JPH10168105A - Method for adjusting specific gravity of granular article for immobilizing enzyme or microorganismic cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To adjust specific gravity of the subject article to be lower than that of water by adding inorganic-based fine hollow beads in a specific aqueous liquid composition, dropping them into a specific aqueous medium to be gelled to granules and hardening the granules by irradiating active rays. SOLUTION: This method for adjusting the specific gravity of granular articles for immobilizing enzymes or microorganismic cells is to drop an aqueous liquid composition containing (A) a hydrophilic optically hardening resin having at least two ethylene-based unsaturated bonds in a molecule, (B) a photopolymerization initiator, (C) a water soluble high molecular weight polysaccharide capable of gelling by contacting with alkali (polyvalent) metal ions and (D) inorganic fine hollow beads consisting of hollow glass beads or hollow Celite, etc., having 1-200μm average particle size and 0.3-0.7 specific gravity to bring the composition to be gelled to granules, then irradiate active rays to the granulated gel to harden the component A in the gel to obtain the granular articles for immobilizing enzymes or microorganismic cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for adjusting the specific gravity of a granular molded product for immobilizing enzymes or microorganisms.

[0002]

2. Description of the Related Art As methods for immobilizing enzymes or microorganisms, many methods such as an entrapment method, a physical adsorption method, and a covalent bonding method have been known. When used in a microbial reaction or an enzymatic reaction, the solid or sheet-like immobilized product obtained by these methods is usually cut into small pieces or ground, and then packed into a column. However, in this case, the immobilized substance often comes into close contact with each other, resulting in poor microbial reaction and enzymatic reaction efficiency, and often has a drawback that a channeling phenomenon occurs and the column is clogged.

[0003] For this reason, recently, by immobilizing enzymes or microbial cells as granular moldings, they are easy to flow, easy to pack into columns, have a small contact area between particles, and have a microbial reaction or enzyme. It has been proposed to increase the efficiency of the reaction (for example, Japanese Patent Publication No. Sho 62-198).
No. 37). However, a granular molded product mainly composed of a conventional photocurable resin for immobilizing enzymes or microbial cells is generally 1.0 to 1.03 as water even after immobilization.
Because of having a specific gravity within the range, when fluidized for reaction in a fluidized bed reactor, particularly a large reactor or a reactor having a complicated structure, it is easy to move downward,
There is a problem that it is difficult to make it flow uniformly.

[0004] In order to solve this problem, there is a method of adding plastic beads having a low specific gravity to the particles. However, the particles cannot be used as they are because the particles aggregate in an aqueous medium, and the surface of the plastic beads becomes hydrophilic. Treatment was required. For this reason, the cost is increased and it is not practical at present.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to reduce the specific gravity of a granular molded product for immobilizing enzymes or microbial cells to 1.0 or less, which is lower than that of water, so that the uniformity of the granular molded product in a reactor can be improved. It is an object of the present invention to provide a method capable of arbitrarily adjusting the specific gravity of a granular molded product in order to ensure excellent fluidity.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object, and as a result, added inorganic micro hollow beads to the composition of a granular molded product for immobilizing enzymes or microorganisms. As a result, it has been found that the specific gravity of the granular molded product can be easily adjusted, and the present invention has been completed.

Thus, according to the present invention, (a) a hydrophilic photocurable resin having at least two ethylenically unsaturated bonds in one molecule, (b) a photopolymerization initiator, and (c) an alkali metal An aqueous liquid composition containing a water-soluble polymer polysaccharide capable of gelling by contact with ions or polyvalent metal ions is dropped into an aqueous medium containing alkali metal ions or polyvalent metal ions. The composition is gelled in a granular form, and then the obtained granular gel is irradiated with actinic rays to cure the photocurable resin in the granular gel to produce a granular molded article for immobilizing enzymes or microbial cells. In this case, there is provided a method for adjusting the specific gravity of a granular molded product for immobilizing an enzyme or a microbial cell, characterized by adding inorganic micro hollow beads (d) to the liquid composition.

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

(A) Photo-curable resin In the present invention, a photo-curable resin having at least two ethylenically unsaturated bonds in one molecule used for producing a granular carrier for immobilizing enzymes or microbial cells. Is generally 300 to 30,000, preferably 500 to 2,000
An ionic or non-ionic hydrophilic group having a number average molecular weight in the range of 0 and sufficient to be uniformly dispersed in the aqueous medium;
For example, hydroxyl group, amino group, carboxyl group, phosphate group,
It contains a sulfonic acid group, an ether bond, etc., and has a wavelength of about 2
When irradiated with an actinic ray having a wavelength in the range of 50 to about 600 nm, a resin which cures and becomes a water-insoluble resin is preferably used. As such a photocurable resin, those already known as immobilization carriers for entrapment immobilization can be used (for example, JP-B-55-40, JP-B-55-20676, See Japanese Patent Publication No. 62-19837). Representative examples include those described below.

(I) Compound having a photopolymerizable ethylenically unsaturated group at both ends of polyalkylene glycol:
For example, polyethylene glycol di (meth) acrylates obtained by esterifying the hydroxyl groups at both terminals of 1 mol of polyethylene glycol having a molecular weight of 400 to 6000 with 2 mol of (meth) acrylic acid.

Polypropylene glycol di (meth) acrylates having a molecular weight of 200 to 4000, wherein the hydroxyl groups at both terminals of 1 mol of polypropylene glycol are esterified with 2 mol of (meth) acrylic acid.

The hydroxyl groups at both terminals of 1 mole of polyethylene glycol having a molecular weight of 400 to 6000 are urethanized with 2 moles of a diisocyanate compound such as tolylene diisocyanate, xylylene diisocyanate, or isophorone diisocyanate, and then immiscible with 2-hydroxyethyl (meth) acrylate or the like. An unsaturated polyethylene glycol urethane compound to which 2 mol of a saturated monohydroxyethyl compound has been added.

The hydroxyl groups at both terminals of 1 mole of polypropylene glycol having a molecular weight of 200 to 4000 are urethanized with 2 moles of a diisocyanate compound such as tolylene diisocyanate, xylylene diisocyanate, or isophorone diisocyanate. Unsaturated polypropylene glycol urethanates to which 2 moles of a saturated monohydroxy compound are added, and the like.

(Ii) High acid value unsaturated polyester resin:
The acid value obtained by esterification of a polyhydric carboxylic acid component containing an unsaturated polycarboxylic acid with a polyhydric alcohol is 40 to
200 unsaturated polyester salts and the like.

(Iii) High acid value unsaturated epoxy resin: An acid value obtained by adding an acid anhydride to a hydroxyl group remaining in an addition reaction product of an epoxy resin and an unsaturated carboxyl compound such as (meth) acrylic acid. 40 to 200 unsaturated epoxy resins and the like.

(Iv) Anionic unsaturated acrylic resin:
Copolymer containing carboxyl group, phosphoric acid group and / or sulfonic acid group obtained by copolymerizing at least two kinds of (meth) acrylic monomers selected from (meth) acrylic acid and (meth) acrylic acid ester Such as a resin into which a photopolymerizable ethylenically unsaturated group is introduced.

(V) Unsaturated polyamide: An adduct of a diisocyanate such as tolylene diisocyanate or xylylene diisocyanate with an ethylenically unsaturated hydroxy compound such as 2-hydroxyethyl acrylate is subjected to an addition reaction to a water-soluble polyamide such as gelatin. Unsaturated polyamide and the like.

The photo-curable resins as exemplified above can be used alone or in combination of two or more.

Of these photocurable resins, the present invention
And the ones that can be used particularly advantageously are the above-mentioned (i) polymers.
Ethylenic photopolymerizable at both ends of the alkylene glycol
A compound having an unsaturated group.
Is ENT-1000, EN from Kansai Paint Co., Ltd.
T-2000, ENT-4000, ENTG-200
0, sold under trade names such as ENTG-3800
Can be mentioned. (B) Photopolymerization initiator Promotes the photopolymerization reaction of the photocurable resin described in (a) above.
For the purpose, a photopolymerization initiator is added to the liquid composition according to the present invention.
Include. The photopolymerization initiator that can be used is separated by light irradiation.
Dissociated to form radicals, which become polymerization initiation species
Crosslinking reaction between resins containing polymerizable unsaturated groups
For example, α-carbo such as benzoin
Acyls such as benzoin ethyl ether
-Tels: polycyclic aromatic compounds such as naphthol;
Α-substituted acyloins such as rubenzoin; 2-cyano
Azoamide compounds such as -2-butylazoformamide
And the like.

(C) Water-Soluble Polymer Polysaccharide The water-soluble polymer polysaccharide used in the present invention is water-soluble, and becomes water-soluble when it comes into contact with an alkali metal ion or a polyvalent metal ion in an aqueous medium. A polymeric polysaccharide capable of transforming into an insoluble or poorly soluble gel, generally having a number average molecular weight in the range of about 3000 to about 2,000,000, and contacting with an alkali metal ion or a polyvalent metal ion. Usually at least about 10
Those exhibiting a solubility of g / l (25 ° C.) are preferably used.

Specific examples of the water-soluble polysaccharide having such properties include alkali metal salts of alginic acid and carrageenan.

In the case of carrageenan, these water-soluble high molecular weight polysaccharides are dissolved in an aqueous medium, and then contacted with an alkali metal ion such as potassium ion or sodium ion, and the alkali metal salt of alginic acid is dissolved. In case, magnesium ion, calcium ion,
It can be gelled by contact with at least one polyvalent metal ion of alkaline earth metal ions such as strontium ion and barium ion; or other polyvalent metal ions such as aluminum ion, cerium ion and nickel ion. Things. The concentration of the alkali metal ion or polyvalent metal ion at which gelation occurs varies depending on the type of the water-soluble polymer polysaccharide and the like, but is generally 0.01 to 5 mol.
1 / l.

(D) Inorganic Micro Hollow Beads (d) The micro hollow beads (d) used for adjusting the specific gravity of the granular molded product according to the present invention preferably contain silicon as a main component, especially silicate or The particles can be completely closed micro-hollow particles containing alumina silica as a main component and having a hollow body structure in the particles and having a specific gravity in the range of 0.3 to 0.7. Specific examples include hollow glass beads and hollow celite.

The micro hollow beads (d) are generally from 1 to 20
0 .mu.m, preferably can have an average particle size in the range of 3 to 50 [mu] m, also hollow size varies depending desired specific gravity, typically of a diameter of the bead ^_1/2
Or less, preferably advantageously at _^1/3 or less.

The ones hollow diameter is greater than _^1/2, destroy or compression strength immobilization molded particles in the resulting may be lowered.

The micro hollow beads (d) are as described in the above (a),
By adding 0.1 to 10 parts by weight per 100 parts by weight of the component (a) in the aqueous liquid composition comprising the components (b) and (c), the specific gravity of the obtained granular molded product can be reduced. It can be adjusted to fall within the range of 0.90 to 1.00.

As the micro hollow beads, commercially available products can be used. Representative examples of the micro hollow beads are given by trade names, for example, Filite 200/7 and 300/7 (Nippon Ferrite Co., Ltd., alumina / silica system), Sunturite YO
2, YO4, YOC (manufactured by Sanki Kogyo, alumina / silica), Winlight MSB-5021, MSB-501
1, SB-9011, SC-50 (alumina / silica-based, manufactured by Ichi Kasei Kogyo Co., Ltd.), ZEEOSPHERES2
00, 400, 600, 800, 850 (Zeelan Indus
tries INC, alumina / silica-based), Glass Bubbles E
22X, C15 / 250, B23 / 500, B28 / 7
50, B37 / 2000, B38 / 4000, B46 /
4000, S60 / 10000 (Minnesota Mining and MFG, borosilicate glass) Fuji Balloon S-
35, S-40, S-45, H-30, H-35, H-
40 (manufactured by Fuji Silysia Chemical Ltd., borosilicate glass) and the like.

The specific gravity of the granular molded product for immobilizing the enzyme or the microorganism of the present invention is adjusted by adding the aqueous liquid composition comprising the components (a), (b) and (c) to a desired specific gravity. This is performed by adding micro hollow beads (d) and dropping them into an aqueous medium containing an alkali metal ion or a polyvalent metal ion to granulate them.

The proportion of each of the above components (a), (b), (c) and (d) is not strictly limited, and may be varied over a wide range according to the type of each component. In general, it is appropriate to use the components (b) to (d) at the following ratios with respect to 100 parts by weight of the hydrophilic photocurable resin of the component (a). is there).

(B) Photopolymerization initiator: 0.5 to 5 parts by weight (1 to 3 parts by weight) (c) Water-soluble polymer polysaccharide: 0.5 to 15 parts by weight (1
(D) micro hollow beads: 0.1 to 10 parts by weight.

The components (a) to (d) described above are dissolved or dispersed in an aqueous medium to prepare an aqueous liquid composition. Generally, the solid content concentration of the liquid composition is suitably in the range of 5 to 30% by weight.

The aqueous liquid composition thus prepared is then dropped into an aqueous medium containing an alkali metal ion or a polyvalent metal ion of the kind described above, whereby the liquid composition is formed in a granular form. Gelled.

The dropping of the aqueous liquid composition into an aqueous medium containing an alkali metal ion or a polyvalent metal ion can be performed, for example, by dropping the liquid composition from the tip of a syringe or by centrifuging the liquid composition. A method of scattering the composition into particles,
It can be carried out by a method such as a method of atomizing the liquid composition from the tip of the spray nozzle to make it into granules and dropping it. The size of the droplet to be dropped can be freely changed according to the particle size desired for the final particulate immobilized product, but usually the diameter is about 0.1 to about 5 mm, preferably about 0.5 to about 5 mm. 4
It is expedient to drop it as droplets in the range of mm.

The granular gel produced as described above is
By irradiating with an actinic ray in a state of being dispersed in an aqueous medium as it is or after being separated from the aqueous medium, the hydrophilic photocurable resin in the granular gel is cured. As a result, the granular gel is substantially insoluble in water, and a granular molded product for immobilizing enzymes or microorganisms having high mechanical strength can be obtained.

The wavelength of the actinic ray that can be used for the photocuring varies depending on the type of the photocurable resin contained in the granular gel, but is generally within a range of about 250 to about 600 nm. Is advantageously 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 according to the light intensity of the light source, the distance from the light source, and the like, but can be generally in the range of about 0.5 to about 10 minutes.

The granular gel after the irradiation treatment as described above can be washed with water or a buffered aqueous solution and stored as it is or by freeze-drying.

The enzyme- or microbial cell-fixed granular molded article of which specific gravity has been adjusted according to the present invention has a surface structure particularly suitable for adhesion of microorganisms, and can adhere a large amount of microorganisms. The microorganism that can be attached to the carrier is not particularly limited, and either an anaerobic microorganism or an aerobic microorganism can be used. The types of microorganisms include aspargillus, penicillium, fusarium and other molds, Saccharomyces, Phaffia, Candida and other yeasts; Zymomonas, Nitrosomonas, Nitrobacter, Paracoccus, Vibrio, Bacteria such as Methanosarcina and Bacillus can be mentioned.

The above microorganisms and enzymes may be preliminarily mixed and immobilized in an aqueous liquid comprising the components (a), (b), (c) and (d).

Thus, according to the present invention, it is possible to easily convert a particulate immobilized product having a specific gravity substantially equal to that of conventional water to a product having a specific gravity lower than that of water without changing its performance. Moreover, the granular immobilized product provided by the present invention is excellent in fluidity in water, and therefore is most suitable for use in a fluidized-bed bioreactor or a stirred fermenter, etc. It can also be applied to bioreactors, fermenters and the like.

[0040]

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

Example 1 2000 g of polyethylene glycol having a molecular weight of about 4000
And 1 mol of isophorone diisocyanate (222 g) and 1 mol of 2-hydroxyethyl methacrylate (130 mol
Photocurable (resin) prepolymer 1 comprising the mixture of g)
00 parts by weight, 2 parts by weight of benzoin isobutyl ether, 100 parts by weight of a 2% aqueous sodium alginate solution, 6 parts by weight of fine hollow glass beads (trade name S35 beads, true specific gravity 0.35, manufactured by Fuji Serial Co., Ltd.) having an average particle diameter of 40 μm; An aqueous liquid composition obtained by thoroughly mixing 100 parts by weight of distilled water was dropped into a 1 molar calcium chloride solution from a liquid level of 10 cm from the tip of a syringe.
Granules having a particle size of about 2 mm were obtained.

The granules are placed in a Petri dish having a flat bottom, and wavelengths of 300 to 40 are applied from the upper and lower faces of the Petri dish.
Irradiation with an actinic ray of 0 nm for 3 minutes gave a specific gravity of 0.9.
3 and a molded article for fixation having a compressive strength of 20 kg / cm ² were obtained.

Example 2 Polyethylene glycol 2000 (molecular weight 2000) 1
100 g of a photocurable (resin) prepolymer consisting of 000 g and 1 mol of methyl methacrylate is mixed with 100 parts of distilled water.
After the addition of parts by weight, the mixture was heated to about 50 ° C. and mixed well to obtain a uniform aqueous resin solution, to which 2 parts by weight of benzoin ethyl ether was added and mixed and dissolved.

To this resin mixture, 75 parts by weight of a 3% k-carrageenan aqueous solution, 2.5 parts by weight of the same microscopic hollow glass beads used in Example 1, and a 2% glycosyl isomerase enzyme solution (sodium bicarbonate buffer solution pH 8) ) 25 parts by weight were added to prepare a uniform mixed solution. This homogeneous mixture was dropped into a 5% aqueous solution of potassium chloride from the tip of the syringe from a position 20 cm from the liquid surface to obtain granules having a particle size of 1.5 mm.

The granules were transferred to a Petri dish having a flat bottom together with a potassium chloride solution so as to form a single layer, and a depth of 2
Irradiation with an actinic ray having a wavelength of 300 to 400 nm from the upper surface for 3 minutes and from the lower surface for 3 minutes so that the particulate matter is present in the aqueous solution having a specific gravity of 0.95 and a compressive strength of 3
0 kg / cm ² of granular immobilized enzyme was obtained.

Example 3 1 in 500 g of polyvinyl alcohol having 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 0 mol of N-methylolacrylamide,
% K-carrageenan aqueous solution and 5 parts by weight of hollow alumina silica having an average particle diameter of 75 μm (trade name, Filite 300/7, true specific gravity 0.7, manufactured by Nippon Ferrite Co., Ltd.) were uniformly mixed and dispersed. The solution was dropped into a potassium chloride solution having a molar concentration of 0.5 mol and gelled. Put this in a Petri dish, and simultaneously
Irradiate with light of 00 to 400 nm for 3 minutes, particle size 3 mm,
A granular molded product for immobilization having a specific gravity of 0.97 and a compressive strength of 10 kg / cm ² was produced.

[0047]

According to the method of the present invention for adjusting the specific gravity of a granular molded product for immobilizing an enzyme or a microorganism, the specific gravity of the granular molded product can be easily adjusted to 1.0 or less, as is apparent from the above-mentioned examples. Can be.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 11/08 C12N 11/08 A // C08F 299/00 C08F 299/00

Claims (3)

[Claims] (1) a hydrophilic photocurable resin having at least two ethylenically unsaturated bonds in one molecule, (b)
An aqueous liquid composition comprising a photopolymerization initiator and (c) a water-soluble polymer polysaccharide capable of gelling by contact with an alkali metal ion or a polyvalent metal ion is added to the alkali metal ion or the polyvalent metal. The composition is granulated by dropping into an aqueous medium containing ions, and then the resulting granular gel is irradiated with an actinic ray to cure the photocurable resin in the granular gel, thereby producing an enzyme or microorganism. A method for adjusting the specific gravity of an enzyme or microbial cell-immobilized granular molded product, which comprises adding an inorganic micro hollow bead (d) to the liquid composition in producing the bacterial cell-immobilized granular molded product. . 2. The inorganic fine hollow beads (d) are hollow glass beads or hollow celite having an average particle diameter of 1 to 200 μm and a specific gravity of 0.3 to 0.7.
The described method. 3. The amount of the inorganic fine hollow beads (d) added to the hydrophilic photocurable resin (a) 1 in the aqueous liquid composition
3. The method according to claim 1, wherein the amount is in the range of 0.1 to 10 parts by weight based on 00 parts by weight.