JPH0446113B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an oxygen fixation carrier used in bioreactors, biosensors, and the like.

〓Conventional technology〓 General methods for immobilizing physiologically active substances such as enzymes on carriers include physical adsorption to water-insoluble carriers,
The carrier binding method uses ionic and covalent bonds, the crosslinking method uses a crosslinking reagent to form crosslinks between molecules and makes them insoluble, and the enzyme etc. is embedded in a carrier gel lattice or the enzyme is coated with a carrier. Comprehensive laws are known. Various materials have been proposed as carriers for immobilizing physiologically active substances, but such materials need to be water-insoluble, inexpensive carriers, and ensure reliable immobilization. , it is preferable to use chitosan derived from chitin as a carrier having such properties.

As mentioned above, various immobilization methods are known for immobilizing physiologically active substances on carriers, but methods for immobilizing physiologically active substances using chitosan as a carrier include crosslinking with glutaraldehyde and NaNO ₂ A covalent bonding method by treatment (Japanese Patent Publication No. 52-3892), a method using adsorption and peptide bonding using a carbodiimide reagent (Japanese Patent Publication No. 53-1989)
−10151) etc. have been proposed.

〓Problems to be solved by the invention〓 The purpose of the present invention is to provide a carrier that can easily immobilize enzymes without impairing enzyme activity, is resistant to changes in pH, and is unlikely to cause enzymes to fall off. shall be.

Of the above types of immobilization, physical adsorption and ionic adsorption have little undesirable effect on enzyme activity, but there is a high risk that the enzyme will fall off due to ionic strength, pH, etc., while covalent bonding is However, the cross-linking method using glutaraldehyde has a strong pH dependence, and enzymes tend to drop off on the acidic side. Furthermore, when water-soluble carbodiimide is used, it takes time and effort to adjust the pH during the reaction, and there is a drawback that the enzyme is greatly damaged and its activity is reduced. Also
A method of introducing and activating a --CNBr group using a CNBr reagent is known, but CNBr is a dangerous reagent and it is not preferable to use such a reagent.

〓Means for solving the problems〓 The present invention provides porous granular chitosan with the general formula (In the formula, n is an integer of 2 to 8, and R represents any of the following groups.

) The carrier for enzyme immobilization is treated with an excess dicarboxylic acid ester represented by
In the present invention, the strength of the porous granular chitosan can be increased by crosslinking with the dicarboxylic acid ester, and the chitosan can be made to contain unreacted ester. This ester reacts with the amino group of the enzyme in water under mild conditions, making it easy to immobilize the enzyme without impairing the activity of the enzyme.

The porous granular chitosan used in the present invention is produced by the method disclosed in JP-A-61-40337. That is, low-molecular-weight chitosan with an average molecular weight of 10,000 to 230,000 is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, or formic acid alone or as a mixture, and the solution is dropped into a coagulation bath consisting of a basic solution to form porous granular chitosan. obtain.

For the coagulation bath, an alkaline substance such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia, or ethylenediamine is added to water, a polar alcohol such as methanol or ethanol, or a mixture of water and alcohol. Use something similar.

Porous granular chitosan produced by the method described above was prepared using an excess of dicarboxylic acid ester.
The crosslinking reaction is carried out in a solvent for about 2 hours at around 0.degree. C. with stirring. The dicarboxylic acid ester has the general formula as shown above. , where n is an integer from 2 to 8, and R is N
-hydroxysuccinimide group, N-hydroxyphthalic acid imide group, pentachlorophenyl group, p-
A dicarboxylic acid ester of any nitrophenyl group is used.

Dimethylformamide, dimethylacetamide, etc. are used as the solvent for dissolving these crosslinking agents, and the concentration of the dicarboxylic acid ester is set to an excess number of moles per amino residue of chitosan, preferably 2.
Use twice the molar amount or more.

After the crosslinking reaction, the solvent is removed to obtain granular chitosan for enzyme immobilization. The carrier for enzyme immobilization according to the present invention has an unreacted ester, which reacts with the amino group of the enzyme in water under mild conditions, and easily immobilizes the enzyme. Since the bond with the enzyme is an amide bond, it is resistant to changes in pH, making it difficult for the enzyme to fall off. Furthermore, in the present invention, by changing the chain length of the crosslinking agent, the length of the spacer between the carrier and the enzyme can be adjusted, and it is possible to introduce an optimal spacer depending on the type of enzyme, resulting in high The activity expression rate and sustainability can be expected.

〓Example〓 The present invention will be described in detail with reference to Examples below, but the present invention is not limited to these scopes.

Example 1 70 g of chitosan having a degree of deacetylation of 98% and a molecular weight of about 60,000 was dissolved in 930 g of a 3.5% acetic acid aqueous solution to obtain a solution with a concentration of 7%. This solution has a pore size of 0.25m/mφ.
drop it into a 10% NaOH aqueous solution from the nozzle,
Porous granular chitosan was obtained. The particle size of this material is 1
m/mφ and specific surface area of 93.5 m ² /g. After thoroughly washing 100 ml of this granular chitosan with water and once with 500 ml of dimethylformamide to remove moisture, 500 ml of dimethylformamide in which 32 g of adipic acid di-N-hydroxysuccinimide ester had been dissolved by heating was added. Stir at 70°C for 2 hours,
A crosslinking reaction was carried out. After the reaction, the mixture was washed four times with 500 ml of dimethylformamide and further washed with water to remove the dimethylformamide to obtain 90 ml of granular chitosan for enzyme immobilization with a specific surface area of 90.3 m ² /g.

The specific surface area of the porous granular chitosan is determined by rapidly freezing the sample in liquid nitrogen, vacuum drying at 10 ^-4 Torr, -40°C for 8 hours,
After degassing for a minute, the specific surface area was measured using an automatic measuring device (Shimadzu Micromeritics Model 2200) using the BET method.

Application example 1 1 ml of granular chitosan carrier obtained in Example 1 above
To this was added 2 ml of a 1.3% aqueous solution of acylase (Acylase 15000 manufactured by Amano Pharmaceutical Co., Ltd.), and the mixture was shaken for 2 hours to effect immobilization. Thereafter, the immobilized acylase activity was measured after washing thoroughly with water until no enzyme was observed to be removed from the washing solution. For activity measurement, 10 ml of 0.1 M N-acetyl-DL-methionine aqueous solution (PH8.0) was added to 10 ml of granular chitosan for immobilizing acylase, and the mixture was reacted at 37°C for 15 minutes. After stopping the reaction, the expression titer of 1 ml of acylase-immobilized beads was measured from the absorbance at 570 nm using ninhydrin coloring. The titer is 1 U, which is the amount of enzyme that produces 1 μmol of L-methionine in 30 minutes.
The result was 76.7U/g.

Application example 2 Glucose isomerase was immobilized on 30 ml of granular chitosan obtained in Example 1 in the same manner as application example 1, and the amount of fructose produced using 45% glucose as a substrate was measured using high performance liquid chromatography. Measured with E. The rate at which the substrate was passed through the column was 2 c.c./min, and the exchange rate SV = 4.0.

1st day... Conversion rate 30.0% 2nd day... Conversion rate 20.1% 7th day... Conversion rate 20.0% Example 2 The crosslinking agent was converted to adipic acid di-p-nitroph in the same manner as described in Example 1. Particulate chitosan for enzyme immobilization was obtained instead of enyl ester, acylase was immobilized by the method described in Application Example 1, and the activity was measured. The results are as follows; high activity was maintained for 5 days.

1st day...expression titer 262.2U/g 2nd day...expression titer 245.4U/g 3rd day...expression titer 218.75U/g 4th day...expression titer 223.2U/g 5th day...expression titer Value: 228.5 U/g 〓Effects of the Invention〓 As described above, the enzyme immobilization carrier obtained by treating porous granular chitosan with a dicarboxylic acid ester according to the present invention has increased strength by crosslinking. It has a high liquid flow rate when packed into a column and can withstand long-term use. Moreover,
Since the carrier made of chitosan of the present invention has an unreacted ester, it easily binds to the enzyme through an amide bond, is resistant to changes in pH, and is unlikely to fall off.

The activity expression rate and sustainability of the enzyme immobilization carrier of the present invention are extremely high as described in the Examples and Application Examples.

Claims (1)

[Claims] 1 Porous granular chitosan is prepared by the general formula (In the formula, n is an integer of 2 to 8, and R represents any of the following groups. ) An enzyme immobilization carrier treated with an excess dicarboxylic acid ester represented by: