JP2613154B2 - Method for producing carrier for immobilizing microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a carrier for immobilizing microorganisms, which has sufficient strength as a carrier despite having extremely large pores inside the carrier. Since the carrier obtained by this method has large pores, it immobilizes relatively large microorganisms such as yeasts, filamentous fungi, and actinomycetes, and under aerobic conditions, various kinds of physiologically active substances such as enzymes and peptides, and various organic acids. It is suitable for producing a substance on a large scale by microbial conversion.

[0002]

2. Description of the Related Art Immobilization of microorganisms using chitin and chitosan is disclosed in JP-A-53-136584 and JP-A-5-136584.
No. 9-79984, JP-A-1-117787 and the like. However, all of them attempt to entrap and immobilize microorganisms on an immobilization carrier, and are inferior in permeation of substances such as substrates and gases, and are not particularly suitable for immobilizing aerobic microorganisms.

On the other hand, Japanese Patent Application Laid-Open No.
The granular porous chitosan disclosed in Japanese Patent No. 225539 has pores of about 15 μm from the surface to the inside, and exhibits excellent performance as a carrier for immobilizing relatively small microorganisms such as bacteria.

However, a carrier for immobilizing relatively large microorganisms such as yeast, filamentous fungi, and actinomycetes should have a larger pore diameter in order to improve the immobilization performance. When the particle size was increased, the strength of the carrier was reduced, and the support was not practical.

[0005] The strength of the carrier can be improved by cross-linking using a polyfunctional reagent. As a method of cross-linking granular porous chitosan, the applicant of the present invention has previously described Japanese Patent Publication No. 63-54285.
JP-A-63-205144, JP-A-63-284
No. 53 has been disclosed.

However, when a carrier having a large pore volume is treated by the above-mentioned cross-linking method, the carrier is brittle and insufficient in strength, so that vigorous agitation is required in a fluidized-bed culture or when the culture is performed in a packed bed. However, there is a disadvantage that it does not provide sufficient strength.

[0007]

DISCLOSURE OF THE INVENTION The present invention is excellent as a carrier for immobilizing microorganisms such as yeasts, filamentous fungi and actinomycetes despite having a sufficiently large pore diameter and a large pore volume inside. An object of the present invention is to provide a microorganism-immobilizing carrier having strength and high practicality.

[0008]

SUMMARY OF THE INVENTION The present invention relates to a method for dissolving low molecular weight chitosan in an acidic aqueous solution, and dropping the resulting solution into a basic solution. This is a method for producing a microorganism-immobilizing carrier in which N-acylation is carried out in the solution and an organic diisocyanate compound is reacted in a polar solvent.

As a carrier for immobilizing microorganisms such as yeasts, filamentous fungi, and actinomycetes, granular porous chitosan having a sufficiently large pore diameter and a large pore volume inside is disclosed in JP-A-2-22.
It can be obtained by the method disclosed in US Pat.

That is, the average molecular weight is 10,000 to 23.
A low molecular weight chitosan in the range of 0000 is dissolved in the acidic aqueous solution. At this time, a water-soluble polymer substance, such as polyethylene glycol, can be added as a pore regulator to the chitosan acidic aqueous solution. The solution is dropped into a basic solution such as sodium hydroxide or ammonia to solidify and precipitate granular porous chitosan. This is treated for a short time with an aqueous acid solution of an organic acid such as acetic acid or formic acid or a mineral acid such as hydrochloric acid or nitric acid to remove pores on the surface and in the vicinity thereof, which are smaller in diameter than the inside, and remove the inside of the granular material After exposing large pores, wash thoroughly with water.

Then, after replacing water with a polar solvent, the amino group of chitosan is N-acylated with an acid anhydride. As a solvent at the time of the reaction, a solvent inert to an acid anhydride such as dioxane, methanol, ethanol, isopropyl alcohol, dimethylformamide, dimethylsulfoxide, pyridine and the like is selectively used.

As the acylating agent, acid anhydrides of fatty acids such as acetic anhydride, propionic anhydride and butyric anhydride are used.
The reaction conditions for the acylation are as follows: the concentration of the acylating agent is 0.3 to 2 mol / L, the amount of the solution is twice the volume of the carrier, and the reaction temperature is 10 to 60 mol / L.
C. and the reaction time is preferably 1 to 24 hours. After the reaction,
The unreacted acylating agent and the produced fatty acid are removed by thoroughly washing with a solvent.

Next, the acylating agent which has reacted with the hydroxyl group of the N-acylated chitosan is subjected to a saponification treatment at 40 ° C. for 2 hours with 1N sodium hydroxide in the same volume as the carrier, followed by washing with water, Remove the agent thoroughly.

Next, N-acylated chitosan having a large pore volume is reacted with an organic diisocyanate compound in a polar solvent. The water of the N-acylated granular porous chitosan is sufficiently removed with a polar solvent. The polar solvent is selected and used from reaction solvents such as dioxane, methanol, ethanol, isopropyl alcohol, dimethylformamide, dimethylsulfoxide, dimethylacetamide, and pyridine.

As the organic diisocyanate compound,
4,4'-diphenylmethane diisocyanate, 1,4
-Phenylene diisocyanate, 2,4-tolylene diisocyanate, naphthalene diisocyanate, 1,4-
Cyclohexane diisocyanate, 4,4'-dicyclohexylmethane diisocyanate, xylylene diisocyanate, hexamethylene diisocyanate and the like are used.

In the reaction for introducing the organic diisocyanate compound, the concentration of the organic diisocyanate is 0.03 to 1.0.
mol / L, the liquid volume is twice the volume of the carrier, and the reaction temperature is 20
６０60 ° C. and the reaction time is preferably 0.5 to 12 hours. After the reaction, the unreacted organic diisocyanate compound is removed by sufficiently washing with water to obtain a microorganism-immobilizing carrier.

[0017]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these ranges.

The microorganism-immobilizing agent obtained by the method of the present invention.
Physical properties of the body were measured by the following methods.Pore diameter  5 ml sample is quenched and frozen in nitrogen where solid and liquid coexist
After that, -50 ° C, 10^-7After drying at the vacuum of Thor, run
Measured with an electron microscope and calculated the average value of 100 pores
did.Pore volume  After freeze-drying the sample, a mercury intrusion-type pore sizer 9310
(Made by Shimadzu Corporation)
The pore volume was measured.Non-breakage rate  Jar fur with 50 ml of carrier and 750 ml of ion-exchanged water
Put in Menta M-100 (manufactured by Tokyo Rika Kikai), room temperature
The mixture is stirred at 700 rpm for 3 hours. After stirring, the carrier
Is transferred to a 12 mesh wire mesh, and the damaged carrier is
Removed. Transfer the remaining carrier volume on the mesh to the measuring cylinder
And the non-failure rate was calculated by the following equation.

[0019]

(Equation 1)

Example 1 3.5% acetic acid containing 350 g of chitosan having a degree of deacetylation of 80% and an average molecular weight of 35,000 and 500 g of polyethylene glycol (molecular weight: 20,000, manufactured by Sanyo Chemical Industries, Ltd.) Aqueous solution 5,0
Dissolved in 00 ml. The solution is 1% aqueous ammonia 20%
After coagulating and regenerating by dropping a predetermined amount in a mixed solution consisting of ethanol and 79% water, the solid solution is sufficiently washed with water until it becomes neutral, and granular porous chitosan having an average particle diameter of 1.2 mm is prepared.
00 ml (wet) were obtained. After removing water adhering to the granular porous chitosan, 25% in 5,000 ml of 0.5% acetic acid aqueous solution.
After immersion treatment at 30 ° C. for 30 seconds, water washing was immediately performed until the solution became neutral. The volume of granular porous chitosan after washing is 3,
000 ml.

After sufficiently replacing the water contained in 1,000 ml of the granular porous chitosan with ethanol, acetic anhydride 1
Moles of ethanol in 2,000 ml at 25 ° C, 12
After stirring for an hour, the amino group was N-acylated.

Next, in order to remove the acylating agent that has reacted with the hydroxyl group of the N-acylated chitosan, 1N-
After performing a saponification treatment at 40 ° C. for 2 hours with sodium hydroxide, the resultant was washed with water to sufficiently remove the eliminated acylating agent.

Next, the water of the N-acylated granular porous chitosan was sufficiently removed and replaced with dimethylformamide. 0.
One mole of 4,4'-diphenylmethane diisocyanate was diluted with dimethylformamide to prepare 2,000 ml, added to N-acylated granular porous chitosan, and stirred at 25 ° C. for 1 hour to react. Then, unreacted 4,4 '
-Diphenylmethane diisocyanate was washed with dimethylformamide and sufficiently removed. Dimethylformamide was washed with water and removed to obtain 850 ml of a carrier for immobilizing microorganisms. Table 1 shows the measurement results of the pore diameter, the pore volume, and the non-breakage rate of the carrier.

[0024]

[Table 1]

As is clear from Table 1, the pore diameter becomes larger than the conventional pore diameter of about 15 μm, and the non-breakage rate is high.
It is a carrier with excellent strength.

Example 2 Water in 1,000 ml of N-acylated granular porous chitosan obtained in the same manner as in Example 1 was replaced with dimethylformamide and removed. Dilute 0.1 mole of hexamethylene diisocyanate with dimethylformamide to give 2,000
ml of N-acylated granular porous chitosan, and the mixture was stirred and reacted at 25 ° C. for 1 hour.

Thereafter, unreacted hexamethylene diisocyanate was washed with dimethylformamide and sufficiently removed. Dimethylformamide was washed with water and removed to obtain 850 ml of a carrier for immobilizing microorganisms. Table 2 shows the measurement results of the pore diameter, pore volume, and undamaged rate of the carrier.

[0028]

[Table 2]

As is clear from Table 2, excellent carriers were obtained even when the kind of the organic diisocyanate compound was changed.
Compared to the conventional pore diameter of about 15 μm, the pore diameter becomes larger and the non-breakage rate is high. That is, the carrier strength was also excellent.

Comparative Example 1 Water of 1,000 ml of granular porous chitosan obtained in the same manner as in Example 1 was removed and replaced with dimethylformamide. Water contained in the molded product was sufficiently removed. 0.1 mol of 4,4'-diphenylmethane diisocyanate was diluted with dimethylformamide to prepare 2,000 ml, and added to granular porous chitosan,
The mixture was stirred and reacted at 25 ° C. for 1 hour. Thereafter, unreacted 4,4'-diphenylmethane diisocyanate was washed with dimethylformamide and sufficiently removed. Dimethylformamide was washed with water and removed to obtain 850 ml of a carrier for immobilizing microorganisms. Table 3 shows the measurement results of the pore diameter, the pore volume, and the undamaged rate of the carrier.

[0031]

[Table 3]

As is clear from Table 3, when N-acylation is not carried out, the carrier has a lower non-destruction rate as compared with Example 1;
The strength was low.

Comparative Example 2 Water of 1,000 ml of the acid-treated granular porous chitosan obtained in the same manner as in Example 1 was sufficiently removed and replaced with dimethylformamide. Dilute 0.1 mole of hexamethylene diisocyanate with dimethylformamide to give 2,000
ml of chitosan, and add it to the granular porous chitosan at 25 ° C.
The mixture was stirred and reacted for an hour. Thereafter, unreacted hexamethylene diisocyanate was washed with dimethylformamide and sufficiently removed. Dimethylformamide was washed with water and removed to obtain 850 ml of a carrier for immobilizing microorganisms. Table 4 shows the measurement results of the pore diameter, pore volume, and non-breakage rate of each carrier.
It was shown to.

[0034]

[Table 4]

As is apparent from Table 4, when N-acylation was not performed, the carrier had a lower non-destruction rate as compared with Example 2, and had low strength.

[0036]

According to the present invention, a low molecular weight chitosan is dissolved in an acidic aqueous solution, and the resulting solution is dropped into a basic solution. The granular porous chitosan obtained is treated with an acid, and then N-acylated in a polar solvent. The carrier for immobilizing microorganisms obtained by reacting with an organic diisocyanate compound in the medium has excellent strength as a carrier despite having extremely large pores inside the carrier, and is suitable for vigorous stirring in a jar fermenter. Is also tolerable. The carrier for immobilizing microorganisms obtained by this method has pores of a size suitable for immobilizing microorganisms such as relatively large yeasts, filamentous fungi, and actinomycetes. It is suitable as a carrier for producing various substances such as acids on a large scale by microbial conversion.

Claims (1)

(57) [Claims] 1. A method for dissolving low molecular weight chitosan in an acidic aqueous solution, dropping the solution into a basic solution, subjecting the resulting granular porous chitosan to acid treatment, N-acylating the resulting mixture, and adding an organic diisocyanate in a polar solvent. A method for producing a carrier for immobilizing microorganisms, comprising reacting a compound.