JPH0417634B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing a carrier suitable for immobilizing a physiologically active substance such as an enzyme having an amino group.

〓Conventional technology〓 It has been known for a long time that dialdehydes such as glutaraldehyde are used by utilizing the amino groups of chitosan to immobilize physiologically active substances using chitosan. A method using a peptide bond using a carbodiimide reagent is also disclosed in Japanese Patent Publication No. 10150/1983. The inventors previously filed Japanese Patent Application No. 60-210731 (Japanese Unexamined Patent Publication No. 62-70401).
As described in Japanese Patent Application No. 60-218980, porous chitosan particles are N-acetylated to obtain porous chitin particles, treated with a crosslinking agent, carboxyalkylated, and then deacetylated to obtain a carrier. A method for obtaining a carrier by treating porous chitosan particles with an acylating agent in an organic solvent is disclosed in Japanese Patent Application No. 61-188259 (Japanese Patent Application Laid-open No. 63-44884).
No.), crosslinking porous chitosan granules with a dicarboxylic acid derivative and then acetylating them to obtain a carrier;
Japanese Patent Application No. 61-188260 (Japanese Unexamined Patent Publication No. 63-44887) proposes a method of treating porous chitosan particles with an excess of dicarboxylic acid ester.

〓Problems to be solved by the invention〓 When porous chitosan particles are treated with a dialdehyde such as glutaraldehyde and used as a carrier, when immobilizing enzymes, etc., the gap between the amino group and the aldehyde group becomes Schiff base. Because it bonds in the form of , it becomes vulnerable to acids. Further, although reduction strengthens the bond, if the enzyme is easily denatured, there is a drawback that the method of treatment with the reducing agent used is limited. Furthermore, when treated with carposiimide, the bond with the enzyme, etc. becomes an amide bond, which improves the properties against acids and alkalis, but requires time and effort to adjust the pH during immobilization, and there is a high possibility of denaturing the enzyme, etc. . In carriers treated with dicarboxylic acid active esters, enzymes, etc. can be immobilized through amide bonds, and can be immobilized under mild conditions.
Since residual amino groups are inevitably generated, there is a high possibility that non-specific adsorption will occur due to the ionic nature of the amino groups.

The purpose of the present invention is to solve the above-mentioned drawbacks, to obtain a carrier for immobilizing physiologically active substances that has excellent acid resistance, is easy to immobilize, and also solves non-specific adsorption after immobilization. do.

〓Means for solving the problems〓 The present invention provides immobilization of physiologically active substances by treating a granular porous chitosan derivative having a carboxyl group with a carbodiimide binder such as dicyclohexylcarbodiimide and N-hydroxysuccinimide. This is a method for producing carriers for

Porous chitosan granules were first published in Japanese Patent Application Laid-Open No. 1986-
It can be obtained by the method disclosed in No. 40337. The chitosan used in the production of porous toxane granules is not particularly limited, but has an average molecular weight of 10,000 to 10,000.
Preferably, a low molecular weight chitosan of 230,000 is used. Chitosan is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, formic acid, etc. alone or in a mixture to obtain an acidic chitosan aqueous solution. The concentration of chitosan acidic aqueous solution is 2
~20% is preferred. The chitosan acidic aqueous solution is coagulated and regenerated in a basic aqueous solution containing an alkaline substance such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia, and ethylenediamine. At this time, alcohol or the like may be added to the basic aqueous solution.

The chitosan aqueous solution is allowed to solidify and regenerate by dropping a certain amount of the chitosan aqueous solution into the basic aqueous solution under pressure from the discharge hole,
Wash thoroughly with water until it becomes neutral to obtain porous chitosan. Porous chitosan may be further crosslinked using an organic diisocyanate compound in a polar solvent, if necessary.

In order to obtain a granular porous chitosan derivative having a carboxyl group from the porous chitosan granules obtained as described above, acylation using a dicarboxylic acid anhydride, a method using a dicarboxylic acid halide, or carboxyalkyl conversion using a monochloroacetic acid, etc. A method such as that described above may be adopted. Furthermore, in order to block the remaining amino groups of chitosan, the amino groups are acetylated with acetic anhydride. After suspending the granular porous chitosan derivative having a carboxyl group thus obtained in a polar solvent such as dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, etc., N-hydroxysuccinimide is added and further A carbodiimide binder such as dicyclohexylcarbodiimide is added, and the reaction is stirred for several hours.
After the reaction, by thoroughly washing the granules with the polar solvent used, an excellent carrier for immobilizing a physiologically active substance can be obtained.

〓Example〓 The present invention will be explained below with reference to Examples, but the present invention is not limited to the scope described in the Examples. Incidentally, the ion exchange capacity and specific surface area were determined by the following methods.

◎Ion exchange capacity Approximately 50 ml of the sample was treated in 500 ml of 1N HCl for 1 hour with gentle stirring, and thoroughly washed with deionized water until it became neutral, taking care not to absorb carbon dioxide gas in the air. Accurately and quickly measure out 30 ml of the dehydrated sample, add it to 500 ml of 1/5N-NaOH, and leave it for 5 hours with gentle stirring. This supernatant liquid is used as the test liquid. 10 ml of this was taken out and neutralized titrated with 1/10N-HCl using the methylred solution as an indicator to determine the amount using the following formula.

CTW (meq/g) = (b-a) x f x 1/10 x 500/10/V x 16 a: 1/10 N-HCl required to neutralize 10 ml of test solution
Amount b: Amount of 1/10N-NCl required to neutralize 10ml of 1/5N-NaOH before adding the sample f: Titer of 1/10N-HCl V: Amount of wet sample (30ml) ◎Specific surface area: It was measured by the BET method using a specific surface area measuring device.

Example 1 70 g of chitosan with a degree of deacetylation of 77% and an average molecular weight of 42,000 was dissolved in 930 g of a 3.5% acetic acid aqueous solution, and the solution was mixed with 7% caustic soda, 30% ethanol, and 63% acetic acid.
was dropped into a mixed solution of water through a nozzle with a pore size of 0.15 mmφ, solidified and regenerated, and then washed with water until it became neutral to obtain porous chitosan particles with an average particle size of 0.1 mmφ. 5.0 g of hexamethylene diisocyanate was added to 100 ml of the obtained porous chitosan granules (in a wet state), and stirred and reacted in dimethylformamide at room temperature for 1 hour to cause crosslinking. After washing with dimethylformamide, dimethylformamide
Add 12.2 g of acetic anhydride in 100 ml, stir at room temperature for 24 hours to acetylate the amino groups, and add this to 48°B'e.
After soaking with caustic soda at 0°C for 1 hour, the mixture was thoroughly washed with isopropyl alcohol, and 10 g of monochloroacetic acid was added to 100 ml of isopropyl alcohol and reacted for 1 hour to carry out carboxymethylation. After washing with water, 95 ml of a granular porous chitosan derivative having carboxyl groups was obtained. The ion exchange capacity of this is
The specific surface area was 0.35 meq/ml and 94 m ² /g. After suspending 50 ml of the granules in 100 ml of dioxane,
Add dicyclohexylcarbodiimide and N-hydroxysuccinimide to a total concentration of 0.1M,
The reaction mixture was allowed to react for 2 hours at room temperature and thoroughly washed with dioxane and methanol to obtain 48 ml of a carrier for immobilizing a physiologically active substance with a specific surface area of 91 m ² /g. Add 1 ml of the carrier to 2 ml of 1% hemoglobin solution (0.1M borate buffer solution, PH8.3).
In addition, it was fixed by shaking for 2 hours. Immobilization amount
The absorbance at 570 nm was determined to be 3.3 mg/ml. In addition, 1 ml of carrier immobilized with hemoglobin
1% bovine serum albumin solution (0.1M Tris
Pour into 2 ml of HCl buffer (PH7.4) and shake for 2 hours to adsorb bovine serum albumin.
When the adsorption amount was measured from the absorbance at m, it was 1.23
There was little nonspecific adsorption at mg/ml. Furthermore, add 1 ml of the hemoglobin-immobilized carrier to 2 ml of 0.1N-HCl.
After shaking for 15 minutes, the amount of shedding was measured by the absorbance of the supernatant at 570 nm, and it was found to be 0.05.
At mg/ml, there was almost no shedding due to acid.

Example 2 Granular porous chitosan obtained in the same manner as Example 1
Suspend 100ml in 100ml of ethanol and add succinic anhydride.
2.0g was added and reacted at room temperature for 24 hours. After washing thoroughly with ethanol, 12.2 g of acetic anhydride was added to 100 ml of ethanol and stirred for 1 hour to block the amino groups and form porous chitosan with carboxyl groups, ion exchange capacity of 0.25 meq/ml, and specific surface area of 63.5 m ² /g. 95 ml of induced granules were obtained. 50 ml of the granular porous chitosan derivative was esterified with dicyclohexylcarbodiimide and N-hydroxysuccinimide in dioxane in the same manner as in Example 1 to obtain 40 ml of a carrier for immobilizing a physiologically active substance with a specific surface area of 72.3 m ² /g. .

When hemoglobin was immobilized in the same manner as in Example 1 using 1 ml of the carrier, the immobilized amount was 8.4 mg/ml,
Bovine serum albumin adsorption amount is 0.95mg/ml, 0.1N−
The amount of shedding after HCl treatment was 0.06 mg/ml, which confirmed that there was little non-specific adsorption and almost no shedding.

Example 3 70 g of chitosan with an average molecular weight of 45,000 and a degree of deacetylation of 82% was dissolved in 930 g of a 3.5% acetic acid aqueous solution, and the solution was mixed with 10% caustic soda, 30% methanol, and 60%
The porous chitosan particles 1 were dropped into a mixed solution of water through a nozzle with a hole diameter of 0.15 mmφ, solidified and regenerated, and then washed with water until neutralized to obtain porous chitosan particles 1 with an average particle size of 0.3 mmφ.

8.3 g of adipic acid dichloride and 12.0 g of triethinamine were added to 100 ml of the granules in 100 ml of dioxane, and the mixture was stirred and reacted at room temperature for 1 hour. After that, wash thoroughly with ethanol, and 12.2g in 100ml ethanol.
of acetic anhydride, stirred at room temperature for 24 hours to acetylate the remaining amino groups, and washed thoroughly with water to reduce the exchange capacity.
88 ml of a granular porous chitosan derivative having a carboxyl group with a specific surface area of 85.2 m ² /g and a specific surface area of 0.31 meq/ml was obtained.

50 ml of the granular porous chitosan derivative was active esterified with dicyclohexylcarbodiimide and N-hydroxysuccinimide in 100 ml of dioxane in the same manner as in Example 1 to obtain 45 ml of a carrier for immobilizing a physiologically active substance with a specific surface area of 83.8 m ² /g. Obtained.

When hemoglobin was immobilized using 1 ml of the carrier in the same manner as in Example 1, the immobilized amount was 12.3 mg/ml, and the adsorbed amount of bovine serum albumin was 0.75 mg/ml.
The amount of shedding measured by absorbance after treatment with 0.1NHCl was 0.11 mg/ml, which confirmed that non-specific adsorption was small and there was almost no shedding.

Comparative Example 1 1 ml of porous chitosan granules obtained in the same manner as in Example 1 was added to 10% glutaraldehyde aqueous solution 2
ml, shake at room temperature for 2 hours, and then wash thoroughly with water.
To this was added 2 ml of a 1% hemoglobin aqueous solution, and the mixture was shaken at room temperature for 2 hours to fix hemoglobin.
The immobilized amount was 14.3 mg/ml. 1 ml of this carrier
After adding 0.1N-HCl and shaking for 15 minutes, the result was 7.0mg/
ml of hemoglobin was lost.

Comparative Example 2 After washing 100 ml of porous chitosan granules obtained by the method of Example 3 with 100 ml of dimethylformamide, 500 ml of dimethylformamide in which 32 g of adipic acid di-N-hydroxysuccinimide ester had been dissolved in advance by heating was added. was added and stirred at 70°C for 2 hours to carry out a crosslinking reaction. Dimethylformamide after reaction
The carrier was washed four times with 500 ml and further washed with water to remove dimethylformamide to obtain a carrier having a specific surface area of 88.3 m ² /g. When 2 ml of a 1% hemoglobin solution (01M borate buffer, pH 8.3) was added to 1 ml of this carrier and the mixture was shaken at room temperature for 2 hours, 13.4 mg/ml of hemoglobin was immobilized. When 1ml of this hemoglobin-immobilized carrier was placed in 2ml of 1% bovine serum albumin solution (0.1M Tris-HCl buffer solution, PH7.4) and the amount of bovine serum albumin adsorbed was measured from the absorbance of the remaining solution, it was found to be 5.3mg/ ml of bovine serum albumin was adsorbed, indicating a high specific adsorption.

Effects of the Invention The physiologically active substance immobilized carrier according to the present invention is obtained from a granular porous chitosan derivative that is porous and has a large specific surface area.
It is characterized by a large amount of adsorption and particularly excellent acid resistance. Furthermore, the carrier for immobilizing a physiologically active substance according to the present invention is obtained by treating a granular porous chitosan derivative having carboxyl groups with N-hydroxysuccinimide using a carbodiimide binder, and in which residual amino groups are Since it is blocked, specific adsorption can be extremely reduced.

Claims (1)

[Claims] 1. A granular porous chitosan derivative having a carboxyl group is mixed with dicyclohexylcarbodiimide and N-
A method for producing a carrier for immobilizing a physiologically active substance, which comprises treating with hydroxysuccinimide. 2. Claim 1, characterized in that the residual amino groups of the granular porous chitosan derivative having carboxyl groups are acetylated.
A method for producing a carrier for immobilizing a physiologically active substance as described in 2.