JPH0529432B2 - - Google Patents

Description

[Detailed description of the invention]

〓Field of Industrial Application〓 The present invention has excellent performance for immobilizing enzymes or microorganisms by reducing the shrinkage rate of the carrier during dehydration and drying, and by imparting high compressive strength to the carrier. The present invention relates to a method for producing a carrier. 〓Conventional technology〓 Polysaccharide-based carriers are suitable as immobilization carriers and chromatography carriers due to their excellent porosity and hydrophilic properties, but on the other hand, they have low physical strength and
Furthermore, since the water content was high, the volume change of the carrier before and after dehydration and drying was extremely large. As disclosed in Japanese Patent Publication No. 63-54286, Japanese Patent Application Publication No. 63-28453, and Japanese Patent Application Publication No. 63-205144, the present inventors have proposed a method for using porous molded chitosan as a carrier for enzyme immobilization. We have been researching the introduction of functional groups using various cross-linking agents to immobilize enzymes in aqueous solutions, and when performing enzymatic reactions directly in aqueous solutions, they exhibit extremely excellent performance, but in organic solvents When performing an enzyme reaction, dehydration and drying of the enzyme immobilization carrier is essential, and when dehydration and drying is performed, the carrier shrinks significantly and the expression activity of the enzyme is extremely low. In addition, the carrier for immobilizing microorganisms, filed in Japanese Patent Application No. 1-45781, has a porous chitosan molded product having pores suitable for immobilizing relatively large microorganisms such as yeast and filamentous fungi. However, although fluidized bed reactors exhibit excellent performance due to the high porosity of the carrier, fixed bed reactors packed in columns still lack sufficient compressive strength. 〓Problem to be solved by the invention〓 The characteristic of enzymatic reactions in organic solvents is that the equilibrium relationship of the enzyme performing the dehydration condensation reaction is shifted to the product side, and the water content in the reaction solution must be strictly controlled. Must be. On the other hand, enzymes are insoluble in organic solvents, so in order to immobilize enzymes on a carrier, it is necessary to add a carrier to an aqueous enzyme solution, adsorb and immobilize the enzyme on the carrier, and then dehydrate and dry the solution. Therefore,
In order to obtain sufficient expression activity of the immobilized enzyme, it is necessary to minimize the shrinkage of the carrier during dehydration and drying. However, the above-mentioned chitosan-based immobilization carrier is porous and has good diffusion of enzymes and substrates, and is also hydrophilic, so it has the characteristics of less denaturation of the adsorbed enzyme and high expression activity. However, when the carrier is dehydrated and dried, it breaks or shrinks significantly. or,
When used as a carrier for immobilizing microorganisms, it is more elastic and has a lower specific gravity than inorganic carriers, so it has excellent fluidity and can withstand various stirring operations. Providing pores large enough to penetrate the carrier increases the porosity inside the carrier, and especially in fluidized bed reactors, the compaction of the carrier that occurs during passage of liquid is significant. The object of the present invention is to reduce the shrinkage rate of the carrier during dehydration and drying operations, and to improve the carrier to have high compressive strength. <Means for Solving the Problems> The present invention involves coagulating and precipitating an acidic aqueous solution of low molecular weight chitosan in a basic aqueous solution to obtain a porous chitosan molded product, then reacting it with glycidyl ether of an aliphatic polyalcohol, and further producing a functional A method for producing a carrier for immobilizing an enzyme or microorganism into which a group is introduced is characterized in that glycidyl ether of an aliphatic polyalcohol is reacted in dioxane containing potassium hydroxide. In the present invention, low molecular weight chitosan with an average molecular weight of 10,000 to 230,000 is used as chitosan,
The low molecular weight chitosan is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, and formic acid alone or in a mixture to obtain an acidic chitosan aqueous solution. The concentration of chitosan in the chitosan acidic aqueous solution can be freely selected within the range of 2 to 20% (by weight). At this time, water-soluble polymeric substances such as polyvinyl alcohol, polyethylene glycol, etc. may be added alone or in combination as a pore control agent to the chitosan acidic aqueous solution. In order to regenerate chitosan from the chitosan acidic aqueous solution and obtain a porous chitosan molded product, for example, the pore size is 0.1 to 0.25 mmφ.
The chitosan acidic aqueous solution is dropped into a basic aqueous solution in a fixed amount under pressure through a nozzle and solidified and regenerated, thereby obtaining granular bodies of porous chitosan molded articles. As the basic substance added to the basic coagulation solution, alkaline substances such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia, and ethylenediamine are used.To make a basic solution, water or methanol is used. The above-mentioned basic substance is added to a polar alcohol such as ethanol, or a mixture of water and alcohol. The obtained water-containing porous chitosan molded article may be prepared in accordance with Japanese Patent Application No. 1999-1-2010, if necessary, in order to realize fine pores with a larger diameter on the surface of the molded article.
Surface treatment may be performed by the method disclosed in No. 45781. That is, the obtained porous chitosan molded product is surface-treated with an acid aqueous solution by replacing water with an alcohol such as methanol, ethanol, isopropyl alcohol, or a polar solvent such as acrylonitrile, if necessary. The acids used at this time include organic acids such as acetic acid, formic acid, and propionic acid, or hydrochloric acid,
Mineral acids such as nitric acid are used. For surface treatment with acid, if the molded product contains water, an aqueous acid solution is used.
If a polar solvent is included, it is preferable to use a mixture of the polar solvent and an acid. As the concentration of acid
A concentration of 0.5 to 5.0% (by weight) is used, the treatment is carried out for a short time of 10 seconds to 5 minutes, and the acid is thoroughly washed with water after the acid treatment. Next, in order to reduce the shrinkage rate of the final product carrier and give it high compressive strength, the water in the porous chitosan molded product was replaced with dioxane, an aqueous potassium hydroxide solution was added, and a glycidyl ether of aliphatic polyalcohol was added. is added and reacted in the same volume of dioxane as the porous chitosan molded article. At this time, potassium hydroxide having a concentration of 0.01 to 3 normal is added in a volume of 1/100 to 1/10 to dioxane. Examples of the glycidyl ether of aliphatic polyalcohol used in the present invention include ethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, trimethylene glycol diglycidyl ether, tetramethylene glycol diglycidyl ether, and hexamethylene glycol diglycidyl ether. Can be mentioned. The reaction between the porous chitosan molded product and the glycidyl ether of the aliphatic polyalcohol is carried out at a temperature of 20 to 90°C, preferably 25 to 80°C, for 0.5 to 48 hours with gentle stirring. The present invention is characterized in that glycidyl ether of aliphatic polyalcohol is reacted in dioxane containing potassium hydroxide, but potassium hydroxide has better solubility in dioxane than other alkaline substances. In addition, potassium hydroxide has good reactivity with the hydroxyl group at the C-6 position of chitosan,
This has the effect of promoting the crosslinking reaction of aliphatic polyalcohol with glycidyl ether. After the reaction is completed, wash with dioxane and then with pure water until neutral. Functional groups containing various active groups suitable for immobilizing enzymes or microorganisms are further introduced into the porous chitosan molded article thus obtained. The first type of functional group is
anion exchange groups such as primary, secondary, tertiary, and quaternary ammonium groups; cation exchange groups such as sulfonic acid groups and carboxylic acid groups; iminodiacetic acid groups, amidoxime groups, thiol groups, and dithiocarbamine groups. and hydrophobic groups such as aromatic and aliphatic alkyl. Reagents used to react these with the remaining amino groups or hydroxyl groups of chitosan include alkyl halides, epoxides, isocyanates, and epoxides containing the above-mentioned functional groups.
Examples include acid anhydrides, aldehydes, ketones, and acid halides of carboxylic acids and sulfonic acids. 〓Example〓 Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited to the scope described in the Examples. In addition, compressive elastic modulus, specific surface area, pore diameter,
The dry carrier volume after dehydration and drying was measured by the following method. Compressive modulus Rheometer NRM-2010J-CW (Fudou Kogyo Co., Ltd.)
Pack the sample into a sample adapter with a diameter of 6.0 mmφ and a depth of 10 mm using a
The compressive elastic modulus was measured when the sample was pushed in 4 mm at a speed of cm/min. Specific surface area The sample was rapidly cooled in liquid nitrogen and freeze-dried at −50° C. under a vacuum of 10 ⁻⁷ Torr, and then measured by the BET method using a specific surface area measuring device. Pore diameter The sample was rapidly cooled in liquid nitrogen, freeze-dried at −50° C. under a vacuum of 10 ⁻⁷ Torr, and then measured using a scanning electron microscope. Dry carrier volume after dehydration and drying The water contained in 10 ml of the sample was replaced with acetone, and the volume was expressed as the volume after air drying in a desiccator. Example 1 350 g of chitosan having a degree of deacetylation of 80% and an average molecular weight of 52,000 was dissolved in 4,650 g of a 3.5% acetic acid aqueous solution.
After solidifying and regenerating the solution by dropping it into a mixed solution of 8% sodium hydroxide, 20% ethanol, and 72% water through a nozzle with a hole diameter of 0.25 mm,
Wash thoroughly with water until neutral, and the average particle size is approximately 1.2mm.
φ, compressive modulus 1.1×10 ⁶ dyn/cm ² , specific surface area 70
Porous chitosan granules with m ² /g and pore diameter of 0.2μ 5
(Wet) (Sample A) was obtained. The obtained porous chitosan granules 1 (wet) were collected, the water contained therein was sufficiently replaced with dioxane, and then a solution of 100 ml of 0.5N potassium hydroxide added to 900 ml of dioxane was added and stirred. .
Furthermore, ethylene glycol diglycidyl ether
33.0g was added and reacted at 70°C for 3 hours. After the reaction is completed, the compressive modulus of elasticity is 7.0×10 ⁶ dyn/cm ² after thorough washing with water.
A carrier (sample B) with a specific surface area of 82 m ² /g, a pore diameter of 0.1 μm, and a volume of 510 ml (wet) was obtained. The obtained sample B is described below.
Functional groups were introduced by the methods described below. 100 ml of 15% aqueous sodium hydroxide solution was added to 100 ml of sample B (wet), and the mixture was stirred at room temperature for 1 hour. After removing the liquid, add 100 ml of dioxane, 12.9 g of 2-chlorotriethylamine hydrochloride, and 7.6 g of triethylamine, and react at 80°C for 3 hours with gentle stirring. After completion, wash thoroughly with pure water, volume 101 ml (wet). A carrier (sample 1) was obtained. After sufficiently replacing the water contained in 100 ml of sample B (wet) with dimethylformamide, 8 g of hexamethylene diisocyanate was added and reacted for 2 hours at room temperature. After completion, unreacted hexamethylene diisocyanate was removed with dimethylformamide. The carrier was washed thoroughly with ion-exchanged water to obtain a carrier (sample 2) with a volume of 99 ml (wet). After the water contained in 100 ml (wet) of sample B was sufficiently replaced with dimethylformamide, 8 g of 4,4'-diphenylmethane diisocyanate was added and reacted at room temperature for 1 hour. After the reaction was completed, unreacted 4,4'-diphenylmethane diisocyanate was removed with dimethylformamide and thoroughly washed with ion-exchanged water to obtain a carrier (sample 3) with a volume of 98 ml (wet). The compressive modulus, specific surface area, pore diameter, and dry carrier volume after dehydration and drying were measured for each of Samples 1, 2, and 3, and the results are shown in Table 1. Comparative Example 1 Sample A 1 obtained by the same operation as Example 1
After sufficiently replacing the water contained in (wet) with dioxane,
1 dioxane was added, 33.0 g of ethylene glycol diglycidyl ether was added without adding any potassium hydroxide, and the mixture was reacted at 70° C. for 3 hours. After the reaction is complete, wash thoroughly with water and compressive modulus is 2.8×
A carrier (sample C ⁾ with a volume of ⁶⁹⁰ ml (wet) and a specific surface area of 80 m ² /g and a pore diameter of 0.2 μ was obtained. Three samples of 100 ml each (wet) were taken from the sample C obtained, and each was subjected to the treatment for introducing the functional groups and as described in Example 1, each having a volume of 101 ml.
(wet) (sample 4), volume 95ml (wet) (sample 5),
A volume of 95 ml (wet) (sample 6) was obtained. The compressive modulus, specific surface area, pore diameter, and carrier volume after dehydration and drying were measured for the obtained samples 4, 5, and 6, and the results are shown in Table 1.

[Table] As is clear from the results in Table 1, when reacting glycidyl ether of aliphatic polyalcohol,
Samples 4, 5, and 6 without using potassium hydroxide
It is clear that the compressive modulus and dry carrier volume (which prevents shrinkage of the carrier during drying) are inferior to those obtained by the method of the present invention. Example 2 700 g of chitosan with a degree of deacetylation of 78% and an average molecular weight of 12,000 and polyethylene glycol (molecular weight
29000 (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in a 3% acetic acid aqueous solution such that the total amount was 10. The solution,
A fixed amount is dropped into a mixed solution consisting of 2.5% ammonia, 20% ethanol, and 77.5% water from a nozzle with a pore size of 0.25 mmφ to solidify and regenerate, and then washed thoroughly with water until it becomes neutral to form porous particles with an average particle size of approximately 1.2 mmφ. 10 (wet volume) of quality chitosan granules were obtained. The obtained porous chitosan granules 5 (wet) were collected, the water contained therein was removed by suction filtration, and then immersed in a 0.5% acetic acid aqueous solution 5 at 25°C for 30 seconds,
Immediately wash with water until neutral, and check the compressive modulus.
0.6×10 ⁶ dyn/cm ² , specific surface area 20.5 m ² /g, pore diameter
20μ porous chitosan granules 3900ml (wet volume)
(Sample D) was obtained. After collecting the obtained porous chitosan granules 1 (wet) and sufficiently replacing the water contained therein with dioxane, a solution of 100 ml of 0.5N potassium hydroxide in 900 ml of dioxane was added and stirred. did. Further, 28 g of ethylene glycol diglycidyl ether was added, and the mixture was reacted at 70°C for 3 hours. After the reaction is complete, wash thoroughly with water to reduce the compressive modulus to 1.5.
A carrier of 600 ml (wet volume) (sample E) having a density of ×10 ⁶ dyn/cm ² , a specific surface area of 26 m ² /g, and a pore diameter of 16 μm was obtained. Three samples of 100 ml (wet) each were collected from the obtained sample E, and functional groups were introduced by the methods described in Example 1.
100 ml (wet) of the carrier (sample 7), with a volume of
A volume of 99 ml (wet) carrier (sample 8) and a volume of 98 ml (wet) carrier (sample 9) was obtained by treatment. Sample 7,
The compressive modulus, specific surface area, pore diameter, and dry carrier volume after dehydration and drying were measured for each of Samples No. 8 and No. 9, and the results are shown in Table 2. Comparative Example 2 Sample D 1 obtained by the same operation as Example 2
After sufficiently replacing the water contained in (wet) with dioxane,
1 dioxane was added, 28 g of ethylene glycol diglycidyl ether was added without adding any potassium hydroxide, and the mixture was reacted at 70° C. for 3 hours. After the reaction is complete, wash thoroughly with water and compressive modulus is 3.2×
A carrier (sample F) with a volume of 810 ml (wet) having a surface area of 10 ⁶ dyn/cm ² , a specific surface area of 30 m ² /g and a pore diameter of 19 μm was obtained. Three samples of 100 ml each (wet) were taken from the obtained sample F, and the functional groups of and were introduced as described in Example 1. A volume of 96 ml (wet) (sample 12) was obtained. The compressive modulus, surface area, pore diameter, and dry carrier volume after dehydration and drying were measured for the obtained samples 10, 11, and 12, and the results are shown in Table 2.

[Table] As is clear from the results in Table 2, the pore diameter is the
It is clear that the carrier prepared by the method of the present invention in Example 2 has a high compressive modulus of elasticity, even though the values are larger than those in the table, and the dry carrier volume is also excellent. 〓Effects of the Invention〓 The carrier for immobilizing enzymes or microorganisms produced by the production method of the present invention has high stability against the filling pressure during column filling and the drying operation of the carrier, and is suitable for immobilization for enzyme reactions in non-aqueous systems. It exhibits extremely excellent performance as a chemical carrier. In addition, even if it has a large pore diameter suitable for immobilizing microorganisms such as yeast and filamentous fungi, it has high strength against compression, making it suitable for repeated use and long-term continuous use as a fluidized bed reactor for immobilized microorganisms. Furthermore, it is an extremely excellent immobilization carrier for use in packed reactors.

Claims (1)

[Claims] 1. Obtaining a porous chitosan molded article by coagulating and precipitating an acidic aqueous solution of low molecular weight chitosan in a basic aqueous solution,
A method for producing an immobilization carrier in which a glycidyl ether of an aliphatic polyalcohol is then reacted and a functional group is further introduced, characterized by reacting the glycidyl ether of an aliphatic polyalcohol in dioxane containing potassium hydroxide. A method for producing a carrier for immobilizing enzymes or microorganisms.