JPS6019998B2 - Method for producing immobilized enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an immobilized enzyme, and more specifically, a water-swellable pullulan gel comprising a reaction product of pullulan, which is a water-dropping polymer, and a bifunctional substance, which has an ionic functional group. This invention relates to a method for producing an immobilized enzyme using an ionic pullulan gel obtained by reacting a compound as a carrier.

An object of the present invention is to provide a simple method for producing a stable immobilized enzyme with high enzymatic activity, which enables continuous contact reaction of a substrate with an enzyme, and allows the reaction solution to be transferred from an immobilized enzyme. The object of the present invention is to provide a method for producing an immobilized enzyme that enables efficient use in which the reaction is stopped upon separation.

Examples of ionic polygels used as carriers for enzyme immobilization include ionic cellulose and ionic dextran gels.

However, cellulose is highly crystalline and has poor solubility in water, so the ionization reaction does not proceed quickly, making it difficult to obtain products with high ion exchange ability, and it is difficult to immobilize a large amount of enzyme per unit weight of the carrier. It is difficult to use this method, and therefore it cannot be said that it is fully satisfactory as an immobilization carrier.
It also has the disadvantage of low mechanical strength when water swells. On the other hand, ionic dextran compounds can immobilize a relatively large amount of enzyme per unit weight, but it is difficult to directly obtain dextran with the desired degree of polymerization, and after producing a very high molecular weight product, it is partially hydrated. After decomposition, the desired molecular weight is obtained. In addition, since dextran is a polymer in which glucose units are repeatedly bonded through Q-1,6 bonds, it is easy to form ionic gels with large ion exchange chambers by using the highly reactive hydroxyl group at the C-6 position of glucose units. It cannot be said that it is advantageous in terms of getting more.
For these reasons, ionic dextran gels are very expensive, and this type of material cannot be used as an enzyme immobilization carrier on an industrial scale, except in the case of immobilization methods that allow reuse of the carrier. This poses a huge economic burden. The inventors of the present invention have conducted intensive studies in consideration of these problems, and have found that the ionic gel of pullulan (Hakama Gan Sho 52-37787), which allows for control of the molecular weight during production and has various excellent characteristics, is an enzyme-based gel. It was discovered that the immobilized male body of the present invention has various excellent properties, and the present invention was completed.

The pullulan used in the present invention is a linear polymer in which maltotriose, which is a trimer of glucose, is repeatedly bonded by Q-1,6 bonds, which are different from the trimer. A hydrophilic gel having a three-dimensional network structure can be obtained by reaction with a bifunctional substance that forms an ether bond by utilizing the reactivity of the hydroxyl group contained in the unit.

Furthermore, an ionic pullulan gel is obtained by reaction with a compound having an ionic group at one end and a functional group capable of forming an ether bond with a hydroxyl group in the presence of an alkaline compound at the other end. Although pullulan is composed of glucose units, its molecular structure is completely different from conventionally known polysaccharides such as starch and its derivatives, cellulose and its derivatives, etc., and its properties are also significantly different. For example, pullulan is extremely soluble in both cold and hot water;
The viscosity of this aqueous solution is extremely low, and compared to aqueous solutions of other polysaccharides, this aqueous solution is stable for a long time without phenomena such as gelation or aging, and pullulan itself has no plexus and is biocompatible. It has many favorable properties, including very good properties. There are no particular restrictions on the method for producing pullulan.

Currently, it is generally isolated and collected as an extracellular mucilage by culturing a strain of the genus Plullaria, which is a deuteromycete. If necessary, bacterial cells are removed from the culture solution by centrifugation, and purified by precipitation with methanol. The molecular weight of pullulan is not particularly limited, but it is desirable that the average molecular weight is 1×1 pi or more. The "hardness" of the ionic flurane gel as an enzyme immobilization carrier, that is, the water content in the swollen state, is preferably in the range of 1 to 50 mm, and the shape is preferably spherical with a diameter of 10 to 500 mm. desirable.

It is also effective to manufacture spherical pullulan gel according to the patent application No. 76267/1983.

Furthermore, it is most effective to produce ionic pullulan gel according to the method described in Hakamagao Sho 52-37787. Suitable bifunctional substances include epichlorohydrin, epibromohydrin, diglycidyl ether, ethylene glycol-diglycidyl ether, and the like. Spherical water-swellable pullulan gel is produced in the presence of alkaline substances such as sodium hydroxide and potassium hydroxide.
A liquid dispersion medium which is immiscible with the pullulan solution and contains a dispersion stabilizer such as polyvinyl acetate, polystyrene, etc.
This is achieved by reacting in a two-phase system in which droplets are dispersed in (for example, n-hexane, heptane, isooctane, toluene, etc.) while controlling the rate of droplet formation.

Ionic pullulan gel is produced using the general formula Z may be a carboxyl group, a phosphoric acid group, a sulfonic acid group, a guanide group, or a salt thereof, or a general formula or (where R, , R2 and R3 are each hydrogen, methyl group,
They are ethyl group, hydroxyethyl group, hydroxybropyl group or phenyl group.

) is reacted with a spherical water-swellable pullulan gel in the presence of an alkaline substance such as sodium hydroxide or potassium hydroxide at 100 qo or less. This is achieved by Suitable compounds represented by the general formula Examples include 8,y-epoxypropyltriethylammonium chloride and their salts with inorganic acids or alkyl halides. Examples of those having a carboxyl group include chloroacetic acid, bromoacetic acid, and those having a sulfonic acid group. Examples include chloroethanesulfonic acid, bromethanesulfonic acid and salts thereof.

The enzyme can be bound to the ionic Bullulan gel carrier thus prepared by both ion adsorption and covalent bonding methods. In the case of the ion adsorption method, a cationic or anionic ionic pullulan gel may be selected as the rod in consideration of the relationship between the isoelectric point of the enzyme to be immobilized and the action shoe of the enzyme.

For example, in the case of an enzyme that is an acidic protein with an isotropic point around pH 5 and has an optimum pH around pH 7, it is preferable to use a basic pullulan gel such as jethylaminoethylated pullulan gel as a support. When immobilizing an enzyme that acts near neutrality using a basic protein with a constant dew point of about 1 cape, it is preferable to use an acidic pullulan gel such as carboxymethylated pullulan gel as the immobilizer. Thus, the immobilized iQ enzyme can be prepared by the ion adsorption method using conventional methods such as
．． The ionic pullulan gel is treated with a hydrochloric acid solution at a concentration of 0.02 M to IM or a caustic soda solution at a concentration of 0.02 to IM to activate the phosphorus exchange group, or a buffer having a buffering effect on the working pH of each enzyme ( 0.
This can be achieved by buffering the ionic pullulan gel with a solution of the enzyme to be immobilized for a sufficient period of time (02M or IM concentration), immersing it in the solution of the enzyme to be immobilized for a sufficient period of time, and then filtrating it as necessary, filtering it, and washing it with water. Ru.

The temperature at which adsorption and immobilization is performed is preferably 4.0 or lower, especially 4.0 or lower.
Preferably around ℃. It is desirable that the adsorption time be 2 hours or more. The immobilized enzyme thus obtained usually contains 100 or fewer enzyme proteins per dried morsel and is stable unless purified with a highly active, high ionic strength salt solution. On the other hand, in the case of covalent bonding, various bonding methods are used that utilize the reactivity of hydroxyl groups in ionic bullulan gel, introduced ionic groups such as primary amino groups, secondary amino/carboxymethyl groups, etc. It has been found that all of the three commonly known binding methods effective for ionic cellulose and ionic dextran are applicable, and that an immobilized enzyme with high activity and high activity retention can be obtained.

In particular, '11 Coupling method using triazinyl derivatives using cyanuric chloride, '21 Coupling method using azide bond, '3'
Bonding methods using diazo bonds, bonding methods using monohalogen acetyl derivatives, '5) bonding methods using carbodiimide derivatives, and '6) bonding methods with carriers using glutaraldehyde, etc. are used as hydroxyl groups or ionic groups in gels. It is excellent for immobilization by utilizing the reactivity of introduced primary or secondary amino groups, carboxyl groups, etc.

In this way, the immobilization method using the covalent bond method has a more complicated immobilization operation than the immobilization method using the ion adsorption method, but the immobilized IQ enzyme, which is more stable than the immobilized IQ enzyme in the ion adsorption method, is bound. The enzyme to which the immobilization method of the present invention is applied is not particularly limited as long as the enzyme activity is completely eliminated by the above-mentioned binding method.

For example, trypsin, chymotrypsin, lipase, protease of microbial origin, esterase, cholinesterase, urease, promelain, ribonuclease, theoxyliponuclease, penicillin amidase, aminoacylase, P-galactosidase, glucose isomerase, glucose oxidase, creatine kinase, Oxidase, papain, inverdase, pepsin, 8-amylase, isoamylase, maltase, uricase, etc. are effectively immobilized. Next, the present invention will be explained in more detail with reference to examples, but these are merely illustrative and the invention is not limited to the following examples and various modifications can be made as long as they do not exceed the spirit thereof.

Example 1 Pullulan 4 base having an average molecular weight of 72,000 was dissolved in 80 parts of water, and 12.5 parts of sodium hydroxide was added to form a uniform aqueous solution.

This solution was mixed with 1 part of polyvinyl acetate and 16 parts of toluene.
0 parts of solpitan monostearate and 1.2 parts of solpitan monostearate were added to the dispersed soot and dispersed in the form of droplets while rotating at 800 revolutions per minute. After adding epichlorohydrin and reacting at 50 qo for 5 hours, filtering and refining with toluene and methanol, drying under reduced pressure, a spherical hydrophilic product with a moisture content of 2.35 q/q. obtained pullulan gel. 10 parts of this pullulangel were dispersed in an aqueous solution of 48 parts containing 11 parts of sodium hydroxide, and while worshiping at room temperature, 24 parts of 2-jethylaminoethyl chloride hydrochloride was dissolved in the part of water pine. The solution was dropped over 4 hours, reacted for an additional hour after dropping, washed with water and methanol, and the diameter when dried was 3.
A diethylamide/ethylated pullulan gel (abbreviated as DEAE-pullulan gel) was obtained, which was a spherical gel of 7 to 149 mm, had a water content of 2.8 wc/d, and an amine amount determined by conductivity titration of 2.7 wc/d. . The DEA thus obtained
A commercially available enzyme pronase was immobilized as an E-Bullan gel carrier by an ion adsorption method as shown below. That is, the D
EAE-Pullulan gel (when dried) was treated with a 0.1 normal concentration caustic soda solution and thoroughly washed with water to remove excess caustic soda. Commercially available pronase E50 female was dissolved in a phosphate buffer solution with a pH of 7.0 and a concentration of 0.02M.
The DEAE-Pullulan gel activated with caustic soda was poured into the container, and the enzyme was adsorbed to the DEAE-Pullulan gel while slowly anchored at 4°C for 5 hours. It was thoroughly purified with phosphate buffer solution at a concentration of .02M (pH 7.0) and water.

The furnace solution and washing solution were collected, and the amount of enzyme adsorbed and immobilized on the DEAE-Pullulan gel was calculated to be 39 samples based on the ultraviolet absorption intensity of the recovered solution at 28 samples. This immobilization was carried out to determine the specific activity of the enzyme at pH 7.0 using N-benzoyl-L-arginine ethyl ester (BAEE) as a substrate. u 30℃
When measured with a PH stat (Hiranuma PHstat-11), it was 3.2 mmoleg/M min under conditions where the amount of substrate was in excess of the amount of enzyme. It had 35% activity of the enzyme in solution before immobilization. When the activity of Koen Sadai Q enzyme was measured 5 times under the same conditions as above, the activity at the 5th time was about 73% of the activity at the 1st time. Example 2 The enzyme pronase E7o was incubated at 4°C with axis 6.0 and 0.
Dissolve in 25' of phosphate buffer with a concentration of 0.8M.

This solution has the same properties as those used in Example 1.
Add EAE-Puljarangel 1.0 ml (dry),
The enzyme was immobilized by slowly shaking the tube while keeping it at about 7°C. The immobilized pronase E was purified with a 0.08M phosphate buffer (pH 6.5), a 0.1M sodium chloride solution, and pure water until no protein absorption was observed in the washings, and then washed. The knee fluid was collected, and the amount of immobilized enzyme was calculated from the ultraviolet absorption intensity of the protein in the collected fluid to be 49 females. The specific activity of this immobilized enzyme was measured using a pH stat using DL-lysine methyl ester as a substrate.
When measured at 0.040 oo, the specific activity of the enzyme in the form of a solution was 37%. Example 3 Three parts of commercially available papain 100 was dissolved in 30' of a 0.02M phosphate buffer solution with a pH of 6.0 kept at 4°C, and 24.5 parts of epichlorohydrin was added to this solution. A spherical hydrophilic pullulan gel with a water content of 2.5 days/day obtained using the same compound and reaction conditions as in Example 1 except for the following: 3 parts of monochloroacetic acid, Add 2 parts of water and react in 20 parts of methanol at about 10°C for 6 hours to obtain a powder with a dry diameter of 7.
It is a spherical gel with a diameter of 4 to 149 mm, a water content of 2.9 mm, and a carboxyl group content of 3.5 mm as determined by conductivity titration.
1 meq/ta carboxymethyl pullulan gel (CM
- Pullulan gel) 1.0 ml was added, and the enzyme was immobilized by slowly stirring while keeping the temperature at about 4°C.

The immobilized enzyme and enzyme were washed and removed using the same machine as in Example 2, and the amount of protein in the recovered solution was determined by the Lowry method, and the amount of immobilized enzyme was calculated to be 30.1 female. The specific activity of this immobilized enzyme was measured by PH stat at 40°C using BAEE as a substrate and found to be 1.70 moles/
moth/min, which is 2 of the specific activity of the enzyme in solution before immobilization.
It was 3%. When measuring the activity of the fixed IQ enzyme and the enzyme in solution, ethylenediaminetetraacetic acid at a concentration of 2x10-3M, cysteine at a concentration of 5x10-3M, and sodium chloride at a concentration of 0.3M were allowed to coexist. Example 4 5 ml of commercially available purified trypsin was added to 30 ml of 0.02 M Tris-HCl buffer (0.0
CM-Pullulan Gel 1.0 used in Example 3 was added to this solution (containing 2M calcium chloride), and the enzyme was immobilized while being kept at about 4°C and slowly stirred for about 3 hours. It became.

For immobilization, the amount of enzyme was calculated to be 9 from the ultraviolet absorption intensity of the protein in the washed rice bran recovery solution. The specific activity of this immobilized trypsin was determined at pH 7.5 and 30°C using BAEE as a substrate.
In the presence of calcium chloride at a concentration of 0.02 M, the pH value was 5.5 moles/min, which was 21% of the specific activity of the enzyme in solution. Example 5
Crush the Streptomyces phaechromogenes viable cells,
Glucose disomerase with an activity of 750 skin nits and a protein amount of 84 o, which was determined by the Lowry method, was purified by the acetone precipitation method using a refrigerated centrifuge. Salt buffer solution 25 [
dissolved in it.

Hydrophilic pullulan gel 32 prepared in Example 3 was added to this solution.
35 parts of sodium hydroxide and 15 parts of water were dissolved in an alkaline aqueous solution, and 2 parts of Isobe was dissolved in 4 parts of water and 1 part of water.
- A spherical gel with a dry diameter of 39 μm to 1494 μm obtained by dropping jetylaminoethyl chloride hydrochloride and reacting at room temperature for 18 hours, with a water content of 30 μm, determined by conductometric titration. DEAE whose basic amount of ethylaminoethyl (DEAE) is 2.8 heq/night
- Pullulan Gel 3.0 was added, and while the solution was kept at about 15° C., it was shaken and fixed in a PM at 6 rotations for 10 hours. After immobilization, the immobilized enzyme was separated in a furnace and incubated at pH 7.5 to 0.
After thorough washing with a phosphate buffer solution of 0.8M concentration, the immobilized activity was calculated to be 6 points, and the protein amount was calculated to be 71/9 by measuring the activity and protein of the furnace solution. The thus obtained immobilized glucose isomerase was packed into a column with a jacket tube having a diameter of 1.0 mm, and while the temperature of the jacket tube was maintained at 600m, a 54W/V% crystalline glucose aqueous solution (5 x 10-3M
(containing concentrated MgH ions and adjusted to pH 7.5) was pumped down from the top of the column to perform an isomerization reaction. The flow rate was kept at SV = r-1. As a result of continuous reaction day and night, the isomerization rate to flatose was maintained at 51.5% for about 35 hours after the start of the reaction, after which the activity slowly decreased. The half-life was approximately 50 days. Note 1
) Glucose isomerase 1 skin it was prepared using a phosphate buffer solution with a concentration of 0.08M and MgS04 with a concentration of 0.003M.
This is the amount of enzyme that will produce 1 ounce of fructose when reacted for 1 hour with 0.1 M concentration of D-glucose as a substrate at pH 7.0 and 70° C. in the presence of 7 days and 20 days.

Note 2) Fructose was determined by the cysteine-carbazole sulfuric acid method according to the JAS regulations.

In the following examples, glucose
t and fructose were determined according to Note 1) and Note 2). Example 6 Glucose isomerase purified in the same manner as in Example 5 and having an activity of 760 and a protein amount of 70 was fed to bait 7.
．． 5 and dissolved in phosphate buffer at a concentration of 0.08M for 25 minutes.

Soak the hydrophilic pullulan gel prepared in Example 3 in this solution for 1 and 3 dollars. An alkaline aqueous solution of B-0-hydroxypropyltrimethylammonium chloride epoxidized with 6 parts of sodium hydroxide.
A spherical gel with a dried diameter of 39 mm to 149 mm is obtained by adding 1.5 mm to 149 mm in dry diameter.
8-HPTMA, which is a spherical gel of 49 μm and has a water content of 2.8 meq, and a base amount of r-hydroxypropyltrimethylamine/(8-HPTMA) determined by conductivity titration method is 0.57 meq/unit. - Pullulan gel 3.0 was added, and while maintaining the temperature from 15q0 to 20q0, it was shaken for 10 hours at a PM rotation rate of 8 to immobilize it. After immobilization, Example 5
The immobilized enzyme was washed in the same manner as above, and the immobilized activity was calculated to be 711 m and the miL immobilized protein was calculated to be 65 o from the washed rice bran recovery liquid.

The thus obtained immobilized glucose isomerase was packed into a column with a jacket tube of 15 ribs in diameter and the temperature of the jacket tube was kept at 65°C.
54 W/V% crystalline glucose aqueous solution (5
x10-3M concentration of MgSO4・7days20, pH
7.5) from the top of the column with a pump.
V] Water was allowed to flow down at a rate of r-1 to perform a continuous isomerization reaction. After the start of the reaction, the spheres remained for 35 q hours. The heterosexual IG rate was maintained at 5%. Example 7 Obtained as an extracellular enzyme of Asparagillus oryzae,
8-galactosidase 100c partially purified by alcohol precipitation was mixed with citric acid at pH 5.5 at a concentration of 0.02M.
25'' of phosphate buffer was dissolved.

To this solution, the same DEAE-Pullulan gel 1.0 as used in Example 5 was added, and while keeping the liquid at about 4°C,
The face was shaken at a rotation speed of 60 RPM and fixed. After immobilization, the amount of immobilized enzyme protein was calculated to be 40 females from the ultraviolet absorption intensity of the protein in the rice bran water washed with a 0.02M concentration pH 5.5 citric acid-phosphate buffer and pure water. The amount of immobilized iQ enzyme was also calculated by the Lowry method and was found to be 43o. The activity of this Enzyme was measured at pH 4.0 using 5W/V% lactose as a substrate. When measured at 30°C, the specific activity was 1.74 rmoles/min. The activity was measured by a colorimetric method in which the amount of glucose produced by the reaction was reacted with a mixed reagent of glucose oxidase dye. Example 8 The same DEAE-Pullangel 2 used in Example 1
In the evening, add 25% of caustic soda to an IN concentration of 1% at room temperature.
After lighting for 8 minutes, excess alkaline solution was removed by filtering.

The DEAE-Bullan gel was then soaked for 5 minutes at room temperature in 25% dioxane, and a previously prepared 20% dioxane solution containing 40% cyanate chloride was added and stirred at room temperature. After 1 minute, the reaction was stopped by adding 25' of ice-cold water followed by 25' of acetic acid.

After filtering the mixture, the DEAE-pullulan gel was quickly washed with cold acetone and ice-cold water, and immediately subjected to a fixation reaction. That is, 170 female pronase was dissolved in 20% phosphate buffer and the S-triazinylated DEAE obtained by the above method was added to a solution kept at pH 80.4°C.
- Add pulluranger and add 0. The pH was maintained at 8.0 by the addition of caustic soda at a concentration of 100.degree. C., and fixation was carried out for 5 hours while being careful not to raise the temperature above 4.degree. After 5 hours, the immobilized enzyme was pierced in an oven and mixed with 8 M sodium chloride solution and 0.1 M phosphate buffer (pH
6.0) and cold water until protein was found in the washing solution and no protein was removed.

The amount of immobilized enzyme, calculated from the ultraviolet absorption intensity of the collected solution at 28 meters, is 6 per evening of dry gel.
It was 6c. The specific enzyme activity of the immobilized enzyme thus obtained was determined to be 2.94 mmoles/mole/mole when measured using DL-lysine methyl ester as a substrate at a substrate concentration of 2% by weight at 30°C and pH 6.0. The specific activity of the enzyme in solution was 60%. Example 9 DEAE-Bullanger 1.0, which had exactly the same properties as used in Example 1, was immersed in 35 cm of water, and Bromcyan 1.
．． Added 0 evenings.

While cooling to 4°C, add a solution of caustic soda at a concentration of 1.10 to the mixed solution while keeping the pH at 1.10. After the pH has stopped decreasing, the gel is immediately heated to a furnace and heated to a concentration of 0.1M. It was quickly purified with phosphoric acid buffer (pH 8.0).

DEA activated with Promcyan thus obtained
0.1M containing E-Pullangel and Bronase E8
Pronase E was immobilized by transferring it to a borate buffer solution (pH 80) with a concentration of 10 Z and gently shaking it back and forth at room temperature for 2 hours. After washing the pronase-immobilized gel with 10 times more water to immobilize unreacted active groups, 1. ■
The fins were immersed in an ethanolamine solution (pH 8.0) with a concentration of about 100 ml, and the fins were kept at room temperature for 2 hours. The enzyme-immobilized gel was separated into I
It was washed repeatedly with a 0.1 M acetate buffer solution (pH 4.0) containing an M concentration of sodium chloride, a 0.1 M borate buffer solution (pH 80) containing an IM concentration of sodium chloride, and cold water. Collect the washing bran liquid, and add 2 of the collected liquid.
The amount of immobilized enzyme calculated from the ultraviolet absorption intensity in blood was 31.3 per 1.0 of dry gel. The specific activity of the immobilized enzyme was determined using DL-lydimethyl ester as a substrate.
312 mmoles/mole as measured by State Stat at 40°C and sleeve 6.0 at a substrate concentration of 2% by weight.
n, which was 52% of the specific activity of the enzyme in solution. Example
10 8-HPTMA-Pullulan gel 2.0 having exactly the same properties as in Example 6 was reacted with cyanate chloride under the same conditions as in Example 8, washed with rice bran, and S-triazinylated HPTMA-
I got Pullulanger.

An o. s- in 25' of phosphate buffer with a concentration of 08M.
A triazinylated hydrophilic gel was added, and immobilization and washing were performed in the same manner as in Example 8. Phosphate buffer solution containing glucose at a concentration of 0.1 M (0.09 M concentration, fence 7
．． Contains magnesium ions at a concentration of 0.08M) 50
Using '? When the activity of this immobilized iQ enzyme was measured after being incubated at 0°C for 1 hour, the immobilized enzyme was 627
It was a mountain hit.

This immobilized enzyme was packed into a column with a jacket tube with a diameter of 12 ribs, and while circulating hot water at 60°C through the jacket tube, Hiro W/V% (
$M) concentration of crystalline glucose solution (pH 7.0.0)
SV=3.08M concentration of magnesium ions). When the amount of fructose in the effluent was determined by the cysteine-carbazole sulfuric acid method, the conversion rate from glucose to 7-lactose was 52%. Example 11 The same CM-Purlangel 2 as used in Example 3 was used for 4
The mixture was immersed in 0.0 methanol and methylesterified by a conventional method using hydrogen chloride gas, and further hydrazidated with foamy hydrazine.

Thereafter, azidation was performed with 3% sodium nitrate solution, and immediately 25% of phosphate buffer containing 1000 Sumner units of commercially available urease was added.
After immobilization reaction was carried out with slow shaking at 4°C for 12 hours, a NaC solution with an OM concentration of 0.1
The immobilized enzyme was purified with M phosphate buffer (pH 6.7) and distilled water. The enzyme activity of the obtained immobilized enzyme was determined by the colorimetric method of Van Slice et al. and was found to be 500 Sumner units. Example 12 DEAE-Pullulan gel 2.0 having exactly the same properties as used in Example 5 was reacted with cyanate chloride under the same conditions as in Example 8, washed in the same manner as in Example 8, and S-triazinyl DEAE-pullulan gel was obtained.

Claims (1)

[Claims] 1. Crosslinked water-swellable pullulan gel obtained by reacting pullulan with a bifunctional compound and 1. General formula X-R-Z [
However, X is a halogen or an epoxy group, and R is a hydrocarbon group containing an aliphatic group or (and) an aromatic group having 1 to 20 carbon atoms, which may contain one or more hydroxyl groups, and one or more hydroxyl groups. or more heteroatoms such as oxygen atoms may be interposed, and Z may be a carboxyl group, a sulfonic acid group, a phosphoric acid group, a guanide group, or a salt thereof, or a general formula▲Mathematical formula, chemical formula, table, etc.▼ Or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (Here, R_1, R_2 and R_3 are hydrogen, respectively.
These are an alkyl group having 1 to 3 carbon atoms, a hydroxyalkyl group, or a phenyl group. ) or a salt thereof. A method for producing an immobilized enzyme, which comprises immobilizing the enzyme by an ion adsorption method or a covalent bond method using an ionic pullulan gel obtained by reacting a compound represented by the following as a carrier.