JPS6051485B2 - Method for manufacturing carrier for enzyme immobilization - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing a carrier for enzyme immobilization.

Traditionally, enzymatic reactions have generally been carried out by reacting a substrate with an aqueous solution of the enzyme, but this method has many drawbacks.

In particular, it is not easy to separate the reaction product from the enzyme after the reaction is complete, and even if the enzyme still has activity, a method of denaturing it and separating it from the product is usually adopted. Since it is a batch method, it is not economical.
0, which is inferior to productivity ← Kotsukuda Junior High School 14-1
A method has been proposed in which the enzyme is converted to i-7' to facilitate separation of the reaction product and the enzyme, and the enzymatic reaction is carried out continuously.

Various methods have already been proposed for immobilizing this enzyme, but in all of them, the binding strength between the enzyme and the carrier and the activity of the enzyme in the obtained immobilized enzyme are insufficient, and the manufacturing of the carrier used is complicated. There were various problems such as the need for additional steps. The present invention was made to solve the above problems, and provides an enzyme that has excellent physical strength and can be immobilized with high activity by covalent bonding, ionic bonding, etc. The object of the present invention is to provide a simple method for producing an immobilization carrier. The method for producing a carrier for enzyme immobilization according to the present invention involves reacting a polyaldehyde having at least two aldehyde groups in one molecule with polyvinyl alcohol, crosslinking it to make it water-insoluble, and then using the crosslinked polyvinyl alcohol. It is characterized by reacting the free aldehyde groups possessed by a polyamine having at least two primary amino groups in one molecule. F In the present invention,
Polyaldehyde is a compound (including a polymer compound) having at least two free aldehyde groups in one molecule.
), specifically dialdehyde compounds such as glyoxal, malondialdehyde, maleic dialdehyde, succinic dialdehyde, 5-adipine dialdehyde, glutaric dialdehyde, pimeline dialdehyde, speringaldehyde, terephthalic dialdehyde, etc. High-molecular polyaldehydes such as, dialdehyde starch, and polyacrolein can be used, but glutardialdehyde and terephthaldialdehyde are preferably used because they have excellent crosslinking properties, are stable, and are convenient to handle. Polyvinyl alcohol (hereinafter referred to as PVA).

), the degree of polymerization is 300 to 2500 and the degree of saponification is 8.
Generally commercially available compounds containing 0 mol % or more can be used. The reaction between PVA and polyaldehyde may be carried out by a conventionally known method, and is usually carried out using hydrochloric acid, sulfuric acid, p-
This can be carried out in the presence of an acid catalyst such as toluenesulfonic acid.

Specifically, for example, PVA and polyaldehyde are dissolved or dispersed in water, methanol, ethanol, propanol, cyclohexane, benzene, dimethyl sulfoxide, or a mixture of two or more thereof, and if necessary, in the presence of an acid catalyst. Heat and react. In addition, PVA molded products,
For example, a method such as immersing the film in a solution containing polyaldehyde and a catalyst may be used. No matter what reaction method is adopted, in the present invention, PVA is crosslinked with a polyaldehyde and made water insoluble to form crosslinked PV
When obtaining A, it is necessary to leave free aldehyde groups in this crosslinked PVA.

Therefore, when dialdehyde compounds are used, some of the aldehyde groups are PVA. is acetalized, but the other aldehyde group must remain unreacted. In addition, when polyaldehyde having three or more aldehyde groups in one molecule is used, a part of it is bonded to PVA through at least one aldehyde group, and even if PVA is cross-linked, , but at least one aldehyde group must remain unreacted. The required amount of polyaldehyde depends on the reaction method of polyaldehyde and PVA.
It can be easily determined experimentally. Generally, when a certain amount of PVA is reacted with polyaldehyde, when the polyaldehyde exceeds a certain amount, the amount of free aldehyde in the crosslinked PVA becomes approximately constant. In the present invention, it is sufficient to react PVA with the minimum amount of polyaldehyde that makes the aldehyde groups in the crosslinked PVA almost constant, but usually crosslinking is carried out by reacting PVA in an excessive amount of polyaldehyde solution. All you have to do is get PVA. In the present invention, the water-insoluble crosslinked PVA obtained as described above is reacted with a polyamine having at least two primary amino groups in one molecule to obtain a carrier having free primary amino groups.

Polyamines include ethylene diamine, propylene diamine, butylene diamine, hexamethylene diamine,
Aliphatic polyamines such as polyethyleneimine, phenylenediamine, diaminodiphenyl ether, diaminodiphenylmethane, diaminodiphenylsulfone, diaminodiphenylamine, benzidine, xylylenediamine,
Aromatic polyamines such as triaminobenzene and triaminotoluene are used. When the polyamine is water-soluble, the reaction between the crosslinked PVA and the polyamine is conveniently carried out in water.

However, when it is sparingly soluble in water, such as 4,4''-diaminodiphenyl ether, it may be reacted with crosslinked PVA under acidic conditions as an amine hydrochloride, or it may be reacted in an appropriate organic solvent that dissolves the amine. Cross-linked PVA
The amount of polyamine to be reacted is an equimolar amount to the free aldehyde group in the crosslinked PVA, but usually an excess amount may be reacted.

Incidentally, the azomethine group of the Schiff base formed by the bridge PVA and the polyamine may be stabilized by reducing it with a reducing agent such as sodium borohydride.

Next, in order to immobilize the enzyme on the carrier thus obtained, preferably a covalent bonding method or an ionic bonding method is employed.

As a covalent bonding method, for example, a crosslinking reagent having two or more aldehyde groups in one molecule, such as glutardialdehyde, is reacted with the carrier, and the amino group of the enzyme is bonded to the free aldehyde group of the generated Schiff base. , O- on the carrier
A method is used in which bromoacetyl-N-hydroxysuccinimide is reacted and then the enzyme is reacted and then immobilized, and a method in which the carrier is reacted with succinic anhydride and then the enzyme is bonded in the presence of water-soluble carbodiimide. Examples of ionic bonding methods include a method in which a carrier is reacted with 4-(diethylamino)benzaldehyde and then the enzyme is ionically bonded; a method in which the carrier is reacted with 3-diethylaminopropyl chloride and then the enzyme is ionically bonded. is used. As described above, enzymes can be bound to the carrier of the present invention by various methods, but enzymes having free amino groups are preferably immobilized by a covalent bonding method using glutardialdehyde as a reagent. be able to.

Further, in this method, preferable results can be obtained by using a carrier which is made into a Schiff base by reacting crosslinked PVA with p- or m-xylylene diamine. The enzyme that can be immobilized on the carrier of the present invention is not particularly limited as long as it is an enzyme that can be immobilized by a bonding method that utilizes the amino groups of the carrier, but preferably, as described above, the enzyme may be immobilized with glutardialdehyde as a reagent. It is an enzyme with a free amino group that can be obtained.

Specific examples of such enzymes include oxidoreductases such as glucose oxidase and catalase, transferases such as aspartate aminotransferase and histamine transferase, lipase, α-amylase, β-amylase, urease, and α-chymotrypsin. Examples include hydrolytic enzymes such as, glucose isomerase, isomerase such as alanine racemase, and ligases such as asparagine synthase and glutamine synthase. The immobilized enzyme, in which the enzyme is covalently immobilized on the carrier as described above, can be easily separated from the reaction medium such as a buffer by means such as filtration or centrifugation. may be washed, dried, or stored in a buffer solution at low temperature.

According to the method of the present invention, as described above, since PVA is crosslinked with polyaldehyde, the obtained carrier has excellent physical strength, stability, durability, etc.
By bonding a polyamine to the free aldehyde group of
Since the enzyme is bound to the PVA main chain via this spacer group, the resulting immobilized enzyme has a large degree of freedom and is highly active.

Examples of the present invention are listed below, but the present invention is not limited to these Examples.

Example 1 A film with a thickness of 80μ manufactured from an aqueous solution of PVA (Kuraray PVA-H Co., Ltd.) by a casting method was coated with a film containing 5% by weight of glutardialdehyde and 1% by weight of boronic acid.
PVA was immersed in N-hydrochloric acid aqueous solution at 40'C for 3 hours.
was crosslinked.

This crosslinked film was thoroughly washed with an aqueous sodium carbonate solution, distilled water, methanol, and distilled water in this order, and dried. Next, this crosslinked PVA film was immersed in a 5% by weight aqueous m-xylylene diamine solution at 40°C for 2 hours, and then washed with methanol and distilled water in that order to obtain a carrier according to the present invention.

This film-like carrier was pH adjusted with 0.1M phosphate buffer.
6. 1. to a 5% by weight aqueous glutardialdehyde solution adjusted to O at room temperature. After being immersed for a period of time to react, the pH was 7. O
The cells were thoroughly washed with phosphate buffer.

Next, this film-like carrier was coated with StreptOmyce
1 ml of partially purified glucose isomerase originating from the genus S (activity 6000 U/Mt) was dissolved in phosphate buffer (201T1M
．． pH 7.0) in an aqueous solution of 20 mL at room temperature.
Glucose isomerase was immobilized on the carrier by immersion for a period of time. 0.1M maleic acid, 0.1M Tris, 0.1MM
gS04・7H20 and 2rr1MC0C12, PH7
．． The immobilized enzyme was thoroughly washed with a tris-maleate buffer solution adjusted to a pH of 5.5 using tris-maleate buffer.

The activity of this immobilized enzyme was 110 U/q.

The method for measuring activity is as follows. That is, glucose in the above tris-maleate buffer? 5 mt of this substrate solution was reacted with the immobilized enzyme at a temperature of 60° C. for 3 minutes, and the activity to produce 1 μM of fructose per minute was defined as unit activity U. Incidentally, the fructose concentration was measured by the cysteine-sulfuric acid-carbazole method. Comparative Example 1 Using the cross-linked PVA film obtained in Example as a carrier, it was immersed in an aqueous solution in which the same glucose isomerase 1Tnt used in Example 1 was dissolved in phosphate buffer at room temperature for a period of time to remove glucose isomerase. It was immobilized on a crosslinked PVA film.

The activity of this immobilized enzyme was measured in the same manner as in Example 1 and was found to be 8 U/V. Example 2 0.0 parts of terephthalaldehyde was added to 10 parts of a 5% by weight aqueous solution of PVA (Kuraray PVA-117). After adding the heel portion and dissolving it by heating it to 60℃,
5 parts by weight of 2N hydrochloric acid was mixed to gel the PVA.

Using a homogenizer, mix this gel with a diameter of 0.2 to 0.6
The resulting gel was prepared into a granular gel of 1.0 mm in size, and thoroughly washed in the order of aqueous sodium hydroxide solution, water, methanol, and water. This crosslinked PVA25y was immersed in 200 ml of a 5% by weight aqueous m-xylylene diamine solution at 600C for 1 hour to form a Schiff base, and then thoroughly washed in the order of methanol and water to obtain a carrier. This granular carrier was added to a phosphate buffer (a). 1M) and pH 6. After immersion in a 5% by weight aqueous glutardialdehyde solution adjusted to O for 2 hours at room temperature, phosphate buffer (a). 1M,,PH7
．． 0) was thoroughly washed.

Next, the polyaldehyde-treated carrier was added to a phosphate buffer (4). 1M. , pH 7.0), and an enzyme solution in which urease 2y was similarly dissolved in 100 ml of a buffer solution was added to the above carrier dispersion, and the mixture was gently stirred at a temperature of 5°C for 24 hours. Thereafter, unimmobilized urease was removed by washing with a buffer solution to obtain immobilized urease. The amount of uretase immobilized was 32 m9 per y of carrier, and the activity was 40 U/y per carrier, and the activity yield was 84%.
It was hot. The unit activity U was defined as the activity that produced 1 m9 of ammonia per 5 minutes at 20° C. and pH 6.7 using a 0.03 ISv4 urea aqueous solution as a substrate solution. Comparative Example 2 Using the crosslinked PVA particles obtained in Example 2 as a carrier, urease was immediately immobilized under the same conditions as in Example 2.

Claims (1)

[Scope of Claims] 1. Polyaldehyde having at least two aldehyde groups in one molecule is reacted with polyvinyl alcohol, crosslinked to make it water-insoluble, and then the free aldehyde groups of this crosslinked polyvinyl alcohol are added to the free aldehyde groups in one molecule. A method for producing a carrier for immobilizing an enzyme, which comprises reacting a polyamine having at least two primary amino groups with a polyamine having at least two primary amino groups. 2. The method for producing a carrier for enzyme immobilization according to claim 1, wherein the polyaldehyde is glutardialdehyde or terephthalaldehyde, and the polyamine is xylylenediamine.