JPH0661265B2 - Method for producing granular fixed molded article of enzyme or microbial cell - Google Patents

Description

The present invention relates to a method for immobilizing an enzyme or a microbial cell, and more particularly to a method for immobilizing an enzyme or a microbial cell as a granular immobilization molding.

As an enzyme or microorganism immobilization method, many methods such as entrapment method, physical adsorption method, covalent bond method and the like have been conventionally obtained. 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. However, in that case, the immobilized product often adheres to each other face-to-face, and the efficiency of microbial reaction and enzyme reaction becomes poor, and there are drawbacks such that the channeling phenomenon frequently occurs and the column is blocked.

If the present inventors were able to immobilize the enzyme or microbial cell as a granular molded product, the flowability and the filling work into the column would be easy, and the contact area between the particles would be small and the efficiency of microbial reaction and enzyme reaction would be small. It is believed that the mixture of hydrophilic photocurable resin and water-soluble polymer polysaccharide is used, and the mixture is added dropwise to an aqueous medium containing polyvalent metal ions and granulated. The present inventors have succeeded in easily obtaining a granular material on which an enzyme or a bacterium has been fixed by photocuring (see JP-A-59-11182). However, it is difficult to obtain a particulate immobilization product by the above method using a hydrophobic photocurable resin, and it has been conventionally used in the form of a sheet or a lump.

Therefore, as a result of further research on obtaining a granular immobilizate using a hydrophobic resin, the present inventors have found that a certain kind of hydrophobic photocurable resin as an immobilization carrier and a surfactant are contained. It has been found that a stable granular immobilization product can be produced by dropping the resin in an aqueous medium and forming the micelle on the surface of the resin, and the present invention has been completed.

Therefore, according to the present invention, (a) a hydrophobic photocurable resin having at least two ethylenically unsaturated bonds in one molecule (b) a photopolymerization initiator (c) a hydrophobic photocurable resin A liquid composition comprising a solvent that can be diluted and (d) an enzyme or a microbial cell is dropped into an aqueous medium containing a surfactant to granulate the composition, and then the resulting granules are obtained. There is provided a method for producing a granular fixed molded article of an enzyme or a microbial cell, which comprises irradiating an actinic ray to the article to cure the photocurable resin in the particulate matter.

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

(A) Hydrophobic photocurable resin (i) At least one kind of unsaturated polycarboxylic acid such as maleic anhydride, maleic acid, fumaric acid, itaconic acid, and itaconic anhydride, or these and trimellitic acid, trimellitic anhydride Unsaturated acid having an acid value of less than about 40 obtained by esterification of a polyvalent carboxylic acid component composed of at least one saturated polycarboxylic acid such as acid, pyromellitic acid and pyromellitic dianhydride, and a polyhydric alcohol Polyester (number average molecular weight is about 1000 or more, preferably about 300
0 or more).

(ii) at least one unsaturated polyvalent carboxylic acid such as maleic anhydride, maleic acid, fumaric acid, itaconic acid, and itaconic anhydride, and at least 5 polyhydric alcohols having more than 3 hydroxyl groups in one molecule. An unsaturated polyester having an acid value of less than 40 (number average molecular weight of about 1000 or more, which is obtained by reacting an acid anhydride with a hydroxyl group in the esterified product or a polyhydric alcohol containing 50% by weight,
Preferably about 3000 or more).

(iii) Unsaturated epoxides; polyvalent glycidyl compounds such as Epicoat 828,1001,1004 (trade name, manufactured by Shell Chemical Co.) having n moles of glycidyl group, and maleic acid having (n-2) moles of carboxyl group. ,
Polycarboxylic acids such as adipic acid and trimellitic acid and 2
Moles of an addition reaction product with an unsaturated carboxyl compound such as (meth) acrylic acid or unsaturated epoxides having an acid value of less than 40 in which an acid anhydride is added to the residual hydroxyl group; An unsaturated glycidyl compound such as glycidyl (meth) acrylate is added to a compound obtained by adding an acid anhydride to a hydroxyl group remaining in an addition reaction product of a polyvalent glycidyl compound and the polyvalent carboxylic acid having (n + 2) moles of a carboxyl group. Unsaturated epoxides having an acid value of less than 40 reacted with a compound.

(iv) Anionic unsaturated acrylic resin; the anionic unsaturated acrylic resin here is a copolymer of (meth) acrylic acid and (meth) acrylic acid ester, and C + 5P + 10S = A (1) [Where 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). ] In the above formula (1), A is less than 0.8 (mol / kg), and the concentration of the photopolymerizable ethylenically unsaturated group in the resin is 0.1 to 5 (m
ol / kg). The copolymer of (meth) acrylic acid and (meth) acrylic acid ester is synthesized by a known method. To introduce a carboxyl group into the resin,
To introduce an unsaturated carboxyl compound such as (meth) acrylic acid into a phosphoric acid group, phosmer M and phosmer C1
In order to introduce a sulfonic acid group into an unsaturated phosphoric acid ester (trade name, manufactured by Oil and Fat Products Co., Ltd. with both) (meta)
2-Sulfoethyl acrylate, (meth) acrylic acid-
In order to introduce an ethylenically unsaturated group capable of copolymerizing an unsaturated sulfonic acid ester such as 3-sulfopropyl, the (meth) carboxyl group, phosphoric acid group or sulfonic acid group present in the copolymer is introduced. It is possible by reacting an unsaturated glycidyl compound such as glycidyl acrylate.

(v) Cationic unsaturated acrylic resins; an unsaturated amino compound such as 2-diethylaminoethyl (meth) acrylate, -tert-butylaminoethyl (meth) acrylate, vinylpyridine or the like in an amount of less than 5% by weight. Unsaturated acrylic resin obtained by reacting an unsaturated glycidyl compound such as glycidyl (meth) acrylate with a copolymer of (meth) acrylic acid ester containing, chloromethylated polystyrene, and then quaternized with an unsaturated amino compound. Examples thereof include saturated acrylic resins and adducts of polyethyleneimine with unsaturated glycidyl compounds.

(vi) Polyesters of polypropylene glycol and (meth) acrylic acid; diesters of polypropylene glycol having a molecular weight of 400 to 10,000 and containing less than 30% by weight of ethylene oxide groups with unsaturated monocarboxylic acids such as (meth) acrylic acid.

(vii) Urethane adduct of polypropylene glycol and 2-hydroxyethyl (meth) acrylate; diisocyanates such as n mol tolylene diisocyanate, xylylene diisocyanate, hexamethylene diisocyanate and isophorone diisocyanate, n-1 mol polypropylene glycol. And a urethane compound with 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, 2 mol of polypropylene glycol and 2 Carboxylated urethanization by reacting a mole of a urethane compound with an unsaturated monohydroxy compound such as 2-hydroxyethyl (meth) acrylate with an acid anhydride such as maleic anhydride or succinic anhydride And the like.

The hydrophobic photocurable resins as exemplified above can be used alone or in combination of two or more kinds. Among these photocurable resins, those which can be particularly advantageously used in the present invention include the above (vii)
Polypropylene glycol and (meth) acrylic acid 2-
The urethane addition product with hydroxyethyl can be mentioned.

(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 and α-hydroxyisobutylphenone; benzoin methyl ether, benzoin ethyl ether, benzoin propyl ether,
Acryloin ethers such as anisoin ethyl ether and piparoin ethyl ether; polycyclic aromatic compounds such as naphthol and hydroxyanthracene; α-substituted acylloins such as methylbenzoin and α-methoxybenzoin; 2-cyano-2- Azoamide compounds such as butylazoformamide; metal salts such as lauryl nitrate and ferric chloride; mercaptans; disulfides; halogen compounds; dyes and the like.

These photopolymerization initiators can be used singly or in combination of two or more kinds at a ratio of usually 0.01 to 10 PHR (parts by weight / 100 parts by weight of resin).

(C) Solvent capable of diluting hydrophobic photocurable resin Among hydrophobic photocurable resins, those having high viscosity (10 poise
The above) can be granulated, but the granulation operation is difficult. Therefore, the hydrophobic photocurable resin can be dissolved, but it can be easily granulated by diluting with a solvent that does not dissolve the immobilized polymer. Examples of the solvent include organic solvents such as petroleum benzine, hexane, heptane, benzene, petroleum ether, toluene, ethyl acetate, acetone, alcohol, dioxane, ethylene glycol, formamide, pyridine, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide; oleic acid. , Fatty acids such as linoleic acid and linolenic acid; fats and oils such as olive oil, palm oil, and coconut oil; and the like can be used. These may be used alone or as a mixed solution of two or more kinds. good. Preferably, it is desirable to dilute with the substrate in the reaction. For example, when the hydrolysis reaction of olive oil is performed, the hydrophobic photocurable resin is diluted with olive oil to reduce the viscosity.

(D) Enzymes or microbial cells There are various types of enzymes or microbial cells that can be immobilized by the method of the present invention, and the types are not particularly limited. According to the method of the present invention, any type of enzyme or microbial cell can be immobilized without substantially deactivating the enzyme activity. Enzymes and microbial cells to which the method of the present invention can be applied are shown below, but this is only one example.

Examples of microbial organisms are Lactobacillus bulgaris Aerobactor aerogenes Bacillus subtillis Azotobacter vinelandii Proteus vulgaris Proteus vulgaris・ Simplex (Arthrobacter simplex) Nocardia rhodocrous (Curvularia lunata) Candida cylindracea (Aspergills niger) Rhizopus o candidum) Penicillium cyclopium etc. can be mentioned.

Examples of enzymes include glucose oxidase (1, 1, 3, 4) aldehyde oxidase (1, 2, 3, 1) L-amino acid oxidase (1, 4, 3, 2) D-amino acid oxidase (1, 4, 3, 3) Catalase (1, 11, 1, 6) Glucokinase (2, 7, 1, 2) Carboxysterase (3, 1, 1, 1) Aryl esterase (3, 1, 1, 2) lipase (3,1,1,3) Phospholipase A (3,1,1,4) Lysolectin acylhydrase (3,1,1,5) Acetylesterase (3,1,1,6) Pectinesterase (3, 1,1,11) Cholesterol esterase (3,1,1,13) Glucoamylase (3,2,1,3) 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) Amitase (3, 5, 1, 4) Urease (3, 5, 1, 5) Penicillin acitase (3, 5, 1, 11) aminoacylase (3, 5, 1, 14) aldolase (4, 1, 2, 13) aspertase (4, 3, 1, 1) glycol-6-phosphate isomerase (5, 3, 1,
9) Steroid Δ-isomerase (5, 3, 3, 1) and the like can be mentioned.

Preparation of Liquid Composition The components (a), (b), (c) and (d) described above can be made into a liquid composition by sufficiently mixing them. The enzyme or microbial cell of (d) may be dispersed as a bulk powder or a dried microbial cell, or may be used as an enzyme solution or a microbial suspension in the form of an emulsion. In addition, an enzyme or a microorganism adsorbed in advance; for example, Celite, quartz sand, clay, activated carbon, colloidal silica, alumina or the like may be dispersed and used.

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,
In general, it is appropriate to use the following proportions with respect to 100 parts by weight of the hydrophobic photocurable resin as the component (a) (the preferred range is in parentheses).

(B) Photopolymerization initiator: 0.1 to 5 parts by weight (0.5 to 2 parts by weight) (c) Solvent capable of diluting the hydrophobic photocurable resin: 0 to 1000 parts by weight (10 to 200) (Parts by weight) (d) Enzyme or microbial cells: 0.001 to 50 parts by weight (0.01 to 20 parts by weight) Granulation The liquid composition prepared as described above is then an aqueous solution containing a surfactant. Granulate by dropping into the medium. Granulation is possible without adding a surfactant to the aqueous medium, but if the surfactant is not added, most of it spreads in the aqueous medium and granulation is extremely difficult. In addition, even granulated materials may be fused with each other.
However, when a surfactant is added to the aqueous medium, stable granules are formed due to the surface tension of the dropped liquid composition and the action of forming micelles on the surface of the liquid composition.
That is, by adding the surfactant to the aqueous medium, neither the liquid composition is granulated in the aqueous medium nor the granulation products are fused together, and the liquid composition can be easily granulated.

The surfactant can be used without particular limitation. Examples thereof include sodium lauryl sulfate, glycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, soybean phospholipid, propylene glycol fatty acid ester, sodium alkylbenzene sulfonate, sodium alkyl ether sulfate, and polyethylene glycol octyl phenyl ether.

At this time, the concentration of the surfactant in the aqueous medium is generally 0.
0001 to 10% by weight, preferably 0.01 to 3% by weight
Is the range.

The dropping of the liquid composition into the aqueous medium containing the surfactant is a method of dropping the liquid composition from the tip of a thin tube such as an injection needle. It can be carried out by a method such as a method of atomizing the liquid composition and dropping it in the form of particles from the tip of the spray nozzle. The size of the droplets to be dropped can be freely changed according to the desired particle size of the final particulate immobilized product, but usually the diameter is about 0.1
Conveniently it is applied as a droplet of about 5 mm, preferably about 0.5 to about 3 mm.

The dropped liquid composition is immediately granulated when dropped in an aqueous medium containing a surfactant. The temperature in this granulation is usually room temperature, but if necessary, the granulation may be carried out under heating to such an extent that the enzyme or microbial cell is not inactivated, or may be carried out under cooling.

Photocuring The granular product produced as described above is irradiated with an actinic ray in a state of being dispersed in an aqueous medium as it is, thereby curing the hydrophobic photocurable resin in the granular product to form a granular gel. . As a result, a granular immobilized product of an enzyme or microbial cell having high mechanical strength can be obtained.

The wavelength of the actinic ray that can be used for the photo-curing varies depending on the type of the photo-curable resin contained in the granular material,
It is generally advantageous to use a light source which emits light with a wavelength in the range from about 250 to about 600 nm for irradiation. 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. If the irradiation is performed in an inert gas atmosphere, the irradiation time may be shortened.
Irradiation should be such that the actinic rays are distributed as evenly as possible over all the granules produced.

In this way, the particulate immobilization product after the irradiation treatment is separated and collected from the aqueous medium, washed with water, an aqueous buffer solution or an organic solvent that does not dissolve the granular immobilization product, and stored as it is, or the granular product is freeze-dried and stored. can do.

Thus, according to the present invention, a granular immobilization product of enzyme or microbial cells having a particle size of about 0.1 to about 5 mm can be produced by an extremely simple operation, and continuous production is also possible.

According to the method of the present invention, it is possible to easily carry out the granular immobilization of the enzyme or the microbial cell by the hydrophobic photocurable resin carrier, which has been difficult so far. It is possible to achieve an improvement in reaction rate based on an increase in the permeation / diffusion property of the substrate in the microbial cell reaction, and it can be conveniently used particularly in a reactor in which the scale of the reaction apparatus is not so large, a fluidized bed reactor, and the like.

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

Example 1 Polypropylene glycol 2 having a number average molecular weight of about 4000
000 g and 1 mol of isophorone diisocyanate (222
g) and 100 parts by weight of a photocurable (resin) prepolymer consisting of a mixture of 1 mol (130 g) of 2-hydroxyethyl methacrylate, 0.5 parts by weight of α-hydroxyisobutylphenone and 60 parts by weight of coconut oil. To do. To this, 5 parts by weight of enzyme lipase (as a raw powder) was added and well dispersed, and the resulting photocurable resin-enzyme mixture solution was added to 1 part.
When the mixture was continuously dripped into a wt% -sodium lauryl sulfate aqueous solution from a syringe needle at the tip of the syringe from a liquid surface height of 10 cm, a granule having a particle diameter of about 2 mm was obtained.

Wavelength 300-400 nm from top and bottom
When irradiated with the actinic rays of 5 minutes, the compressive strength is 50 kg / cm ²
The granular immobilized enzyme was obtained.

In a mixed solution of 80 parts by weight of coconut oil and 20 parts by weight of water, 5 parts by weight of the particulate immobilized product was added, and after 7 hours at 30 ° C. and 600 rpm, 95% of coconut oil was hydrolyzed.

Example 2 n-Butyl acrylate 619 parts by weight, acrylic acid 136
A copolymer of 1 part by weight and 103 parts by weight of styrene was reacted with 142 parts by weight of glycidyl methacrylate to obtain 10 parts by weight of a photocurable resin having a resin acid value of 45, a degree of unsaturation of 10 and a number average molecular weight of 30,000, and benzoin ethyl ether. After adding 0.1 part by weight, the solution was neutralized with 1.0 equivalent of aqueous ammonia, to which 3 parts by weight of benzene and 3 parts by weight of n-heptane were added and well stirred. To this, 1.5 parts by weight of Nocardia rhodoclass suspended in 0.1 M phosphate buffer was added and well stirred to obtain a photocurable resin-microbial cell mixture. When this mixed solution was dropped into a 0.5% by weight polyethylene glycol octylphenyl ether aqueous solution from an injection needle at the tip of a syringe from a liquid level of 10 cm, the particle size was about 2
Granules of mm were obtained.

Wavelength 300-400 nm from top and bottom
When irradiated with active light for 5 minutes, the compressive strength is 30kg / cm ²
Granules of immobilized microorganisms were obtained.

Claims (1)

[Claims] 1. A method of diluting (a) a hydrophobic photocurable resin having at least two ethylenically unsaturated bonds in one molecule, (b) a photopolymerization initiator, and (c) a hydrophobic photocurable resin. A liquid composition comprising a solvent capable of (d) an enzyme or microbial cells is dropped into an aqueous medium containing a surfactant to make the composition into particles, and then the resulting particles are provided with an actinic ray. To cure the photocurable resin in the particulate matter by irradiating with the enzyme.