JP3775706B2 - Method for producing granular molded product for enzyme or microbial cell immobilization - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a granular molded product for immobilizing enzymes or microbial cells.
[0002]
[Prior art]
As an enzyme or microorganism immobilization method, conventionally, many methods such as a comprehensive method, a physical adsorption method, and a covalent bond method are known. In the case of using in a microbial reaction or an enzyme reaction, a block or solid immobilization product obtained by these methods is usually finely cut or ground and then packed into a column. However, in this case, the immobilized product often comes into close contact with each other, and the efficiency of the microbial reaction and the enzymatic reaction is deteriorated, and the channeling phenomenon is often caused to block the column.
[0003]
For this reason, recently, by immobilizing enzymes or microbial cells as granular products, it is easy to flow, the column can be easily packed, the contact area between the particles is small, and the efficiency of microbial and enzymatic reactions Has been proposed (see, for example, Japanese Examined Patent Publication No. 62-19837). However, the conventional granular molded product mainly composed of a photocurable resin that immobilizes enzymes or microbial cells needs to irradiate light uniformly to the inside of the granular molded product at the time of curing. There is a problem that productivity is reduced because an apparatus is required and there is a limit to the amount of particulate matter that can be processed by one irradiation apparatus.
[0004]
Furthermore, when trying to produce a granular molded product having a large particle size with an average particle size of 5 mm or more, there is also a problem that the center of the granular material exists in an uncured liquid state.
[0005]
[Problems to be solved by the invention]
The object of the present invention is to improve the productivity of granular moldings for immobilizing enzymes or microbial cells, and to produce granular moldings that are completely cured even when the granular moldings have a large particle size. It is to provide a way that can be done.
[0006]
[Means for Solving the Invention]
As a result of intensive studies in order to achieve the above-mentioned object, the present inventors have found that a hydrophilic thermosetting resin as a constituent material of a granular carrier for immobilizing an enzyme or microbial cells using a water-soluble polymeric polysaccharide. It was found that the above-mentioned problems caused by using a photocurable resin can be solved at once by adding a redox-based thermal polymerization initiator to this and curing by heating, and the present invention has been completed. It was.
[0007]
Thus, according to the present invention,
(A) a hydrophilic thermosetting resin having at least two ethylenically unsaturated bonds in one molecule;
(B) a redox thermal polymerization initiator comprising an oxidizing agent and a reducing agent or a part thereof, and (c) a water-soluble polymeric polysaccharide capable of gelling by contact with an alkali metal ion or a polyvalent metal ion. The aqueous liquid composition is dropped into an aqueous medium containing alkali metal ions or polyvalent metal ions to gel the composition into a granular form or continuously on the surface of the aqueous medium for a predetermined time. The droplets are allowed to grow to a desired particle size, and then the droplets are allowed to settle and gel, and then the resulting granular gel is thermally polymerized to thermoset in the granular gel. There is provided a method for producing a granular molded product for immobilizing an enzyme or microbial cell, characterized by curing a resin.
[0008]
Hereinafter, the present invention will be described in more detail.
[0009]
(A) Thermosetting resin In the present invention, thermosetting having at least two ethylenically unsaturated bonds in one molecule used for the production of a granular carrier for immobilizing enzymes or microbial cells. The resin generally has a number average molecular weight in the range of 300 to 30000, preferably 500 to 20000, and is an ionic or nonionic hydrophilic group sufficient to be uniformly dispersed in an aqueous medium, such as a hydroxyl group. , An amino group, a carboxyl group, a phosphoric acid group, a sulfonic acid group, an ether bond and the like are preferably used. As such a thermosetting resin, those already known as a photocurable resin for entrapping immobilization can be used in the same manner (for example, Japanese Patent Publication Nos. 55-40 and 55-20676). Gazette, Japanese Patent Publication No. 62-19837). Typical examples include those described below.
[0010]
(I) a compound having an ethylenically unsaturated group capable of thermal polymerization at both ends of the polyalkylene glycol:
(1) Polyethylene glycol di (meth) acrylates obtained by esterifying both terminal hydroxyl groups of 1 mol of polyethylene glycol having a molecular weight of 400 to 6000 with 2 mol of (meth) acrylic acid.
[0011]
(2) Polypropylene glycol di (meth) acrylates obtained by esterifying both terminal hydroxyl groups of 1 mol of polypropylene glycol having a molecular weight of 200 to 4000 with 2 mol of (meth) acrylic acid.
[0012]
(3) Both moles of hydroxyl groups at 1 mol of polyethylene glycol having a molecular weight of 400 to 6000 are urethanated with 2 mol of diisocyanate compounds such as tolylene diisocyanate, xylylene diisocyanate, isophorone diisocyanate, Unsaturated polyethylene glycol urethanized product added with 2 mol of saturated monohydroxyethyl compound.
[0013]
(4) 1 mol of polypropylene glycol having a molecular weight of 200 to 4000 is urethanated with 2 mol of diisocyanate compounds such as tolylene diisocyanate, xylylene diisocyanate, isophorone diisocyanate, Unsaturated polypropylene glycol urethanized product added with 2 mol of saturated monohydroxy compound.
[0014]
(Ii) High acid value unsaturated polyester resin:
Salts of unsaturated polyesters having an acid value of 40 to 200 obtained by esterification of a polyvalent carboxylic acid component containing an unsaturated polycarboxylic acid and a polyhydric alcohol.
[0015]
(Iii) High acid value unsaturated epoxy resin:
An unsaturated epoxy resin having an acid value of 40 to 200 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.
[0016]
(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 Resin with an ethylenically unsaturated group that can be thermally polymerized.
[0017]
(V) Unsaturated polyamide:
Unsaturated polyamides obtained by addition reaction of diisocyanates such as tolylene diisocyanate and xylylene diisocyanate and ethylenically unsaturated hydroxy compounds such as 2-hydroxyethyl acrylate with water-soluble polyamides such as gelatin.
[0018]
The thermosetting resins as exemplified above can be used alone or in combination of two or more.
[0019]
Among these thermosetting resins, those that can be used particularly advantageously in the present invention are compounds having an ethylenically unsaturated group capable of being thermally polymerized at both ends of the polyalkylene glycol of the above (i). Examples of such products include those sold by Kansai Paint Co., Ltd. under trade names such as ENT-1000, ENT-2000, ENT-4000, ENTG-2000, and ENTG-3800.
(B) Redox thermal polymerization initiator For the purpose of promoting the thermal polymerization reaction of the thermosetting resin described in (a) above, the liquid composition according to the present invention contains a redox thermal polymerization initiator. Make it. As the redox thermal polymerization initiator, conventionally known ones can be used. For example, the redox thermal polymerization initiator comprises a combination of an oxidizing agent and a reducing agent that can perform radical polymerization at a relatively low temperature of about -10 ° C to 50 ° C. A polymerization initiator is preferably used.
[0020]
Examples of the oxidizing agent include organic peroxides such as benzoyl peroxide, methyl ethyl ketone peroxide, dicumyl peroxide, t-butyl perbenzoate and cumene hydroperoxide; peroxodisulfates such as ammonium peroxodisulfate and potassium peroxodisulfate; Hydrogen peroxide etc. are mentioned. Examples of the reducing agent include bisulfites such as sodium bisulfite; divalent iron salts such as ferrous sulfate and ferrous chloride; amines such as N, N-dimethylaniline and phenylmorpholine; Naphthenic acid metal salts such as cobalt naphthenate, manganese naphthenate, and copper naphthenate;
[0021]
Among these redox thermal polymerization initiators, those that can be used particularly advantageously in the present invention are those in which the oxidizing agent is made of peroxodisulfate or hydrogen peroxide, and the reducing agent is made of bisulfite or divalent iron salt. Is a combination of
[0022]
(C) Water-soluble polymer polysaccharide The water-soluble polymer polysaccharide used in the present invention is water-soluble and water when contacted with alkali metal ions or polyvalent metal ions in an aqueous medium. A high molecular weight polysaccharide having the ability to turn into a gel that is insoluble or sparingly soluble, generally having a number average molecular weight in the range of about 3000 to about 2,000,000, and in contact with alkali metal ions or polyvalent metal ions A water-soluble state before being used usually exhibits a solubility of at least about 10 g / l (25 ° C.).
[0023]
Specific examples of the water-soluble polymeric polysaccharide having such properties include alkali metal salts of alginic acid, carrageenan and the like.
[0024]
These water-soluble polymeric polysaccharides are dissolved in an aqueous medium, in the case of carrageenan, by contacting with an alkali metal ion such as potassium ion or sodium ion, and in the case of an alkali metal salt of alginic acid, Contact with at least one polyvalent metal ion of alkaline earth metal ions such as magnesium ion, calcium ion, strontium ion and barium ion; or other polyvalent metal ions such as aluminum ion, cerium ion and nickel ion By doing so, it can be gelled. The concentration of alkali metal ions or polyvalent metal ions that cause gelation varies depending on the type of water-soluble polymer polysaccharide, but is generally in the range of 0.01 to 5 mol / l.
[0025]
The mutual use ratios of the components (a), (b), and (c) are not strictly limited, and can be changed over a wide range according to the type of each component. It is appropriate to use the components (b) and (c) in the following proportions with respect to 100 parts by weight of the hydrophilic thermosetting resin as component a) (the parenthesized values are in a preferred range).
[0026]
(B) Redox thermal polymerization initiator: 0.1 to 5 parts by weight (0.3 to 3 parts by weight)
(C) Water-soluble polymer polysaccharide: 0.5 to 15 parts by weight (1 to 8 parts by weight)
The redox thermal polymerization initiator (b) is used in combination of an oxidizing agent and a reducing agent, and the mixing ratio of the two is generally 5: 1 to 1: 5, preferably 2.5. : It is suitable to be within the range of 1-1 to 2.5.
[0027]
The granular molded article produced by the present invention generally has a specific gravity within the range of 1.0 to 1.03 which is almost the same as that of water, but flows in a fluidized bed large reactor or a reactor having a complicated structure. When used in such a manner, it may be difficult to move uniformly and easily move downward. In such a case, it is necessary to lower the specific gravity of the granular molded product, and the specific gravity of the granular molded product can be lowered by adding inorganic micro hollow beads to the composition of the granular molded product.
[0028]
The inorganic fine hollow beads used for adjusting the specific gravity of the granular molded product are preferably composed mainly of silicon, particularly silicate or alumina silica, and have a specific gravity having a hollow body structure in the particles. Fully closed microhollow particles in the range of 0.3 to 0.7 can be used. Specific examples include hollow glass beads and hollow celite.
[0029]
The micro hollow beads can generally have an average particle size in the range of 1 to 200 μm, preferably 3 to 50 μm, and the size of the hollow depends on the specific gravity desired, but is usually diameter of _^1/2 or less, preferably advantageously at _^1/3 or less.
[0030]
Those hollow diameter is greater than _^1/2, or disrupted by immobilizing molded particles in the resulting compression strength may be lowered.
[0031]
The micro hollow beads should be added at a ratio of 0.1 to 10 parts by weight with respect to 100 parts by weight of the component (a) in the aqueous liquid composition comprising the components (a), (b) and (c). The specific gravity of the obtained granular molded product can be adjusted to be in the range of 0.90 to 1.00.
[0032]
On the other hand, when it is desired to increase the specific gravity of the granular molded product, 0.1 to 30 parts by weight of inorganic particles such as glass beads and fine barium sulfate having a specific gravity of 1 or more with respect to 100 parts by weight of component (a). The specific gravity can be adjusted so that the specific gravity of the granular composition falls within the range of 1 to 1.24 by adding it to the composition of the granular molded product at a ratio.
[0033]
The aqueous liquid composition is prepared by dissolving or dispersing the components (a) to (c) described above and the above-mentioned additives for adjusting the specific gravity, which are added as necessary, in an aqueous medium. . The solid content concentration of the liquid composition is generally in the range of 5 to 30% by weight. In addition, any one of the oxidizing agent or the reducing agent constituting the redox thermal polymerization initiator of the component (b) is, for example, 0% in an aqueous medium containing an alkali metal ion or a polyvalent metal ion. You may make it contain by the density | concentration of 0.05 to 5 weight%, Preferably 0.1 to 2 weight%.
[0034]
The aqueous liquid composition thus prepared is then dropped into an aqueous medium containing alkali metal ions or polyvalent metal ions of the kind described above, or a granular material having an average particle diameter of 5 mm or more. In the case of obtaining the liquid composition, the liquid composition is continuously poured onto the surface of the aqueous medium for a predetermined time to grow the droplets to a desired particle size, and then the droplets are allowed to settle. Granular and gelled.
[0035]
The dropping of the aqueous liquid composition into the aqueous medium containing the alkali metal ion or the polyvalent metal ion is performed by, for example, a method of dropping the liquid composition from the tip of a syringe or the liquid composition using centrifugal force. It can be carried out by a method such as a method of scattering in a granular form, a method of atomizing the liquid composition from the tip of a spray nozzle and dropping it into a granular form. The aqueous liquid composition can be poured onto the surface of the aqueous medium by continuously supplying it as a thin liquid flow from a nozzle opening having a desired pore diameter. The size of the droplets can be freely changed depending on the particle size desired for the final granular immobilization product. However, in the dropping method, the diameter is usually about 0.1 to about 5 mm, preferably about 0.5. It is convenient to drop the liquid droplets in the range of about 4 mm to about 4 mm, and in the case of the pouring method, the liquid droplets are in the range of about 0.5 to 3 cm.
[0036]
The granular gel formed as described above is cured in a state in which it is dispersed in an aqueous medium as it is or after it is separated from the aqueous medium and then thermally polymerized to cure the hydrophilic thermosetting resin in the granular gel. Let me. Thereby, the granular gel is substantially insoluble in water, and a granular molded product for immobilizing microbial cells can be obtained.
[0037]
Since the granular gel contains a redox thermal polymerization initiator, it can be cured until the required mechanical strength is obtained by thermal polymerization even if it is left at room temperature. Accordingly, curing may be performed in a constant temperature atmosphere. The temperature of the constant temperature atmosphere is generally 0 ° C. to 50 ° C., particularly preferably in the range of 20 ° C. to 40 ° C. In order to obtain the required mechanical strength, it is desirable to spend at least 10 minutes to 30 minutes for thermosetting.
[0038]
The granular gel thus cured by thermal polymerization can be washed with water or an aqueous buffer solution and stored as such or freeze-dried.
[0039]
The granular molded product for immobilizing enzymes or microbial cells produced according to the present invention has a surface structure that is particularly suitable for adhesion of microorganisms, and can attach 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. As types of microorganisms, Aspargillus genus, Penicillium genus, Fusarium genus fungi, Saccharomyces genus, Phaffia genus, Candida genus yeasts; Examples include bacteria such as Methanosarcina and Bacillus.
[0040]
The microorganisms and enzymes described above are comprehensively mixed with an aqueous liquid comprising the components (a), (b) and (c) in advance if the thermal polymerization temperature is a low temperature such as room temperature. It may be fixed.
[0041]
Thus, according to the method for producing a granular molded product for immobilization of the present invention, a product having the same performance as that of a conventional photocurable granular fixed product is more productive in the range of an average particle size of 0.1 mm to 30 mm. Can be obtained. Moreover, since the granular molded product provided by the method of the present invention is excellent in fluidity in water, it is most suitable for use in a fluidized bed bioreactor or a stirred fermenter, etc. It can also be applied to floor-type bioreactors, fermenters, and the like.
[0042]
【Example】
Next, the present invention will be described more specifically with reference to examples.
[0043]
Example 1
2000 g of polyethylene glycol having a number average molecular weight of about 4000 and 1 mol (222 g) of isophorone diisocyanate were placed in a reaction vessel and reacted at 80 ° C. for 2 hours. Further, 1 mol (116 g) of 2-hydroxyethyl acrylate and 1 g of hydroquinone were put into a reaction vessel, reacted for 3 hours at 80 ° C. while blowing air, and hydrophilic heat having ethylenically unsaturated bonds at both ends of one molecule. A curable resin was obtained.
[0044]
Aqueous liquid composition obtained by thoroughly mixing 100 parts by weight of the above thermosetting resin, 1 part by weight of ammonium peroxodisulfate, 1 part by weight of anhydrous sodium hydrogen sulfite, 100 parts by weight of 2% aqueous sodium alginate solution and 100 parts by weight of distilled water. When the product was dropped into a 5% calcium chloride aqueous solution from the tip of the syringe from a liquid surface height of about 10 cm, a granular material having a particle size of about 2 mm was obtained. This granular material was allowed to stand at 30 ° C. for 30 minutes to obtain a fixed granular molded product having a specific gravity of 1.03 and a compressive strength of 20 kg / cm ² .
[0045]
Example 2
100 parts by weight of the hydrophilic thermosetting resin prepared in Example 1, 1 part by weight of ammonium peroxodisulfate, 0.7 parts by weight of ferrous sulfate, 100 parts by weight of 2% sodium alginate aqueous solution and 100 parts by weight of distilled water were mixed well. The aqueous liquid composition thus obtained was dropped into a 5% calcium chloride aqueous solution from the tip of the syringe from a liquid surface height of about 10 cm, and a granular material having a particle size of about 2 mm was obtained. This granular material was allowed to stand at 30 ° C. for 30 minutes to obtain a fixed granular molded product having a specific gravity of 1.02 and a compressive strength of 20 kg / cm ² .
[0046]
Example 3
100 parts by weight of hydrophilic thermosetting resin prepared in Example 1, 1 part by weight of benzoyl peroxide, 0.5 part by weight of N, N-dimethylaniline, 100 parts by weight of 2% aqueous sodium alginate solution and 100 parts by weight of distilled water When an aqueous liquid composition obtained by mixing well was dropped into a 5% calcium chloride aqueous solution from the tip of the syringe from a liquid surface height of about 10 cm, a granular material having a particle size of about 2 mm was obtained. This granular material was allowed to stand at 30 ° C. for 30 minutes to obtain a fixed granular molded product having a specific gravity of 1.04 and a compressive strength of 20 kg / cm ² .
[0047]
Example 4
An aqueous liquid composition obtained by thoroughly mixing 100 parts by weight of the hydrophilic thermosetting resin prepared in Example 1, 0.7 part by weight of ferrous sulfate, 100 parts by weight of 2% aqueous sodium alginate solution and 100 parts by weight of distilled water. When the product was dropped into a 5% aqueous calcium chloride solution (containing 0.5% ammonium peroxodisulfate) from the tip of the syringe at a liquid surface height of about 10 cm, a granular material having a particle size of about 2 mm was obtained. This granular material was allowed to stand at 30 ° C. for 30 minutes to obtain a fixed granular molded product having a specific gravity of 1.01 and a compressive strength of 20 kg / cm ² .
[0048]
Example 5
After adding 100 parts by weight of distilled water to 100 parts by weight of the thermosetting resin prepared in Example 1, it was heated to about 50 ° C. and mixed well to obtain a uniform resin aqueous solution. An aqueous liquid composition obtained by thoroughly mixing 200 parts by weight of this resin mixture with 1 part by weight of ammonium peroxodisulfate, 1 part by weight of anhydrous sodium hydrogen sulfite and 100 parts by weight of 2% sodium alginate aqueous solution was obtained using a peristaltic pump. The solution was continuously supplied to the surface of a 5% calcium chloride aqueous solution for 3 seconds at a flow rate of 1 ml / second. The supplied composition spontaneously settled in an aqueous solution, and a granular material having a particle size of about 10 mm was obtained. This granular material was allowed to stand at 30 ° C. for 30 minutes to obtain a fixed granular molded product having a specific gravity of 1.03 and a compressive strength of 30 kg / cm ² .
[0049]
Example 6
200 parts by weight of the thermosetting resin aqueous solution prepared in Example 4, 1 part by weight of ammonium peroxodisulfate, 1 part by weight of sodium hydrogensulfite anhydrous salt, 75 parts by weight of 3% k-canaginan aqueous solution, and 2% glycolose isomerase bacterial enzyme solution ( Sodium bicarbonate buffer pH 8) 25 parts by weight was added to prepare a uniform mixture. When this uniform mixed solution was dropped into a 5% potassium chloride aqueous solution from the tip of the syringe from a position about 20 cm above the liquid level, a granular material having a particle size of about 1.5 mm was obtained. This granular material was allowed to stand at 30 ° C. for 30 minutes to obtain a fixed granular molded product having a specific gravity of 1.01 and a compressive strength of 30 kg / cm ² .
[0050]
Example 7
500 g of polyvinyl alcohol having a polymerization degree of 500, 1 mol (101 g) of N-methylolacrylamide, 0.5 g of hydroquinone and 1803 g of deionized water are placed in a reaction vessel and kept at 70 ° C. for 7 hours to carry out an addition reaction. A 25% aqueous solution of a curable resin was obtained. 400 parts by weight of this thermosetting resin aqueous solution, 1 part by weight of ammonium peroxodisulfate, 1 part by weight of anhydrous sodium hydrogen sulfite, 100 parts by weight of 2% aqueous sodium alginate solution and hollow alumina silica having an average particle size of 75 μm (trade name, manufactured by Nippon Ferrite Co., Ltd.) , Filite 300/7, true specific gravity 0.7) When an aqueous liquid composition obtained by thoroughly mixing 5 parts by weight is dropped into a 5% calcium chloride aqueous solution from the tip of the syringe from a liquid surface height of about 10 cm. A granular material having a particle diameter of 2 mm was obtained. This granular material was allowed to stand at 30 ° C. for 30 minutes to obtain a fixed granular molded product having a specific gravity of 0.97 and a compressive strength of 10 kg / cm ² .
[0051]
As mentioned above, the granular moldings for immobilization obtained in Examples 1 to 5 and Example 7 were separately taken into 500 mL Erlenmeyer flasks, respectively, and then GY-10 medium (yeast extract 1 g / L, glucose 100 g / L), respectively. L) was added. Zymomonas mobilis IFO 13756 was added thereto at a concentration of 2%, and stationary activated culture was performed at 30 ° C. for 24 hours.
[0052]
After activation, the surface of each immobilization granular molding was washed with distilled water, and then the activated fermentation broth was replaced with a new medium, followed by stationary culture for 24 hours, and the ethanol concentration was measured. The results are shown below.
[0053]
Granular molding ethanol concentration Example 1 7.5%
Example 2 7.1%
Example 3 6.7%
Example 4 7.6%
Example 5 6.6%
Example 7 6.4%
[0054]
【The invention's effect】
According to the method for producing a granular molded product for immobilizing an enzyme or microbial cell of the present invention, as is apparent from the above examples, a granular molded product equivalent to a conventional photocurable granular product is obtained, and light Unlike the case where a curable resin is used, no light irradiation device is required, the productivity is high, and the production cost of the granular molded product can be greatly reduced.

Claims (3)

(A) a hydrophilic thermosetting resin having at least two ethylenically unsaturated bonds in one molecule;
(B) a redox thermal polymerization initiator comprising an oxidizing agent and a reducing agent or a part thereof, and (c) a water-soluble polymeric polysaccharide capable of gelling by contact with an alkali metal ion or a polyvalent metal ion. The aqueous liquid composition is dropped into an aqueous medium containing alkali metal ions or polyvalent metal ions to gel the composition into a granular form or continuously on the surface of the aqueous medium for a predetermined time. After the droplets are grown to a desired particle size, the droplets are allowed to settle and gel, and the resulting granular gel is thermally polymerized to thermoset resin in the granular gel A method for producing a granular molded product for immobilizing an enzyme or a microbial cell, characterized by curing the material. The method according to claim 1, wherein one of an oxidizing agent and a reducing agent constituting the redox thermal polymerization initiator (b) is contained in an aqueous medium containing an alkali metal ion or a polyvalent metal ion. The redox thermal polymerization initiator (b) comprises a combination of an oxidizing agent comprising peroxodisulfates or hydrogen peroxide and a reducing agent comprising bisulfites or a divalent iron salt. 2. The method according to 2.