the photocurable resin described in (a) above, the liquid composition according to the present invention contains a photopolymerization initiator (photosensitizer). The photopolymerization initiator that can be used is one that decomposes upon irradiation with light to generate a radical, which acts as a polymerization initiation species to cause a crosslinking reaction between resins having a polymerizable unsaturated group, such as benzoin, Α-Carbonyl alcohols such as acetoin; benzoin methyl ether, benzoin ethyl ether, benzoin propyl ether, anisoin ethyl ether, pivaloin ethyl ether and other acyloin ethers; naphthol, hydroxyanthracene and other polycyclic aromatic compounds Α-substituted acyloins such as methylbenzoin and α-methoxybenzoin; azoamide compounds such as 2-cyano-2-butylazoformamide; uranyl nitrate
Metal salts such as ferric chloride; mercaptans; disulfides; halogen compounds; dyes and the like.

These photopolymerization initiators may be used alone or in combination of two or more kinds, usually in a proportion of 0.01 to 10 pHR (per handred resin).

(C) Water-Soluble Polymer Polysaccharide The method of the present invention is described in the above (a) as an immobilization carrier in order to achieve gelation of the immobilization carrier of an enzyme or microbial cell in an aqueous medium. One major feature is the use of water-soluble polymeric polysaccharides in combination with photocurable resins.

The water-soluble polymeric polysaccharide used in the present invention is a polymeric polysaccharide that is water-soluble and has the ability to transform into a water-insoluble or sparingly soluble gel when contacted with a polyvalent metal ion in an aqueous medium. , Generally about 3,000 to about 2,000,000
It has a molecular weight of 0 and is usually at least about 10 g / (25
Those exhibiting a solubility of (° C.) are preferably used.

Specific examples of the water-soluble polymer polysaccharide having such characteristics include alkali metal salts of alginic acid, carrageenan, mannan, chitosan and the like.

These water-soluble polymeric polysaccharides, in a state of being dissolved in an aqueous medium, are polyvalent metal ions such as alkaline earth metal ions such as magnesium ion, calcium ion, strontium ion, and barium ion, or aluminum ion, cerium ion, nickel ion. Gelation may occur upon contact with at least one polyvalent metal ion of other polyvalent metal ions such as The water-soluble polymer polysaccharide that can be used in the present invention does not need to have a gelling ability for contact with all polyvalent metal ions, and at least one polyvalent metal ion, preferably It suffices to have the ability to gel when contacted with alkaline earth metal ions. The concentration of the polyvalent metal ion that causes gelation varies depending on the type of the water-soluble polymer polysaccharide and the like, but is generally at least 0.1 mol /.

(D) Enzyme or microbial cell There is no particular limitation on the type of enzyme or microbial cell that can be immobilized by the method of the present invention, and according to the method of the present invention, any type of enzyme or microbial cell, It can be immobilized without substantially deactivating its enzymatic activity.

The typical examples of enzymes and microbial cells that can be immobilized by the method of the present invention are as follows.

(A) Examples of enzymes Lactate dehydrogenase (1.1.2.3), lactate oxidase (1.1.3.2), glucose oxidase (1.1.3.4), formate dehydrogenase (1.2. 1.2), aldehyde dehydrogenase (1.2.1.3), aldehyde oxidase (1.2.3.1), xanthine oxidase (1.2.3.2), pyruvate oxidase (1.2.3) .3), pyruvate reductase (1.2.4.1), cortisone-α-reductase (1.3.1.4), acyl CoA-dehydrogenase (13.99.3), 3-ketosteroid Δ ^1. -Dehydrogenase (1.3.
99.4), 3-ketosteroid Δ ⁴ -dehydrogenase (1.3.
99.5), L-alanine dehydrogenase (1.4.1.1), L-glutamate dehydrogenase (1.4.1.
3), L-amino acid oxidase (1.4.3.2), D-amino acid oxidase (1.4.3.3), pyridoxal phosphate oxidase (1.4.
5), catalase (1.11.1.6), catechol methyltransferase (2.1.1.
6), carnitine acetyltransferase (2.3.1.
7), acetyl-CoA acetyltransferase (2.3.
1.9), aspertate aminotransferase (2.6.
1.1), alanine aminotransferase (2.6.1.
2), pyridoxamine pyruvate transferase (2.
6.1), hexokinase (2.7.11), glucokinase (2.7.1.2), flucotokinase (2.7.1.4), phosphoglucokinase (2.7.1.10) Phosphofructokinase (2.7.1.11), pyruvate kinase (2.7.1.10), carboxyesterase (3.1.1.1), arylesterase (3.1.1.2) ), Lipase (3.1.1.3), phospholipase A (3.1.1.4), acetyl esterase (3.1.1.6), cholesterol esterase (3.1.1.13), gluco Amylase (3.2.1.3), Cellulase (3.2.1.4), Inulase (3.2.1.7), α-Glucosidase (3.2.1.20), β-Glucosidase ( 3.2.1.21), α-gala Tosidase (3.2.1.22), β-galactosidase (3.2.1.23), invertase (3.2.1.26), pepsin (3.4.4.1), trypsin (3. 4.4.4), chymotrypsin A (3.4.4.5), cathepsin A (3.4), papain (3.4.4.10), thrombin (3.4.4.13), amidase (3.5.1.4), urease (3.5.1.5), penicillin acidase (3.5.1.11), aminoacylase (3.5.1.14), adenine deaminase (3 5.4.2), A. T. P. ASE (3.6.1.3), pyruvate decarboxylase (4.1.1.1), oxalate decarboxylase (4.1.1.2), tryptophan decarboxylase (4.1.1. Two
7), aldolase (4.1.2.13), malate statase (4.1.3.2), tryptophan synthase (4.2.1.20), aspertase (4.3.1.1) , Lysine racemase (5.1.1.5), Glucose-6-phosphate isomerase (5.3.1.
9), steroid Δ-isomerase (5.3.3.1), maxinyl CoA synthetase (6.2.1.5), and the like.

(Note) The numbers in parentheses represent enzyme numbers.

(B) Examples of microbial cells: Lactobacillus bulgari
cus), Aerobacter aerogene
s), Bacillus subtilis, Azotobacter vinelandi
i), Proteus vulgaris, Arthrobacter simp
lex), Escherichia coli, Pseudomonas putida, Achromobacter liquidu
m), Curvularia lunata, Corynebacterium glutamicum
lutamicum), Nocardia rhodcrous, Saccharomyces for Saccharomyces for
mosensis), Saccharomyces cerevisia
e), Saccharomyces c.
arlsbergensis), Saccharomyces robustu
s), Saccharomyces rouxii, Zygosaccharomyces
japonicus), Zygosaccharomyces m
ajar), Zygosaccharomyces soya, Chizosaccharomyces pom
bo), Chizosaccharomyces octosporus (Schizosaccharom)
yces octosporus), Schizosaccharomyces m.
ellacei), etc.

Preparation of liquid composition: The components (a), (b), (c) and (d) described above can be made into a liquid composition by sufficiently mixing them in an aqueous medium. . The aqueous medium that can be used is preferably water or a buffered aqueous solution, but in some cases, a mixed solution of water-soluble alcohols and water or a buffered aqueous solution,
It is also possible to use a mixed solution of water-soluble ketones and water or a buffer aqueous solution, an ester solvent solution which can be uniformly mixed with water or a buffer aqueous solution, and the like.

The mutual use ratios of the respective components (a), (b), (c) and (d) are not strictly limited and can be widely varied depending on the type of each component,
Generally, it is suitable to use the following proportions with respect to 100 parts by weight of the hydrophilic photocurable resin as the component (a) (the preferred range is in parentheses).

(B) Photopolymerization initiator: 0.5-5 parts by weight (1-3 parts by weight) (c) Water-soluble polymeric polysaccharide: 0.5-15 parts by weight (1-
8 parts by weight) (d) Enzyme or microbial cell: 0.001 to 50 parts by weight (0.01 to 20 parts by weight) Further, the aqueous medium is 1 with respect to the total of the above (a) to (d).
It can be used in the range of 0 to 1500 parts by weight (50 to 900 parts by weight).

Gelation: The liquid composition prepared as described above is then gelled into particles by dropping into an aqueous medium containing polyvalent metal ions and ascorbic acid or sodium ascorbate.

The polyvalent metal ion that can be contained in the aqueous medium,
A substance having the ability to gel the water-soluble polymer polysaccharide in the liquid composition is selected. At that time, whether or not the polyvalent metal ion has the ability to gel the water-soluble polymer polysaccharide is determined by, for example, a mixed aqueous solution containing a hydrophilic photocurable resin and the water-soluble polymer polysaccharide uniformly. Can be easily determined by dripping into the polyvalent metal ionic liquid and observing whether or not it gels into particles.

Further, ascorbic acid contained in the aqueous medium, or sodium ascorbate, hydroquinone contained in the photocurable resin in the liquid composition to be dropped, strong polymerization inhibition due to oxidation by dissolved oxygen in the aqueous medium. It acts to prevent deterioration of photocurability by changing to benzoquinone.

Preparation of an aqueous medium containing a selected polyvalent metal ion and ascorbic acid or sodium ascorbate, a water-soluble compound of the polyvalent metal, such as halides, carbonates, carbonates of the polyvalent metal in the aqueous medium. It can be carried out by dissolving hydrogen salt, sulfate, nitrate or the like, and then dissolving ascorbic acid or sodium ascorbate.
At that time, the concentration of the polyvalent metal ion in the aqueous medium can be generally in the range of 0.01 to 5 mol /, preferably 0.1 to 2 mol /. On the other hand, the concentration of ascorbic acid or sodium ascorbate is generally 0.001.
It may be in the range of ˜1 mol / preferably 0.005 to 0.1 mol / p.

Dropping of the liquid composition into an aqueous medium containing such polyvalent metal ions and ascorbic acid or sodium ascorbate, for example, a method of dropping the liquid composition from the tip of a narrow tube such as an injection needle, It can be carried out by a method in which the liquid composition is scattered in a granular form by utilizing a centrifugal force, a method in which the liquid composition is atomized from the tip of a spray nozzle and dropped as a granular form, and the like. The size of the droplets to be dropped can be freely changed according to the particle size desired for the final particulate immobilization product, but usually the diameter is about 0.1 to 5 m.
Conveniently, it is added as a droplet of m, preferably about 0.5-3 mm.

The dropped liquid composition comes into contact with polyvalent metal ions in an aqueous medium and immediately gels to form a granular gel. Although the mechanism of gelation at that time is not exactly known, the water-soluble polymer polysaccharide is aggregated by the halide or salt solution, and then the anionic group contained in the water-soluble polymer polysaccharide is mediated by the polyvalent metal ion. , It is presumed that ionic bonding causes three-dimensional crosslinking and gelation.

The above-mentioned gelation temperature is usually room temperature, but if necessary, the gelation may be carried out while being heated to such an extent that the enzyme or microbial cell is not inactivated, or may be carried out under cooling.

Photocuring: The granular gel produced as described above is directly dispersed in an aqueous medium and is irradiated with an actinic ray to cure the hydrophilic photocurable resin in the granular gel. At this time, ascorbic acid or sodium ascorbate contained in the aqueous medium, hydroquinone contained in the photocurable resin, is oxidized by dissolved oxygen in the aqueous medium, it is changed into benzoquinone having strong polymerization inhibition property. Prevents deterioration of photocurability. This is considered to be because ascorbic acid or sodium ascorbate reduces benzoquinone and the like to hydroquinone and the like, which has a weak polymerization inhibition property, due to its reducing property. When hydroquinone contained in the photocurable resin is contained in the granular gel by being oxidized to benzoquinone during storage of the resin or compounding of the liquid composition, benzoquinone is used as a polymerization inhibitor of the photocurable resin to form granular particles. Similarly, when it is contained in the gel, it is reduced by ascorbic acid or sodium ascorbate in an aqueous medium to hydroquinone, which has a weak polymerization inhibiting effect, and the photocurability is improved. By curing the photocurable resin, the granular gel is substantially insoluble in water and a granular immobilized product of enzyme or microbial cells having high mechanical strength is obtained.

The wavelength of the actinic ray that can be used for the photo-curing varies depending on the type of the photo-curing resin contained in the granular gel, etc., but is generally irradiated with a light source that emits light having a wavelength in the range of about 250 to about 600 nm. It is advantageous to use Examples of such a light source include a low pressure mercury lamp, a high pressure mercury lamp, a fluorescent lamp, a xenon lamp, a carbon arc lamp, and sunlight. The irradiation time needs to be changed according to the light intensity of the light source, the distance from the light source, etc., but it can generally be within the range of about 0.5 to about 10 minutes. Irradiation should be such that the actinic rays are distributed as evenly as possible into the whole particle gel that is produced.

The thus-irradiated granular gel can be washed with water or a buffered aqueous solution and then stored as it is, or the granular gel can be freeze-dried and stored.

Thus, according to the present invention, a granular immobilization product of enzyme or microbial cells having a particle size of about 0.5 to about 5 mm can be produced by an extremely simple operation, and continuous production is also possible.

The advantage of the present invention is that when a granular gel dropped into an aqueous medium as a carrier for immobilizing a photocurable resin is irradiated with light to immobilize an enzyme or microbial cells in a granular manner, a polymerization inhibitor or an aqueous solution of the photocurable resin is used. The purpose is to prevent curing failure due to dissolved oxygen in the medium. This makes it possible to remarkably reduce the leakage of the entrapped and immobilized enzyme or bacterial particles and the turbidity of the aqueous medium, and has the effect of improving the activity of the particulate immobilized enzyme or bacterial cells.

The present invention will be further described with reference to examples.

Example 1 100 parts by weight of a photocurable resin containing 2000 g of polyethylene glycol having a number average molecular weight of 4000 and 2 mol of hydroquinone, 1 mol (222 g) of isophorone diisocyanate and 1 mol (130 g) of 2-hydroxyethyl methacrylate, and benzoin isobutyl ether. Mix 2 parts by weight and 100 parts by weight of distilled water well. To this, 100 parts by weight of a 2% sodium alginate aqueous solution and 100 parts by weight of an enzyme invertase (0.1% concentration) dissolved in 0.1 M phosphate buffer (pH 5) were added and well mixed to obtain a photocurable resin- Add the enzyme mixture to 0.
When dropped into a 1: 1 mixture of a 5M calcium chloride solution and a 0.05M ascorbic acid solution from an injection needle at the tip of a syringe from a liquid surface height of 10 cm, granules having a particle diameter of about 2 mm were obtained.

This granular material was transferred with a calcium chloride solution containing ascorbic acid so as to form a further single layer of petri having a flat bottom surface, and the granular material was present in an aqueous solution having a depth of 1 cm so that the wavelength of 300 nm was measured from the upper and lower surfaces of the Petri dish. Irradiation with actinic rays of ˜400 nm for 3 minutes gave a granular immobilized enzyme having a compressive strength of 30 kg / cm ² . When the turbidity of the dropped calcium chloride-ascorbic acid solution was measured by the absorbance at 375 nm, it was 0.01.

When the invertase activity of these particles was measured using sucrose as a substrate, the specific activity to invertase not immobilized was 85%.

For comparison, the compressive strength of the granular immobilized enzyme obtained by dropping the above photocurable resin-enzyme mixed solution into a 0.5 M calcium chloride solution containing no ascorbic acid and irradiating with light was 20 kg /
The cm ² and invertase specific activity were 65%. Also,
The turbidity of the dropped calcium chloride solution showed an absorbance at 375 nm of 0.1.

Example 2 100 parts by weight of a photocurable resin containing 0.1 g of benzoquinone and 1000 g of polyethylene glycol having a number average molecular weight of 1000 and 2 mol of methacrylic acid was added to 100 parts by weight of distilled water.
After adding 1 part by weight, the mixture was heated to about 50 ° C. and mixed well to obtain a uniform resin aqueous solution. To this, 2 parts by weight of benzoin ethyl ether was added and mixed and dissolved.

Add 75% of 3% sodium alginate solution to this resin mixture.
By weight, 25 parts by weight of 2% glucose isomerase bacterial enzyme solution (sodium bicarbonate buffer solution pH 8) was added to prepare a uniform mixed solution. When this homogeneous mixed solution was dropped from the tip of the injection needle into an aqueous solution containing 5% calcium chloride and 2% sodium ascorbate from a position 20 cm from the liquid surface, a granular material having a particle diameter of 1.5 mm was obtained.

This granular material was transferred to a Petri dish having a flat bottom surface together with a calcium chloride solution containing sodium ascorbate so as to form a single layer, and the granular material was present in an aqueous solution having a depth of 1 cm for 3 minutes from the upper surface and the lower surface. 3 minutes wavelength 300 ~
Irradiation with actinic light of 400nm gives compressive strength of 40kg
A granular immobilized cell enzyme of cm ² was obtained. When the turbidity of the dropped calcium chloride-ascorbic acid solution was measured by the absorbance at 375 nm, it was 0.05.

When the activity of this granular glucose isomerase was measured at 60 ° C. in a sodium bicarbonate buffer solution of pH 8 using glucose as a substrate, the specific activity to glucose isomerase that was not immobilized was 90%.

For comparison, the photocurable resin-bacterial enzyme mixture solution was added dropwise to a 5% calcium chloride solution containing no sodium ascorbate and irradiated with light to obtain a granular immobilizing bacterial enzyme having a compressive strength of 25 kg / cm ² , Glucose isomerase specific activity is 8
It was 0%. The turbidity of the dropped calcium chloride solution showed an absorbance at 375 nm of 0.5.

Example 3 25% of a photocurable resin obtained by adding 1.0 mol of N-methylolacrylamide to 500 g of polyvinyl alcohol containing 0.2 g of hydroquinone and having a degree of polymerization of 500.
% Benzoin isobutyl ether 0.5 parts by weight is uniformly mixed with 100% by weight aqueous solution, and 100 parts by weight of 3% K-carrageenan aqueous solution and 10 parts by weight of yeast paste of Saccharomyces cerevisiae (ATCC 10275) (10% concentration). Were uniformly mixed and dispersed. After that, the dispersion was dropped from the tip of the syringe into an aqueous solution containing 5% potassium chloride and 2% sodium ascorbate, and a spherical gel was formed. The spherical gel was transferred to a Petri dish together with a potassium chloride solution containing sodium ascorbate, and irradiated with actinic rays having a wavelength of 300 to 400 nm for 3 minutes from the upper surface and the lower surface, respectively, to obtain spherical immobilized yeast having a diameter of 3 mm. When the total number of yeasts in this spherical gel was measured, it was 1 × 10 ⁷ cells / ml.

To 3 g of the granular immobilized yeast, add 30 ml of glucose medium consisting of 20% glucose and 0.5% yeast extract,
When statically cultivated at 30 ° C. for 24 hours, the total number of yeast in the granular gel grew to 6 × 10 ⁹ cells / ml, and the concentration of produced alcohol was 10%. The number of bacteria leaked into the medium is 2 × 10 ^7.
It was a piece / ml.

In comparison with this, the total number of yeasts in the granular immobilized yeast obtained by dripping the photocurable resin-yeast mixed solution into a 5% potassium chloride solution containing no sodium ascorbate and irradiating with light was 1 × 10. It was ⁷ cells / ml. This granular immobilized yeast 3
When 30 ml of glucose medium consisting of 20% of glucose and 0.5% of yeast extract was added to g, and static culture was carried out at 30 ° C. for 24 hours, the total number of yeast in the granular gel was 5 × 10 ⁸ cells / ml, The produced alcohol concentration was 5%. The number of bacteria leaked into the medium was 9 × 10 ⁷ cells / ml.

Claims (1)

[Claims] 1. A hydrophilic photocurable resin having (a) at least two ethylenically unsaturated bonds in one molecule, (b) a photopolymerization initiator, and (c) contact with at least one polyvalent metal ion. A liquid composition comprising a water-soluble polymer polysaccharide capable of gelation by (d) and (d) an enzyme or microbial cells in an aqueous medium containing a polyvalent metal ion and ascorbic acid or sodium ascorbate. Granular immobilization of enzyme or microbial cells characterized by dripping to gel the composition into particles and then irradiating the resulting granular gel with actinic rays to cure the photocurable resin in the granular gel. A method for producing a molded article.