JPH0654686A - Production of carrier for immobilizing microorganism - Google Patents

Abstract

(57) [Summary] [Objective] To obtain a carrier for immobilizing microorganisms, which has excellent strength despite having a large pore diameter and a large pore volume inside. [Structure] A low molecular weight chitosan is dissolved in an acidic aqueous solution, and the granular porous chitosan obtained by dropping the dissolved solution into a basic solution is acid-treated, then N-acylated, and reacted with an organic diisocyanate compound to produce a microorganism. It is used as an immobilization carrier.

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 even though it has extremely large pores inside the carrier. Since the carrier obtained by this method has large pores, it immobilizes relatively large microorganisms such as yeast, filamentous fungi, and actinomycetes, and under aerobic conditions, various physiologically active substances such as enzymes and peptides, and various organic acids. Suitable for producing substances on a large scale level 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-5-36584.
No. 9-74984, JP-A No. 1-117787, and the like. However, all of these are intended to entrap and immobilize microorganisms on an immobilization carrier, and are inferior in substance permeation of substrates and gases, and are not particularly suitable for immobilization of aerobic microorganisms.

On the other hand, Japanese Patent Application Laid-Open No. 2-
The granular porous chitosan disclosed in 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, in a carrier for immobilizing relatively large microorganisms such as yeasts, filamentous fungi, and actinomycetes, it is desirable to have a larger pore diameter in order to improve immobilization performance. When the value is increased, the strength of the carrier is lowered, which is not practical.

The strength of the carrier can be improved by cross-linking using a polyfunctional reagent, and as a method for cross-linking granular porous chitosan, the present applicant has previously mentioned that Japanese Patent Publication No. 63-54285.
No. 63-205144, 63-284.
No. 53 and the like have been disclosed.

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

[0007]

The present invention is excellent as a carrier for immobilizing microorganisms such as yeast, filamentous fungi, and actinomycetes, despite having a sufficiently large pore diameter and a large pore volume inside. The present invention provides a carrier for immobilizing microorganisms, which has strength and is highly practical.

[0008]

The present invention relates to a granular porous chitosan obtained by dissolving low molecular weight chitosan in an acidic aqueous solution and dropping the dissolved solution into a basic solution. It is a method for producing a carrier for immobilizing a microorganism, which is N-acylated in the medium and reacted with an organic diisocyanate compound in a polar solvent.

As a carrier for immobilizing microorganisms such as yeast, filamentous fungi, and actinomycetes, a 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 No. 5539.

That is, the average molecular weight is 10,000 to 23.
Low molecular weight chitosan in the range of 10,000 is dissolved in acidic aqueous solution. At this time, a water-soluble polymeric substance such as polyethylene glycol can be added as a pore control agent to the chitosan acidic aqueous solution. This dissolved solution is dropped into a basic solution such as sodium hydroxide or ammonia to coagulate and separate 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 and pores on the surface and in the vicinity of which the diameter is smaller than the inside. After exposing the large pores of, wash thoroughly with water.

Next, after substituting water with a polar solvent, the amino group of chitosan is N-acylated with an acid anhydride. As the solvent during the reaction, a solvent inert to acid anhydrides such as dioxane, methanol, ethanol, isopropyl alcohol, dimethylformamide, dimethylsulfoxide, pyridine is selected and 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 acylating agent concentration is 0.3 to 2 mol / L, the liquid amount is twice the carrier volume, and the reaction temperature is 10 to 60.
The reaction time at 0 ° C. is preferably 1 to 24 hours. After the reaction,
The unreacted acylating agent and the produced fatty acid are thoroughly washed with a solvent to remove them.

Then, the acylating agent which has reacted with the hydroxyl group of N-acylated chitosan is saponified with 1N sodium hydroxide in the same volume as the carrier at 40 ° C. for 2 hours, washed with water, and the acyl eliminated. Sufficiently remove the agent.

The N-acylated chitosan, which then has 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 thoroughly 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 of introducing the organic diisocyanate compound, the concentration of the organic diisocyanate is 0.03 to 1.0.
mol / L, liquid volume is twice the carrier volume, reaction temperature is 20
It is preferable to carry out at -60 ° C and a reaction time of 0.5-12 hours. After completion of the reaction, the unreacted organic diisocyanate compound is removed by sufficiently washing with water to obtain a carrier for immobilizing microorganisms.

[0017]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to this range.

[0018] For immobilizing microorganisms obtained by the method of the present invention
The physical properties of the body were measured by the following methods. Pore size Freeze by cooling 5 ml of sample rapidly in nitrogen where solid and liquid coexist.
Then, after drying at -50 ° C and a vacuum degree of 10-7 Torr,
Measure with a scanning electron microscope and measure the average value of 100 pores.
I calculated.Pore volume After freeze-drying the sample, mercury press-in type porosizer 9310 type
The amount per 1 g of dried sample by Shimadzu Corporation
Pore volume was measured.Non-breakage rate Jar fur 50ml carrier and 750ml ion-exchanged water
Put in Mentor M-100 (made by Tokyo Rikakikai), room temperature
Medium, stirring at 700 rpm for 3 hours. After stirring, carrier
On a 12-mesh wire mesh and remove the damaged carrier with running water.
Removed. Measure the volume of the carrier remaining on the mesh
, And the non-breakage rate was calculated by the following formula.

[0019]

[Equation 1]

Example 1 3.5% acetic acid containing 350 g of chitosan having a deacetylation degree of 80% and an average molecular weight of 35,000 and 500 g of polyethylene glycol (molecular weight of 20,000, manufactured by Sanyo Chemical Industry Co., Ltd.) Aqueous solution 5,0
It was dissolved in 00 ml. The solution is 1% aqueous ammonia 20%
After a certain amount was dropped into a mixed solution consisting of ethanol and 79% water to coagulate and regenerate, it was thoroughly washed with water until it became neutral, and granular porous chitosan 5,0 having an average particle diameter of 1.2 mm was used.
00 ml (wet) was obtained. After removing water adhering to the granular porous chitosan, 25
After immersion treatment at 30 ° C. for 30 seconds, it was immediately washed with water until it became neutral. The volume of granular porous chitosan after washing with water is 3,
It was 000 ml.

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

Next, in order to remove the acylating agent which has reacted with the hydroxyl group of N-acylated chitosan, 1 N- of the same volume as the carrier is removed.
After saponification treatment with sodium hydroxide at 40 ° C. for 2 hours, the product was washed with water to sufficiently remove the desorbed acylating agent.

Next, the water of the N-acylated granular porous chitosan was thoroughly removed and replaced with dimethylformamide. 0.
1 mol of 4,4'-diphenylmethane diisocyanate was diluted with dimethylformamide to prepare 2,000 ml, which was added to N-acylated granular porous chitosan and stirred at 25 ° C for 1 hour for reaction. Then unreacted 4,4 '
Diphenylmethane diisocyanate was washed thoroughly with dimethylformamide and 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, pore volume, and non-breakage rate of this carrier.

[0024]

[Table 1]

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

Example 2 1,000 ml of N-acyl granular porous chitosan obtained by the same method as in Example 1 was replaced with dimethylformamide to remove water. 0.1 mol of hexamethylene diisocyanate was diluted with dimethylformamide to prepare 2,000 ml, which was added to N-acylated granular porous chitosan and stirred at 25 ° C. for 1 hour for reaction.

After that, 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 non-breakage rate of this carrier.

[0028]

[Table 2]

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

Comparative Example 1 Granular porous chitosan obtained by the same method as in Example 1, 1,000 ml of water, was removed and replaced with dimethylformamide. The 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, which was added to granular porous chitosan,
The reaction was carried out by stirring at 25 ° C for 1 hour. 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 3 shows the measurement results of the pore diameter, pore volume and non-breakage rate of this carrier.

[0031]

[Table 3]

As is clear from Table 3, in the case where N-acylation is not carried out, the carrier has a poor non-breakage rate as compared with Example 1,
The strength was low.

Comparative Example 2 1000 ml of granular porous chitosan obtained in the same manner as in Example 1 was sufficiently removed and replaced with dimethylformamide. 0.1 mol of hexamethylene diisocyanate was diluted with dimethylformamide to prepare 2,000 ml, which was added to granular porous chitosan and stirred at 25 ° C. for 1 hour for reaction. Then, 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.

[0034]

[Table 4]

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

[0036]

Industrial Applicability The low molecular weight chitosan is dissolved in an acidic aqueous solution, and the granular porous chitosan obtained by dropping the solution into a basic solution is acid-treated and then N-acylated in a polar solvent to obtain a polar solvent. The carrier for immobilizing the microorganism obtained by reacting with an organic diisocyanate compound in the medium has excellent strength as a carrier even though it has extremely large pores inside the carrier and is suitable for vigorous stirring in a jar fermenter. Can withstand. 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, and physiologically active substances such as enzymes and peptides and organic substances. It is suitable as a carrier for producing various substances such as acids on a large-scale level by microbial conversion.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

[0018] For immobilizing microorganisms obtained by the method of the present invention
The physical properties of the body were measured by the following methods. Pore size Freeze by cooling 5 ml of sample rapidly in nitrogen where solid and liquid coexist.
After that, -50 ℃, 10^-7After drying at the vacuum degree of Tall, run
Measure with an electron microscope and calculate the average value of 100 pores
did.Pore volume After freeze-drying the sample, mercury press-in type porosizer 9310 type
The amount per 1 g of dried sample by Shimadzu Corporation
Pore volume was measured.Non-breakage rate Jar fur 50ml carrier and 750ml ion-exchanged water
Put in Mentor M-100 (made by Tokyo Rikakikai), room temperature
Medium, stirring at 700 rpm for 3 hours. After stirring, carrier
On a 12-mesh wire mesh and remove the damaged carrier with running water.
Removed. Measure the volume of the carrier remaining on the mesh
, And the non-breakage rate was calculated by the following formula.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

Example 2 1,000 ml of N-acylated granular porous chitosan obtained by the same method as in Example 1 was replaced with dimethylformamide to remove water. 0.1 mol of hexamethylene diisocyanate was diluted with dimethylformamide to prepare 2,000 ml, which was added to N-acylated granular porous chitosan and stirred at 25 ° C. for 1 hour for reaction.

Claims (1)

[Claims] 1. A low molecular weight chitosan is dissolved in an acidic aqueous solution, and the granular porous chitosan obtained by dropping the dissolved solution into a basic solution is acid-treated and then N-acylated, followed by organic diisocyanate in a polar solvent. A method for producing a carrier for immobilizing a microorganism, which comprises reacting a compound.