JPH0654687A - Production of carrier for immobilizing microorganism - Google Patents

Abstract

PURPOSE:To obtain a carrier for immobilizing a microorganism having excellent strength in spite of a large pore diameter and a large pare volume in the interior thereof. CONSTITUTION:The objective carrier for immobilizing a microorganism is obtained by dissolvinig low-molecular weight chitosan in an acidic aqueous solution, dropping the resultant solution containing the chitosan dissolved therein into a basic solution, treating the formed granular porous chitosan with an acid, then N-acylating the granular porous chitosan and reacting the N-acylated product with glycidyl ether of an aliphatic polyalcohol.

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 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 microorganisms in the 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 a glycidyl ether of an aliphatic polyalcohol in the presence of an alkali.

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 a solvent at the time of reaction, a solvent inert to an acid anhydride 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. The unreacted acylating agent and the produced fatty acid are thoroughly washed with a solvent to remove them.

Next, the acylating agent reacted with the hydroxyl group of 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 eliminated acylating agent is removed. Remove enough.

Next, for the purpose of enhancing the reactivity between the hydroxyl groups of N-acylated chitosan and the glycidyl ether of aliphatic polyalcohol, N-acylated granular porous chitosan is immersed in an alkaline aqueous solution. As the alkaline treatment conditions at this time, sodium hydroxide and potassium hydroxide are used, and the concentration thereof is preferably in the range of 0.02-1 normal.
When it is less than 0.02N, the crosslinking reaction does not proceed, and when it is higher than 1N, the granular porous N-acylated chitosan forms a block in the polar solvent and the reaction does not proceed well. The amount of the liquid is twice the volume of the carrier, and the processing temperature is 10 to 5
The treatment time is preferably 0 ° C. and 0.5 to 5 hours. After treatment,
The excess aqueous alkaline solution is separated by filtration and separated from the particulate porous N-acylated chitosan and removed.

Next, the glycidyl ether of the aliphatic polyalcohol is reacted in a polar solvent. Examples of the glycidyl ether of aliphatic polyalcohol include polyethylene glycol diglycidyl ether such as ethylene glycol diglycidyl ether and diethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether such as propylene glycol diglycidyl ether and dipropylene glycol diglycidyl ether, and glycerol polyglycol. Glycidyl ether or the like is used. Isopropyl alcohol, dioxane, etc. are used as the polar solvent.

In the reaction of introducing the glycidyl ether of the aliphatic polyalcohol into the granular porous N-acylated chitosan, the concentration is 0.01 to 0.5 epoxy equivalent / L, the liquid amount is twice the carrier volume, The reaction temperature is preferably 20 to 90 ° C and the reaction time is preferably 1 to 24 hours. After completion of the reaction, the unreacted glycidyl ether of the aliphatic polyalcohol is removed 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% in 5,000 ml of 0.5% acetic acid aqueous solution.
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.

The obtained N-acylated granular porous chitosan was diluted with 1,000 ml of 0.2N potassium hydroxide to obtain 2 parts thereof.
Perform alkali treatment at 5 ° C for 2 hours. After the treatment, the excess alkali is separated from the N-acylated granular porous chitosan by filtration and removed.

Next, 2,000 ml of dioxane containing 0.06 epoxy equivalent / L of ethylene glycol diglycidyl ether is added and reacted at 70 ° C. for 3 hours. After completion of the reaction, wash well with water to remove unreacted reagents and
Thus, ml of the microorganism-immobilized carrier was obtained. Table 1 shows the measurement results of the pore diameter, pore volume and non-breakage rate of this carrier.

[0025]

[Table 1]

As is apparent from Table 1, the conventional pore diameter is about 15 μm, but the pore diameter is large and the non-breakage rate is high. That is, the carrier strength was also excellent.

Example 2 1,000 ml of N-acyl granular porous chitosan obtained by the same method as in Example 1 was used.
Alkali treatment is performed in 5N potassium hydroxide at 25 ° C. for 2 hours. After the treatment, the excess alkali is separated from the N-acyl granular porous chitosan by filtration.

Then, 2,000 ml of dioxane containing 0.06 epoxy equivalent / L of ethylene glycol diglycidyl ether is added, and the mixture is reacted at 70 ° C. for 3 hours. After completion of the reaction, wash well with water to remove unreacted reagents and
Thus, ml of the microorganism-immobilized carrier was obtained. Table 2 shows the measurement results of the pore diameter, pore volume and non-breakage rate of this carrier.

[0029]

[Table 2]

As is apparent from Table 2, the conventional pore size is about 1
The diameter is larger than 5 μm, and the non-breakage rate is high.
That is, the carrier strength was also excellent.

<Comparative Example 1> The 0.2N potassium hydroxide of Example 1 was changed to 2N, and the 0.05N potassium hydroxide of Example 2 was changed to 0.01N. 1. Each carrier for immobilizing microorganisms was obtained by the same operation as in Example 2. Table 3 shows the measurement results of the pore diameter, pore volume and non-breakage rate of each carrier.

[0032]

[Table 3]

As is apparent from Table 3, when the potassium hydroxide concentration is outside the range of 0.02 to 1 normal, the non-breakage rate is low as compared with Examples 1 and 2, which is not preferable.

Comparative Example 2 To 1,000 ml of granular porous chitosan obtained by the same method as in Example 1, 2,000 ml of dioxane containing 0.06 epoxy equivalent / L of ethylene glycol diglycidyl ether was added, and the mixture was heated to 70 ° C. React for 3 hours. After the reaction was completed, it was washed thoroughly with water to remove unreacted reagents 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 this carrier.

[0035]

[Table 4]

As is apparent from Table 4, the non-breakage rate is 0 even when the pore diameter can be increased without N-acylation. That is, there was no carrier strength.

[0037]

Industrial Applicability The low molecular weight chitosan is dissolved in an acidic aqueous solution, and then the solution is dropped into a basic solution to obtain a granular porous chitosan, which is then acid-treated and then N-acylated in a polar solvent to obtain an alkali solution. The carrier for immobilizing a microorganism obtained by reacting a glycidyl ether of an aliphatic polyalcohol in the presence of a bacterium has sufficient strength as a carrier even though it has extremely large pores inside the carrier. It did not break even with vigorous stirring in the fermentor. The carrier obtained by this method has a pore size suitable for immobilizing relatively large microorganisms such as yeasts, filamentous fungi, and actinomycetes, and various substances such as enzymes, peptides, physiologically active substances, organic acids, etc. It is suitable as a carrier produced on a large scale level by microbial conversion.

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[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

[Name of item to be corrected] 0027

[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 added to 0.2 wt.
Alkali treatment is performed in 05 normal potassium hydroxide at 25 ° C. for 2 hours. After the treatment, the excess alkali is separated from the N-acylated granular porous chitosan by filtration.

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 solution into a basic solution is acid-treated and then N-acylated to obtain an aliphatic compound in the presence of an alkali. A method for producing a carrier for immobilizing a microorganism, which comprises reacting a glycidyl ether of polyalcohol.