JPS6219837B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immobilizing enzymes or microbial cells, and more particularly to a method for immobilizing enzymes or microbial cells in the form of granular molded products.

Many methods have been known for immobilizing enzymes or microorganisms, such as entrapment methods, physical adsorption methods, and covalent bonding methods. When using a lump or sheet-like immobilized product obtained by these methods in a microbial reaction or an enzyme reaction, it is usually cut or ground into small pieces and then packed into a column. However, in this case, the immobilized substances often come into close contact with each other, which reduces the efficiency of microbial reactions and enzymatic reactions, and also has drawbacks such as often causing a channeling phenomenon and clogging the column.

The present inventors believe that if enzymes or microbial cells can be immobilized in the form of granular moldings, they will be fluid and easy to fill into columns, and the contact area between particles will be small, increasing the efficiency of microbial reactions and enzyme reactions. As a result of intensive research into methods of immobilizing enzymes or microbial cells as particulate compositions, we found that a certain type of hydrophilic photocurable resin and polyvalent metal ions were used as immobilization carriers. By using a combination of water-soluble polymeric polysaccharides that have the ability to gel when contacted with water, it is possible to easily produce granular immobilized materials with high mechanical strength from an aqueous medium without loss of enzymes or microbial cells. The present inventors have discovered that it can be manufactured, and have completed the present invention.

Therefore, according to the present invention, (a) a hydrophilic photocurable resin having at least two ethylenically unsaturated bonds in one molecule, (b) a photopolymerization initiator, (c) a polyvalent metal ion, and A liquid composition comprising a water-soluble polymeric polysaccharide capable of gelling upon contact with (d) an enzyme or a microorganism other than yeast having the ability to ferment alcohol, containing a polyvalent metal ion. An enzyme or microorganism characterized by dropping the composition into a granular gel by dropping it into an aqueous medium, and then irradiating the resulting granular gel with actinic rays to cure the photocurable resin in the granular gel. A method for producing a granular immobilized molded body is provided.

The method of the present invention will be explained in more detail below.

(a) Hydrophilic photocurable resin In the method of the present invention, a hydrophilic photocurable resin having at least two ethylenically unsaturated bonds in one molecule is used as one of the immobilization carriers. The photocurable resin is generally 300
~30,000, preferably in the range of 500 to 20,000, and sufficiently ionic or nonionic to disperse uniformly in the aqueous medium in which the enzyme or microbial cells are suspended. Contains hydrophilic groups, such as hydroxyl groups, carboxyl groups, phosphoric acid groups, sulfonic acid groups, amino groups, ether bonds, etc., and is cured when irradiated with actinic light having a wavelength in the range of about 250 to about 600 nm. Alternative resins that are substantially insoluble in water are preferably used. Such photocurable resins are already known as immobilization carriers for enzymes or microorganisms (for example, Japanese Patent Publication No. 1983-40, Japanese Patent Publication No. 1983-1983).
(Refer to Publication No. 20676, etc.), typical examples include those described below: (i) High acid value unsaturated polyesters: For example, maleic anhydride, maleic acid, fumaric acid, itaconic acid, anhydride At least one unsaturated polyhydric carboxylic acid such as itaconic acid, trimellitic acid, trimellitic anhydride, pyromellitic acid,
Salts of unsaturated polyesters having an acid value within the range of 40 to 200 obtained by esterifying a polycarboxylic acid component consisting of at least one saturated polycarboxylic acid such as pyromellitic anhydride with a polyhydric alcohol; At least 5% by weight of at least one unsaturated polyhydric carboxylic acid such as maleic anhydride, maleic acid, fumaric acid, itaconic acid, and itaconic anhydride, and a polyhydric alcohol having more than 3 hydroxyl groups in one molecule. Unsaturated polyesters with an acid value within the range of 40 to 200 obtained by reacting an acid anhydride with the residual hydroxyl group in the esterified product with a polyhydric alcohol component.

(ii) Highly oxidized unsaturated epoxides: polyglycidyl compounds such as Epicote 828, Epicote 1001, and Epicote 1004 manufactured by Ciel Chemical Co., Ltd.
Hydroxyl residue remaining in the addition reaction product of n equivalents such as and (n-1) equivalents of a polyhydric carboxylic acid, such as maleic acid, adipic acid, trimellitic acid, etc., and 2 equivalents of an unsaturated carboxyl compound such as (meth)acrylic acid. Unsaturated epoxides with an acid value within the range of 40 to 200 obtained by adding an acid anhydride to the group; addition reaction between n equivalents of the above polyglycidyl compound and (n+2) equivalents of the above polyhydric carboxylic acid Unsaturated epoxides with an acid value of 40 to 200 are obtained by reacting an unsaturated glycidyl compound such as glycidyl (meth)acrylate with a compound obtained by adding an acid anhydride to the hydroxyl groups remaining in a substance.

(iii) Anionic unsaturated acrylic resins: Anionic unsaturated acrylic resins are at least two types of (meth)acrylic resins selected from (meth)acrylic acid and (meth)acrylic esters.
It is a resin in which a photopolymerizable ethylenically unsaturated group is introduced into a copolymer containing a carboxyl group, a phosphoric acid group and/or a sulfonic acid group obtained by copolymerizing an acrylic monomer, and it has the following formula: C+5P+10S=A (1) [In the formula, C is the concentration of carboxyl groups in the resin (mol/Kg), P is the concentration of phosphoric acid groups in the resin (mol/Kg), and S is the concentration of sulfonic acid groups in the resin. (mol/Kg)] The value of A calculated by is 0.8 to 5 (mol/Kg).
kg), and the concentration of photopolymerizable ethylenically unsaturated groups in the resin is from 0.1 to 5.
(mol/Kg). Such a copolymer can be synthesized by a known method. At that time, if an unsaturated carboxylic acid such as acrylic acid or methacrylic acid is used as a comonomer, a copolymer containing a carboxyl group can be obtained, and a copolymer containing a carboxyl group can be obtained by using a phosmer as a comonomer. M,
A copolymer containing a phosphoric acid group can be obtained by using an unsaturated phosphoric acid ester such as Phosmer Cl (both manufactured by Yushi Products Co., Ltd.), and 2-sulfoethyl (meth)acrylate, ( If an unsaturated sulfonic acid ester such as 3-sulfopropyl meth)acrylate is used, a copolymer containing sulfonic acid groups can be obtained. In order to introduce a photopolymerizable ethylenically unsaturated group into the copolymer thus obtained, glycidyl (meth)acrylate or the like is added to the carboxyl group, phosphoric acid group or sulfonic acid group present in the copolymer. This is possible by reacting unsaturated glycidyl compounds.

(iv) Cationic unsaturated acrylic resin: e.g.
Copolymers of (meth)acrylic acid esters containing more than 5% by weight of unsaturated amino compounds such as 2-diethylaminoethyl (meth)allylic acid, tert-butylaminoethyl (meth)acrylate, and vinylpyridine. unsaturated acrylic resin obtained by reacting unsaturated glycidyl compounds such as glycidyl (meth)acrylate; unsaturated acrylic resin obtained by chloromethylating polystyrene and then quaternizing it with an unsaturated amino compound; polyethylene Adducts of imines and unsaturated glycidyl compounds, etc.

(v) Polyesters of polyethylene glycol and (meth)acrylic acid: e.g.
Diesters of unsaturated monocarboxylic acids such as (meth)acrylic acid of polyethylene glycol containing from 400 to 10,000 and less than 30% by weight of propylene oxide groups; molecular weight of n moles of a dibasic acid such as maleic anhydride and (n+1) moles. 600～
Esterified product of unsaturated monocarboxylic acid such as 10000 polyethylene glycol and 2 mol (meth)acrylic acid; molecular weight of n mol trichloride cold acid such as trimellitic acid and n+2 mol
600-10000 polyethylene glycol and 3 mol esters of unsaturated carboxylic acids such as (meth)acrylic acid, etc.

(vi) Urethane adducts of polyethylene glycol and 2-hydroxyethyl (meth)acrylate: for example, n moles of diisocyanate such as tolylene diisocyanate, xylylene diisocyanate, hexamethylene diisocyanate and (n-1) moles of diisocyanate; Molecular weight 800~
10,000 polyethylene glycol and 2 mol of an unsaturated monohydroxy compound such as 2-hydroxyethyl (meth)acrylate; n mol of a triisocyanate such as Desmodyur L (Bayer AG) and (n-1) mol. polyethylene glycol with a molecular weight of 800 to 10,000 and (n+2) mol of an unsaturated monohydroxy compound such as 2-hydroxyethyl (meth)acrylate; 1 mol of a trifunctional hydroxy compound such as trimethylolpropane and 4 mol of diisocyanate and 2 mol of molecular weight 400-10000
of polyethylene glycol and 2 moles of an unsaturated monohydroxy compound such as 2-hydroxyethyl (meth)acrylate, etc.

(vii) Unsaturated celluloses: For example, water-soluble cellulose such as cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, hydroxyethyl cellulose, unsaturated glycidyl compounds 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 is added to a water-soluble polyamide such as gelatin. Unsaturated polyamide subjected to addition reaction.

The photocurable resins exemplified above can be used alone, or in combination of two or more. Among these photocurable resins, those which can be particularly advantageously used in the present invention include polyesters of polyethylene glycol and (meth)acrylic acid (v) and polyesters of polyethylene glycol and (meth)acrylic acid (vi). Mention may be made of urethanized adducts with 2-hydroxyethyl acid.

(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 initiators that can be used are those that decompose by light irradiation to generate radicals, which act as polymerization initiation species to cause a cross-linking reaction between resins having polymerizable unsaturated groups, such as benzoin, α-Carbonyl alcohols such as acetoin; Acilloin ethers such as benzoin methyl ether, benzoylethyl ether, benzoin propyl ether, anisoin ethyl ether, pivaloin ethyl ether; Polycyclic aromatic compounds such as naphthol and hydroxyanthracene α-substituted acyloins such as methylbenzoin and α-methoxybenzoin; 2-cyano-
Examples include azoamide compounds such as 2-butylazoformamide; metal salts such as launyl nitrate and ferric chloride; mercaptans; disulfides; halogen compounds; dyes and the like.

These photopolymerization initiators are used alone or in combination of two or more, and usually have a concentration of 0.01 to 10 pHR (per hundred pHR).
Resin) can be used.

(c) Water-soluble polymeric polysaccharide In order to achieve gelation of an enzyme or microbial cell immobilization carrier in an aqueous medium, the method of the present invention uses the immobilization carrier as described in the preceding paragraph (a). One major feature is the use of a water-soluble polymeric polysaccharide in combination with a photocurable resin.

The water-soluble polymeric polysaccharide used in the present invention is a polymeric polysaccharide that is water-soluble and has the ability to change into a gel that is insoluble or poorly soluble in water when it comes into contact with polyvalent metal ions in an aqueous medium. , generally has a molecular weight of about 3,000 to about 2,000,000, and
Those which normally exhibit a solubility of at least about 10 g/(25° C.) in a water-soluble state before being brought into contact with polyvalent metal ions are preferably used.

Specific examples of water-soluble polymeric polysaccharides having such characteristics include alkali metal salts of alginic acid, carrageenan, konjac mannan, and the like.

These water-soluble polymeric polysaccharides, when dissolved in an aqueous medium, contain polyvalent metal ions, such as alkaline earth metal ions such as magnesium ions, calcium ions, strontium ions, and barium ions, or aluminum ions, cerium ions, and nickel ions. When it comes into contact with at least one polyvalent metal ion among other polyvalent metal ions, such as polyvalent metal ions, it can gel. It should be noted that the water-soluble polymeric polysaccharide that can be used in the present invention does not need to have gelation ability upon contact with all polyvalent metal ions, but at least one type of polyvalent metal ion, preferably It is sufficient that it has the ability to gel when contacted with alkaline earth metal ions. The concentration of polyvalent metal ions at which gelation occurs varies depending on the type of water-soluble polymeric polysaccharide, but is generally at least 0.01 mol/.

(d) Enzymes or microbial cells There are no particular restrictions on the types of enzymes or microbial cells that can be immobilized by the method of the present invention. Even microorganisms selected from the following can be immobilized without substantially deactivating their enzymatic activity.

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

(b) Examples of enzymes Lactate hydrogenase (1, 1, 2,
3.), lactate oxidase (1.1.3.
2), Glucol 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 △ ¹ -dehydrogenase (1, 3, 99, 4), 3-ketosteroid △ ⁴ -dehydrogenase (1, 3, 99, 5), L-alanine dehydrogenase (1, 4,
1.1), L-glitamate 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, 3, 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), fructokinase (2, 7.1.4), phosphoglucokinase (2.7.1.10), phosphofructokinase (2.7.1.
11), pyruvate kinase (2.7.1.40), carboxylesterase (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), glucoamylase (3.2.1.3), cellulase (3.2.1.4), inulase (3.2.1.7), α-glycosidase (3.2.1.20) ), β-glycosidase (3.2.1.21), α-galactosidase (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), ATPase (3.6.1.3), pyruvate decarboxylase (4.1.
1.1), oxalate decarboxylase (4.
1, 1, 2), tryptophan decarboxylase (4,
1, 1, 27), aldolase (4, 1, 2, 13), malate cystase (4, 1, 3, 2), tryptophantase (4, 2, 1,
20), aspertase (4, 3, 1, 1), lysine racemase (5, 1, 1, 5), glycol-6-phosphate isomerase (5, 3, 1, 9), steroid Δ-isomerase (5・３・
3.1), maxinyl-CoA synthetase (6.2.
1.5), etc.

(Note) The number inside the bracket represents the enzyme number.

(b) Examples of microbial cells: Lactobacillus bilgaricus, Aerobacter aerogenes, Bacillus subtilis
subtulis), Azotobacter vinelandii, Proteus vulgaris
vulgaria), Arthrobacter simplex, Escherichia coli
coli), Pseudomonas putida
putida), Achromobacter liquidum, Curvularia lunata
lunata), Corynebacterium glutamicum, Nocardia rhodocras
rhodcrous) etc.

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

The mutual usage ratio of each component (a), (b), (c) and (d) above is not strictly limited and can be varied over a wide range depending on the type of each component, etc. However, in general, 100% of the hydrophilic photocurable resin of component (a)
It is appropriate to use the following proportions based on parts by weight (within the preferred range).

(b) Photopolymerization initiator: 0.5 to 5 parts by weight (1 to 3 parts by weight) (c) Water-soluble polymeric polysaccharide: 0.5 to 15 parts by weight (1 to 3 parts by weight)
(8 parts by weight) (d) Enzymes or microbial cells: 0.001 to 50 parts by weight (0.01 to 20 parts by weight) In addition, the aqueous medium is 10 parts by weight relative to the total of (a) to (d) above.
~1500 parts by weight (50-900 parts by weight) can be used.

Gelation: The liquid composition prepared as described above is then dropped into an aqueous medium containing polyvalent metal ions to be gelled into particles.

As the polyvalent metal ion that can be included in the aqueous medium, one is selected that has the ability to gel the water-soluble polymeric polysaccharide in the liquid composition. At that time, whether or not the polyvalent metal ion has the ability to gel the water-soluble polymeric polysaccharide is determined by, for example, a mixture uniformly containing a hydrophilic photocurable resin and a water-soluble polymeric polysaccharide. This can be easily determined by dropping an aqueous solution into the polyvalent metal ion liquid and observing whether it gels into a liquid.

Preparation of an aqueous medium containing selected polyvalent metal ions involves adding water-soluble compounds of the polyvalent metal, such as halides, carbonates, etc. of the polyvalent metal, into the aqueous medium.
This can be done by dissolving hydrogen carbonate, sulfate, nitrate, etc. At that time, the concentration of polyvalent metal ions in the aqueous medium is generally 0.01 to 5 mol/
, preferably within the range of 0.1 to 2 mol/.

Dropping of the liquid composition into the aqueous medium containing such polyvalent metal ions can be carried out, for example, by dropping the liquid composition from the tip of a tube with a narrow tip such as a syringe needle, or by using centrifugal force. This can be carried out by methods such as scattering the liquid composition into particles, or atomizing the liquid composition into particles and dropping the liquid composition dropwise from the tip of a spray nozzle. The size of the droplets to be dropped can be freely changed depending on the desired particle size of the final granular immobilized product, but usually droplets with a diameter of about 0.1 to about 5 mm, preferably about 0.5 to about 3 mm are used. It is convenient to drip.

The dropped liquid composition contacts polyvalent metal ions in an aqueous medium and immediately gels to form a granular gel. The gelation mechanism at this time is not precisely known, but the water-soluble polymeric polysaccharide is aggregated by a halide or salt solution, and then the anion groups contained in the water-soluble polymeric polysaccharide are aggregated via polyvalent metal ions. ,
It is presumed that the ionic bond causes three-dimensional crosslinking and gelation.

Room temperature is usually sufficient for the above gelation temperature, but
If necessary, gelation may be carried out under heating to an extent that the enzyme or microbial cells are not inactivated, or may be carried out under cooling.

Photo-curing: The granular gel produced as described above can be directly dispersed in an aqueous medium, or after being separated from the aqueous medium, it can be irradiated with active light to photo-cure the hydrophilic properties in the granular gel. harden the resin. As a result, the granular gel can be obtained as a granular immobilized enzyme or microorganism that is substantially insoluble in water and has high mechanical strength.

The wavelength of the active light that can be used for the above photocuring differs depending on the type of photocurable resin contained in the granular gel, but generally a light source that emits light with a wavelength in the range of about 250 to about 600 nm is used. Advantageously, it is used for irradiation. Examples of such light sources include low pressure mercury lamps, high pressure mercury lamps, fluorescent lamps, xenon lamps, carbon arc lamps, sunlight, and the like. The irradiation time needs to be changed depending on the light intensity of the light source, the distance from the light source, etc., but can generally be within a range of about 0.5 to about 10 minutes. Note that the irradiation time may be shortened if the irradiation is performed in an inert gas atmosphere.

Irradiation should be done so that the actinic rays are distributed as evenly as possible over the entire granular gel that has been formed. For example, when irradiating active rays to a granular gel that has been separated from an aqueous medium, the separated granular gel should be irradiated with active rays. It is convenient to arrange the irradiation material in substantially a single layer in a suitable transparent glass plate or glass container and to irradiate the glass plate or glass container both from above and from below.

The granular gel that has been irradiated in this manner can be washed with water or an aqueous buffer solution and stored as is, or the granular gel can be lyophilized and stored.

Thus, according to the present invention, particulate immobilized enzymes or microbial cells having a particle size of about 0.5 to about 5 mm can be produced by extremely simple operations, and continuous production is also possible.

According to the method of the present invention, it is now possible to immobilize enzymes or microbial cells in granular form using synthetic carriers, which has been difficult until now, and the activity of enzymes and microbial cells is achieved by using water-soluble polymeric polysaccharides. It is possible to improve the reaction rate due to the increase in substrate permeability due to the fine porosity imparted to the granular immobilized material when the water-soluble polymeric polysaccharide is decomposed. Benefits can be obtained.

In addition, these granular immobilized products can be easily used particularly in reactors whose scale is not very large, fluidized bed type reactors, and the like.

Next, the present invention will be further explained by examples.

Example 1 2000g of polyethylene glycol with a molecular weight of approximately 4000
and isophorone diisocyanate 1 mol (222 g)
1 mole (130 g) of 2-hydroxyethyl methacrylate and 2 parts by weight of benzoin isobutyl ether and 100 parts by weight of distilled water are thoroughly mixed. Add to this 2% sodium alginate aqueous solution.
Add to 100 parts by weight of the enzyme invertase (0.1% concentration) dissolved in 0.1M phosphate buffer (PH5) and mix well, and mix the resulting photocurable resin-enzyme mixture with a 1M calcium chloride solution. When the solution was dropped from a liquid level of 10 cm from the needle at the tip of the syringe, granules with a diameter of about 2 mm were obtained.

Place this granular material in a Petri dish with a flat bottom, and apply a wavelength of 300 to 400 nm from the top and bottom surfaces of the Petri dish.
Compressive strength was 20 when exposed to active light of m for 3 minutes.
Kg/cm ² of granular immobilized enzyme was obtained.

When the invertase activity of these particles was measured using sucrose as a substrate, the specific activity relative to non-immobilized invertase was 65%.

Example 2 Polyethylene glycol 2000 (molecular weight 2000)
Distilled water to 100 parts by weight of a photocurable (resin) prepolymer consisting of 1000 g and mol of methyl methacrylate.
After adding 100 parts by weight, the mixture was heated to about 50° C. and thoroughly mixed to obtain a uniform aqueous resin solution, and 2 parts by weight of benzoin ethyl ether were added thereto and mixed and dissolved.

Add 3% k-carrageenan aqueous solution to this resin mixture.
75 parts by weight and 25 parts by weight of 2% glycose isomerase cell enzyme solution (sodium bicarbonate buffer pH 8) were added to prepare a homogeneous mixed solution. When this uniform mixture was dropped from the tip of a syringe needle into a 5% potassium chloride aqueous solution from a position 20 cm from the liquid surface, the particle size was 1.5 mm.
of granules were obtained.

The granules were transferred to a Petri dish with a flat bottom in a single layer along with the potassium chloride solution, and placed at a depth of 2
When the particulate matter was present in an aqueous solution of 30 mm in size and was irradiated with actinic light having a wavelength of 300 to 400 nm for 3 minutes from the top surface and 3 minutes from the bottom surface, a granular immobilized enzyme with a compressive strength of 30 Kg/cm ² was obtained.

When the activity of this granular glucose isomerase was measured at 60° C. in a sodium bicarbonate buffer at pH 8 using glucose as a substrate, the specific activity was 80% relative to the non-immobilized glucose isomerase.

Example 3 1 mole of Epicoat 1001 resin (molecular weight: about 900, epoxy equivalent: about 475, manufactured by Shell Chemical Co., Ltd., trade name) was reacted with 1.5 moles of adipic acid, and then esterified with 4.5 moles of succinic anhydride, followed by 2.75 moles of glycidyl methacrylate. Add 1 part by weight of benzoin ethyl ether to 100 parts by weight of a resin liquid (solid content 50%) obtained by neutralizing a photocurable resin with an acid value of 75 (number average molecular weight approximately 4100) with caustic soda. Parts by weight were uniformly mixed, and further 50 parts by weight of a 3% aqueous sodium alginate solution and 0.5 parts by weight of acetone-treated cells of Arthrobacter simplex (ATCC694) were uniformly mixed and dispersed. Thereafter, when the dispersion was dropped into a 1.0M aluminum chloride solution from the tip of a syringe, it gelled into a spherical shape. When the spherical gel was transferred to a Petri dish and irradiated with actinic light with a wavelength of 300 to 400 nm for 3 minutes from the top and bottom of the Petri dish, spherical immobilized microorganisms with a diameter of approximately 3 mm and a compressive strength of 15 Kg/cm ² were obtained. Ta.

When the Δ ¹ -dehydrogenation reaction of hydrocortisone was carried out using the spherical immobilized acetone-treated bacterial cells, prednisolone was produced, with a specific activity of 50% of that without immobilization.

Example 4 300 parts by weight of ethyl acrylate, methacrylic acid
100 parts by weight, 80 parts by weight of styrene, Phosmer M [methacrylate phosphate monoester, oil and fat products]
Co., Ltd.] 90 weight resin liquid (solid content 75%) obtained by neutralizing a photocurable resin with a number average molecular weight of approximately 2500, which is a copolymer of 20 parts by weight and 50 parts by weight of glycidyl methacrylate, with caustic potassium. 2 parts by weight of benzoin isobutyl ether was added to the mixture and mixed uniformly. To this mixture, 50 parts by weight of a 3% sodium alginate aqueous solution and 50 parts by weight of a 0.5% glucose oxidase aqueous solution dissolved in 0.1M acetate buffer (PH5.6) are uniformly mixed, and the resulting resin mixture is inserted into the tip of a syringe. from
When it was dropped into a Petri dish containing a 10% ammonia alum aqueous solution, it gelled into particles. When this granular gel was irradiated with actinic light having a wavelength of 300 to 400 nm from the upper and lower surfaces for 3 minutes each, a granular immobilized enzyme with a diameter of about 2 mm and a degree of compression of 17 kg/cm ² was obtained.

When the activity of this particulate immobilized glycol oxidase was measured using glycose as a substrate, the specific activity was 85% compared to non-immobilized glycol oxidase.

Example 5 500g of polyvinyl alcohol with a degree of polymerization of 500,
0.5 parts by weight of benzoin isobutyl ether was uniformly mixed with 100 parts by weight of a 25% aqueous solution of a photocurable resin obtained by adding 1.0 mol of N-methylolacrylamide, and 100 parts by weight of a 3% aqueous k-carrageenan solution and Ethuselicia coli were added. 10 parts by weight of the bacterial cell suspension was uniformly mixed and dispersed. The thus obtained photocurable resin-microbial cell mixture is supplied onto a rotating disk,
The mixed resin liquid was scattered by the centrifugal force, and the scattered particles were dropped into a 0.5 molar potassium chloride solution and gelled into particles. This was placed in a Petri dish and irradiated with light of 300 to 400 nm from the top and bottom surfaces for 3 minutes to achieve a particle size of 3 mm and a compressive strength of 10 kg/kg.
We were able to produce immobilized bacterial cells measuring ² cm2.

When fermentation was carried out using glucose as a substrate using these granular immobilized E. coli cells, carbon dioxide was produced at a rate 1.5 times faster than in the case without immobilization.

Example 6 100 parts by weight of a photocurable (resin) prepolymer consisting of a mixture of 2000 g of polyethylene glycol with a molecular weight of 4000, 1 mol (222 g) of isophorone diisocyanate, and 1 mol (130 g) of 2-hydroxyethyl methacrylate, and 1 part by weight of benzoin isobutyl ether. parts by weight and 20 parts by weight of distilled water are thoroughly mixed. Add to this 3% sodium alginate aqueous solution 50%
Parts by weight and 0.1M Tris-HCl buffer (PH7.5)
10 parts by weight of a spore suspension of the filamentous fungus Curvularia lunata (ATCC 12017) was uniformly mixed and dispersed. Thereafter, the dispersion was dropped into a 0.2M calcium chloride solution from the tip of a syringe, and the solution turned into a liquid gel. Transfer the spherical gel to a Petri dish, and apply a wavelength of 300 to 300 nm from the top and bottom of the Petri dish.
When irradiated with active light of 400 nm for 3 minutes, granular immobilized spores with a diameter of 3 mm and a compressive strength of 20 Kg/cm ² were obtained.

The granular immobilized spores were cultured aseptically. As a result, spores germinated and grew in the particle gel, and hyphae developed as culture time progressed. This granular immobilized hyphae exhibited hydroxylation activity, and had the same activity as non-immobilized hyphae in the 11β-hydroxylation reaction of steroids.

Claims (1)

[Scope of Claims] 1 (a) a hydrophilic photocurable resin having at least two ethylenically unsaturated bonds in one molecule, (b) a photopolymerization initiator, (c) at least one polyvalent metal A liquid composition comprising a water-soluble polymeric polysaccharide capable of gelling upon contact with ions, and (d) an enzyme, or a microbial cell selected from bacteria and fungi, is mixed with polyvalent metal ions. An enzyme or an enzyme characterized by dropping the composition into a granular gel by dropping it into an aqueous medium containing the enzyme, and then irradiating the resulting granular gel with actinic rays to cure the photocurable resin in the granular gel. A method for producing a granular immobilized molded product of microbial cells.