JPS597436B2 - immobilized enzyme - Google Patents

Description

[Detailed description of the invention] TECHNICAL FIELD The present invention relates to a commercializing enzyme.

Enzyme reactions are used particularly in the food and pharmaceutical industries due to their substrate specificity and high reactivity under normal pressure. Conventionally, enzyme reactions are carried out by dissolving enzymes in an aqueous solution of the substrate. It was set.

Therefore, according to this method, it is very easy to maintain the reaction conditions constant, replenish fresh enzyme, and separate the product and enzyme without deactivating the enzyme after the reaction. This method is difficult, the enzyme is consumed uneconomically, and the reaction is batchwise, resulting in poor productivity.

Therefore, in order to solve these problems, it has been proposed to immobilize enzymes on water-insoluble carriers using various methods and to cause the immobilized enzymes to react continuously with substrates, and some have already put it into practical use. has been done.

As a method for immobilizing enzymes, the covalent bond method, in which the enzyme is bound to the carrier through a covalent bond, is widely used because the enzyme does not easily detach from the carrier. Cellulose, dextran, polysaccharide derivatives such as agarose, polyacrylamide gel, etc., which are commonly used as carriers, are particles with a diameter of several mm and do not have sufficient physical strength, so they cannot be packed in columns for long periods of time. When used, clogging occurs and reaction efficiency is reduced.

On the other hand, regenerated cellulose and ethylene-vinyl alcohol copolymers, which have physical strength and relatively good moldability, can be used as membrane-like carriers, but because the functional groups necessary for immobilizing enzymes are hydroxyl groups, cyanogen bromide In particular, when the carrier is a membrane or film, activation is very difficult, the amount of enzyme immobilized is small, and the activity yield of the resulting immobilized enzyme is The disadvantage was that the rate was low.

The present invention was made to solve the various problems described above, and an object of the present invention is to provide an immobilized enzyme that has excellent physical strength and moldability, and has a higher activity yield.

The present invention mainly focuses on the general formula (However, R represents a divalent organic group.

) has a water-insoluble polyimide molded product having an imide ring opening rate of 1 at least on its surface.
It is characterized by being ring-opened to 0 to 100% and having an enzyme immobilized on the thus formed carboxyl group.

A method for producing a polyimide mainly consisting of repeating units represented by the above general formula I and a film thereof is disclosed in JP-A-54-717.
It is described in Publication No. 85.

The above polyimide is usually prepared by combining 1,2,3,4-butanetetracarboxylic acid with the general formula NH-R-NH2 (where R is the same as above) in a polar organic solvent such as N-methyl-2-pyrrolidone. be.

) in an approximately equimolar ratio of 100 to 3
It is obtained by heating at a temperature of about 00°C for 10 to 50 hours to cause dehydration condensation.

Instead of 1,2,3,4-butanetetracarboxylic acid, its monoanhydride, dianhydride, dialkyl ester, amide, etc. can also be used.

R in the polyimide, that is, R in the diamine, is an aliphatic, aromatic or alicyclic divalent hydrocarbon group, or -CH2-, -CH(CH3)-, monoCO-
, 10-, -SO2-, to S, etc., and preferred representative examples are.

The polyimide used in the present invention has an intrinsic viscosity (measured at 30°C using N-methyl-2-pyrrolidone as a solvent) of 0.55 to 1,00, preferably 060 to 0.85.

If this is converted into an average molecular weight, it is 20,000 to 12,000.
0, preferably 30,000; 80,000.

Furthermore, depending on the reaction conditions, polyimide has an amide bond as a precursor structure before being closed to an imide ring, but the polyimide support used in the present invention has the following number of imide rings: imidization rate = x1oo
(L: fA Number of imide rings + Number of amide bonds Imide ring opening rate Number of amide bonds 2111117-- The imide ring opening rate is 10-10
It is desirable that the imide ring opening rate is 0% and in the substantial portion excluding the surface, that is, the base layer, the imide ring opening rate is 0 to 30%.

That is, the imidization rate is 70% or higher, preferably 90% or higher, and particularly preferably 98 to 100%.

Thus, carriers that can be preferably used in the present invention are:
The surface has an imide ring opening rate of 10 to 100, and the interior is substantially composed of imide repeating units represented by the above general formula.

In order to obtain a carrier having a structure in which the surface and the inside are different, the polyimide substantially represented by the above general formula (2) is molded into a desired shape such as a particle, a film, or a porous model, and then the surface of this molded product is hydrolyzed. obtained by

Examples of repeating units having a precursor structure of an imide structure or an amic acid structure produced by hydrolysis of an imide ring include the following.

The polyimide structure substantially represented by the above general formula (2) has excellent physical strength and chemical properties, so it can be used as a non-porous film or a porous film with selective permeation performance such as ultraviolet permeability or reverse osmosis. When a carrier is formed into a membrane shape, the carrier surface has a high density of carboxyl groups for enzyme immobilization, and this carrier surface is integrally reinforced with a polyimide structure that has excellent physical strength and chemical properties. , a practically excellent carrier can be obtained.

The conditions for hydrolyzing the surface of the molded article depend on the shape of the carrier, but in the case of a porous membrane, it is usually soaked in an alkaline aqueous solution with a pH of 10 to 12, preferably 10.5 to 11.5 for 10 minutes. Several tens of hours, preferably 30 minutes to 5 hours,
It may be immersed at a temperature of 35 to 40°C.

In the case of non-porous films, they may be treated under harsher conditions than porous films.

The degree of hydrolysis on the surface of the molded article, ie, the imide ring opening rate, is appropriately selected in consideration of the required density of carbo and xyl groups, the activity of the resulting immobilized enzyme, the strength of the carrier, and the like.

In addition, from a physicochemical perspective, the degree of hydrolysis can be determined by the change in absorbance of carbonyl groups in the infrared absorption spectrum of the surface of the molded product.

Incidentally, since the main chain of the polyimide hydrolyzed in this manner is not cut, the physical strength hardly decreases.

However, in the present invention, if the carrier does not require relatively strong strength, the entire carrier may have a uniform imide ring opening rate.

The polyimide for such a carrier is preferably 1,
2,3,4-butanetetracarboxylic dianhydride is reacted with the diamine in a polar organic solvent such as N-methyl-2-pyrrolidone at a temperature of 30 to 100°C to obtain an imide ring opening rate of 100. %, that is, a polyamic acid containing no imide structure, and then adjusting the heating depth in the range of 100 to 300°C and arbitrarily controlling the imidization rate.

Therefore, if a polyimide with an imidization rate of, for example, 30 to 70% is obtained, it can be used immediately as a carrier without requiring hydrolysis.

In the present invention, the method for binding the enzyme to the carrier may be either a covalent bonding method or an ionic bonding method.

Since the carrier used in the present invention has a carboxyl group, for example, if the enzyme is reacted with the carrier together with a carbodiimide reagent or Woodward reagent, the enzyme can be easily immobilized by a covalent bond, but the method is not limited to these methods. do not have.

Furthermore, in order to ionically bond the enzyme to the carrier, the carrier may be immersed in an aqueous solution of the enzyme.

When an enzyme is covalently bonded to a carrier, a conventionally used spacer such as hexamethylene diamine or xylylene diamine is bonded to the carboxyl group as a spacer group, and the free amino group of the spacer group is bonded to the enzyme. It is also possible to covalently bond the enzyme to the carboxyl group of the carrier, and in the present invention, the expression "bonding the enzyme to the carboxyl group of the carrier" includes such a case.

Therefore, in the present invention, various enzymes can be immobilized on the carrier, and specific examples include amino acid oxidase, catalase, xanthine oxidase, glucose oxidase, glucose-6-phosphate dehydrogenase,
Redox enzymes such as glutamate dehydrogenase, cytochrome C oxidase, tyrosinase, lactate dehydrogenase, peroxidase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, aspartate acetyltransferase, aspartate aminotransferase, glycine aminotransferase Elase, glutamate monooxamate acetate amine transferase, glutamate monopyruvate amine transferase, creatine phosphokinase, histamine methyltransferase, pyruvate kinase, fructokinase, hexokinase, δ-lysine acetyltransferase, leucine Transferases such as aminopeptidase, asparaginase, acetylcholinesterase, aminoacylase, amylase, alkinase,
L-arginine deiminase, invertase, urease, uricase, urokinase, esterase, β-
Galactosidase, kallikrein, chymotrypsin, trypsin, thrombin, naringinase, nucleotidase, papain, hyauronidase, plasmin, pectinase, hesperidinase, pepsin, penicillinase, penicillin amidase, phosphory; pase, phosphatase, lactose, lipase, ribonuclease,
Hydrolases such as rennin, aspartate decarboxylase, aspartase, citrate lyase, glutamate decarboxylase, histidine ammonia lyase, phenylalanine ammonia lyase, fumarase, fumarate hydratase, malate synthetase, alanine racemase, glucose isomerase, glucose phosphate isomerase,
Examples include isomerases such as glutamate racemase, lactate racemase, and methionine racemase, and gauze such as asparagine synthase, glutathione synthase, and pyruvate synthase.

As described above, since the immobilized enzyme of the present invention has a carboxyl group as a functional group for enzyme immobilization, the enzyme can be easily immobilized regardless of the shape of the carrier, and the immobilized enzyme obtained High activity yield.

Furthermore, in the present invention, since a carrier based on polyimide represented by the above general formula is used, it can be formed into any shape such as particles, a porous membrane having selective permeability, or a non-porous film. The carrier obtained by subjecting the surface of a molded product made of polyimide represented by the above general formula to D-water decomposition has excellent physical strength and chemical properties because the base layer of the carrier is substantially composed of polyimide. An immobilized enzyme is obtained.

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

In the following, parts indicate parts by weight.

Example 1 According to the method described in JP-A-54-71785, the intrinsic viscosity [η] is 0.76 (30°C), the imidization rate is 99% or more, and R in the general formula is 00《
An N-methyl-2-pyrrolidone solution of polyimide having a large size was obtained (solid content 2
5%, 180 poise measured at 30°C with a B-type viscometer).

Add lithium nitrate finely ground in a mortar to the above polyimide solution at a ratio of 100 parts per 100 parts of polyimide.
The mixture was stirred at ℃ for 5 hours to obtain a homogeneous solution.

This solution was applied on an aluminum plate to a thickness of 320μ,
It was immediately put into a 50°C water bath and immersed for 120 minutes to obtain a permselective porous membrane with a thickness of 250 μm.

This membrane was cut into 5cfrL×5crI′L, and Na2H
PO4-NaOH buffer (0.15M Na2HPO
4, 0.1M NaOH (pH 1 1.0) and hydrolyzed at 40°C for 90 minutes with shaking.

After this, the membrane was thoroughly washed with distilled water and used as a carrier.

Put 50 ml of water and the above carrier into a 300 rnl beaker, add liquid glucose isomerase (manufactured by Nagase Seikagaku Kogyo Co., Ltd., 6300 U/ml) 1 hemp while stirring at the edge of the room, and immediately add 1-ethyl-3 (3-dimethyl). 150 m9 of (aminopropyl)carbodiimide hydrochloride was added, followed by 0.01N hydrochloric acid or 0.1N hydrochloric acid. The pH was adjusted to 5.0 by sequential addition of IN aqueous sodium hydroxide solution.

After 2 hours, the mixture was cooled to 4°C and left overnight to complete immobilization of the enzyme.

After this, Trisma L/Iic acid buffer (0.1
M tris(hydroxymethyl)aminomethane, 0,I
M maleic acid, 2T7LM cobalt chloride, 0.1M magnesium sulfate, pH adjusted to 7 with 5N aqueous sodium hydroxide solution.
．． 5) several times to obtain the immobilized enzyme of the present invention.

In a 100 ml volume beaker, add 25 ml of IM glucose tris-maleate buffer and 2.5 ml of the above-mentioned immobilized enzyme.
Add cfrLX2.5CrrL and heat to 60°C while shaking.
After reacting at °C for 30 minutes, 25 ml of 0.5M perchloric acid aqueous solution was added to terminate the reaction.

When the activity of this immobilized enzyme was measured from the amount of fructose produced by the cysteine-rubazol method, it was found that 2
7 7. 8 U\g was one carrier.

However, the enzyme activity that produced 1 μmol of fructose per minute was defined as IU.

Example 2 Laminated carrier 4crrL×4 obtained in the same manner as Example 1
cfrL was placed in a 300 ml beaker with 50 ml of water, then 100 μl of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride and 3.6 μl/! of 1% xylylenediamine aqueous solution were added. and stirred in Murobuchi.
．． IN hydrochloric acid or 0. An aqueous IN sodium hydroxide solution was added to adjust the pH to 4.8 to 5.5.

After 35 hours, it was cooled to 4°C and left overnight.

Thereafter, the carrier was thoroughly washed with distilled water to obtain a carrier having xylylene diamine as a spacer.

Glyose isomerase was reacted and immobilized on the above carrier in the same manner as in Example 1 in the presence of carbodiimide.

The activity of this immobilized enzyme was 308.8 U/y per carrier.

Example 3 N-methyl- of polyimide having an intrinsic viscosity [η] of 0.73, an imidization rate of 99% or more, and in which R in the general formula is
2-pyrrolidone solution (solid content 18%, viscosity 57 poise)
was prepared.

A 15% by weight solution of potassium nitrate in N-methyl-2-pyrrolidone was added to the polyimide solution at a ratio of 5 parts of potassium nitrate per 100 parts of polyimide, and mixed uniformly.

After applying this solution to a thickness of 250μ on a glass plate, it was immediately poured into t-butyl alcohol at 25°C, immersed for 10 seconds, and after 20 seconds, poured into water at 10°C.
Soaked for minutes.

The obtained membrane was hydrolyzed to obtain a carrier in the same manner as in Example 1, and glucose isomerase was immobilized in the same manner as in Example 1.

The activity of this immobilized enzyme was 256 U/g of carrier.

Example 4 The N-methyl-2-pyrrolidone solution of the polyimide obtained in Example 3 was applied onto a glass plate to a thickness of 1 mm, dried at 120°C for 30 minutes, and further dried at 160°C for 1 hour.

After cooling, it was soaked in water overnight to obtain a non-porous film.

This film was treated with Na at pH 11 in the same manner as in Example 1.
The carrier was immersed in 2HPO4-NaOH buffer, shaken at 40°C for 4 hours, and hydrolyzed to obtain a carrier.

Jack bean-derived urease was immobilized on this carrier in the presence of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidometh-p-toluenesulfonic acid to obtain an immobilized enzyme according to the present invention.

The activity yield was 68%. Example 5 Bacterial α-amylase was immobilized on the film carrier obtained in Example 4 in the same manner.

The activity yield was 82%.

Example 6 The carrier obtained in Example 1 was heated to 0. After immersing in IN hydrochloric acid for 30 minutes, it was thoroughly washed with distilled water.

The above carrier was brought into contact with an α-amylase aqueous solution having a concentration of 50 m9/rI′Ll, and α-amylase was immobilized by ionic bonding.

Claims (1)

[Scope of Claims] 1. A water-insoluble polyimide molded product mainly consisting of repeating units represented by the general formula has an imide ring opening rate of 10 at least on its surface.
An immobilized enzyme characterized in that the ring is opened to ~100 degrees, and the enzyme is immobilized on the carboxyl group thus formed. 2 The imide ring opening rate of the base layer of the polyimide molded product is 0 to 3.
The immobilized enzyme according to claim 1, wherein the immobilized enzyme has a coefficient of 0. 3. Claim 1 or 2, characterized in that in the general formula, R is
Immobilized enzymes as described in section.