JPS59216586A - Immobilized enzyme and production thereof - Google Patents

Abstract

PURPOSE:To obtain a stable immobilized enzyme for determining pyruvic acid, by dipping a porous high polymer carrier in a solution containing pyruvic acid oxidase, coenzyme flavin adenine dinucleotide, coenzyme thiamine pyrophosphate and Mg<2+>, etc. CONSTITUTION:A vinyl chloride resin is dissolved in a solvent to give a solution in 2-35wt% resin concentration, which is then dipped in a solvent which is a poor solvent for the vinyl chloride resin and a good solvent for the solvent to prepare a porous high polymer carrier. The resultant carrier is then dipped in a solution containing pyruvic acid oxidase (POP), coenzyme flavin adenine dinucleotide (FAD), coenzyme thiamine pyrophosphate (TPP) and Mg<2+> and/or Mn<2+> ions to afford the aimed immobilized enzyme. The resultant immobilized enzyme membrane is used as an enzymic sensor to measure the pyruvic acid concentration in a standard lithium pyruvate solution from the produced oxidation current of H2O2, and a linear relationship between the concentration and the current value is observed.

Description

Detailed Description of the Invention Technical Field The present invention relates to an immobilized enzyme and a method for producing the same.According to the present invention, an immobilized enzyme with extremely high activity and excellent stability for quantifying pyruvate in biological samples can be obtained. It will be done.

The immobilized enzyme obtained by the present invention is useful as an enzyme sensor for medical analyzers and the like.

Conventionally, various analytical methods using immobilized enzymes have been proposed as methods for selectively quantifying substances in biological samples.

These methods utilize the substrate specificity and high catalytic activity of enzymes and are effective means for detecting trace components in multicomponent solutions.

Measurement of blood pyruvate has been shown to be very useful in assisting in the diagnosis of diseases such as severe liver cirrhosis and hepatic coma, as well as glycogen storage disease, which is an inborn abnormality of glycogen metabolism.

Transaminase is an enzyme that catalyzes the transamination of amino acids, and among these, glutamate pyruvate transaminase (abbreviated as GPT) is contained in large amounts in the liver and kidneys. This GPT measurement is applicable to acute viral hepatitis, chronic hepatitis, liver cirrhosis, liver tumors, liver diseases such as acute alcoholic hepatitis, extrahepatic obstructive rhinopox such as gallbladder, cholangitis, and cholelithiasis, myocardial hard water, and progressive muscular dystrophy. It plays a useful role in diagnosing infectious diseases, etc., and the simple and accurate measurement of GPT has great clinical significance.

Prior Art Conventionally, the method for measuring pyruvate was that pyruvate was α
-Focusing on the fact that it is a keto acid, a method of reacting 2,4-dinitrophenylhydrazine with this ketone group to generate a hydrazone and developing color with an alkali (Friedeman
n-1laugen method). However, α-keto acids other than pyruvic acid, especially α-ketoglutaric acid, exist in the blood, and there are problems such as giving a positive error.To accurately determine pyruvate, it is necessary to quantify 1F using chromatography. It is necessary and the operation is complicated.

In addition, methods for measuring the amount of enzyme activity in biological samples generally involve analyzing and quantifying the components produced by the enzymatic reaction in the presence of the enzyme to be measured, or by combining this product with the enzymatic reaction of other enzymes. A method is used in which the substance is converted into a colorimetric substance through conjugation.

For example, to measure GPT, a sample is added to the substrates alanine and α-ketoglutarate, and pyruvate is conjugated in the presence of lactate dehydrogenase and coenzyme NADH to generate the N
There is the Carmen method, which measures the decrease in absorbance at 340 m of ADII, and the Reitman-Frankel method, which develops color by reacting 2,4-dinitrophenylhydrazine with the pyruvic acid produced as described above.

However, the Carmen method has problems such as the reagents used are unstable and a special spectrophotometer with a constant temperature cell is required. On the other hand, in the Reitman-Frankel method, the α-ketoglutaric acid of group F also develops color and interferes with the color development of the product, so it is necessary to drastically reduce the amount of substrate.
There are disadvantages such as the gland being curved and the measurable range being narrow.

In order to solve these drawbacks, a method for analyzing GPT using pyruvate oxidase (abbreviated as POP) has recently been developed. For example, a method for measuring GPT involves a reaction in which a sample is added to the substrates alanine and α-ketoglutarate to produce pyruvic acid, and a pyruvic acid oxidation reaction in which the produced pyruvic acid is oxidized in the presence of POP and the required substrate. There is a method in which the amount of hydrogen peroxide generated during the pyruvic acid oxidation reaction is determined colorimetrically using a coloring agent.

However, the above-mentioned measurement method using POP has problems such as disposable use of expensive enzymes, long measurement time, and poor reproducibility during measurement because POP itself is unstable in a solution. Furthermore, in order to measure the activity of an enzyme such as GPT in a living body, the initial velocity must be measured in principle12, and a special recording device that can monitor the measurement from the start of the enzyme reaction is required.

Therefore, many studies are being conducted to measure GPT using an enzyme electrode in which POP is immobilized and attached to an electrode for measuring oxidation potential. (Unexamined Japanese Patent Publication No. 55-1117
No. 86, No. 56-124396, Analyt
ica Chimica Acta, 118 (198
G) 65-71, etc.) However, in these immobilization methods, the activity and stability of immobilized POP cannot be said to be sufficient. p m
It is expected that it will be difficult to use it for measurements such as ol /L). Furthermore, as in GPT measurement, the amount of pyruvate produced is closely related to the reaction time in the presence of the enzyme to be measured, so POP immobilization, which has high activity and excellent stability, is necessary for quickly and accurately quantifying pyruvate. There was a great need for the provision of enzymes.

SUMMARY OF THE INVENTION 5 Summary of the Invention In order to meet this demand, the present inventors have conducted extensive research and have developed an immobilized enzyme that has excellent POP enzymatic activity and stability, has good substrate expansion properties, and is easy to wash. Furthermore, by mounting this immobilized enzyme membrane on a electrode for measuring oxidation potential and using it as an enzyme sensor, the drawbacks of conventional measurement methods can be overcome. We have completed the present invention by discovering that pyruvic acid can be measured quickly, accurately, regularly, and over a wide range using a simple method.

That is, the first aspect of the present invention is to combine a porous polymer carrier with pyruvate oxidase (POP), coenzyme frapin adenine dinucleotide (FAD), coenzyme thiamine pyrophosphate (TPP), and M22+ and/or The first method is to provide an immobilized enzyme obtained by immersing a porous polymer carrier in a solution containing Mn''+ ions. ), coenzyme thiamine pyrophosphate (TPP) and %Q 2+ and/or Mn2
The present invention provides a method for producing an immobilized enzyme, which involves immersing it in a solution containing + ions.

(Porous polymer carrier) As the porous polymer carrier used in the present invention, known vinyl chloride resin carriers, acetyl cellulose, polycarbonate, nylon, polypropylene, polyethylene, Teflon, etc. can be used.

Among these, a vinyl chloride resin carrier is preferably used, and the carrier described in JP-A-55-39719 can be used as the carrier.

Particularly preferably, the vinyl chloride resin carrier is produced as follows.

First, a vinyl chloride resin is dissolved in a solvent (5) such as dimethylformamide, and this is formed into a desired carrier shape, and then immersed in a solvent (J3).

Examples of the vinyl chloride resin (PVR) of the present invention include polyvinyl chloride (PVe), vinyl chloride copolymers, and blends of these and other resins. There are binary or ternary copolymers of vinyl acetate, vinylidene chloride, ethylene, acrylic acid, acrylonitrile, etc.

The solvent used is dimethylformamide (DMF), dimethylacetamide (DMA), n-methylpyrrolidone (n-MP),
Examples include hexamethylphosphoramide (IIMPA), tetrahydrofuran (TflF), and a mixed solvent of acetone and benzene. The concentration of the PVR solution used is 1 to 30% by weight. In order for the immobilizer A, such as an enzyme, to exhibit poor adsorption activity and function, it is preferably 6 to 12% by weight when the PvR weight is about 1000.

Next, the PVR solution obtained in this way is formed into a desired carrier shape, and then brought into contact with a solution (1s), which is a poor solvent for PvR and a good solvent for solvent traps, to form a 411 body layer. At this time, the contact with the solvent (F3) is preferably carried out before the PVR solution layer becomes white. Before the bleaching,
This is the period during which the PVR and solvent solution becomes opaque with visible cloudiness.

Examples of solvents include water, alcohol solvents, and ether solvents.

Examples of alcoholic solvents include methyl alcohol, ethyl alcohol, n-7'' lobil alcohol, 1so-propyl alcohol, n-butyl alcohol, 8eC-butyl alcohol, and tert-butyl alcohol.
Polyhydric alcohols such as ethylene glycol, diethylene glycol and glycerin, and glycol monoethers such as ethylene glycol monomethyl ether and ethylene glycol monoethyl ether are used. As the solvent for dipping, these alcoholic solvents may be used alone, or at least 50% by weight of alcoholic solvents.
A mixed solvent in which the vinyl chloride resin is insoluble may be used.

In particular, when methyl alcohol or ethyl alcohol is used, an immobilized product with high immobilized enzyme activity can be obtained, which is preferable.

In addition, in the solution of PVR and solvent (4), 1.0% of PVR such as polyethylene glycol is added in order to adjust the degree of swelling of the carrier, that is, to adjust the water content of the carrier, adjust the pore size, etc. A chemical compound can be added.

The shape of the phase body can be various shapes such as a tau shape, a tube shape, a test tube shape, and a bead shape. For applications such as enzyme electrodes, a membrane-like structure is preferable. The membrane carrier was casted with PVR solution, and then contacted with the solvent (+3), and the tubular carrier formed a solution R membrane layer on the inner wall of the tubular carrier (-1, then the solvent (+3) was coated with f groups: 1t, In the case of a test tube-like carrier, a solution NIK'E layer is formed on the inner wall of the test tube, which is then brought into contact with the solvent (B), and in the case of a bead-like carrier, the PVR solution is sprayed into the air. After that, it is immersed in molten IA color) and formed.

At this time, in order to improve the strength of the carrier, a reinforcing material such as a non-woven fabric may be used in the case of a membrane-like carrier, and a reinforcing material such as a porous spherical body in the case of a bead-shaped carrier.

Note that after forming the carrier, it is preferable to immerse the carrier in water to replace the solvent color (solvent color) with water.

(Preparation of immobilized enzyme) Next, the porous polymer encapsulation was combined with the enzyme POP and the coenzyme flavin adenine dinucleotide (abbreviated as FAD).
Coenzyme thiamine pyrophosphate (abbreviated as TPP)
Then, the immobilized enzyme of the present invention is obtained by immersing it in a solution containing Mg2+ ions and/or Mn''+ ions.

In this case, the solution V containing the enzyme, I) 0 degree of OP is 100 to 100 omv/de, preferably 1 degree is 200 to
so o my/de and the pH of the solution is 6
-8.5, preferably in the range of 6.5-7.0.

The above buffer solution used in the present invention is described by Good et al.
iochemistry, 5.467 (1966))
Known buffering agents having a PK&6 to 8.5 according to the US Pat. For example, N “substituted dipolar amino acids, 1-L
,, N-(z-acetamido)-2-aminoethanesulpone flit, N-(z-acetamido)iminodiacetic acid, N,N-bis(2-hydroxyethyl)-2-
Aminoethanesulfone e, NlN-bis(2-hydroxyethyl)glycine, N-(2-hydroxyethyl)pitarazine-N'-2-ethanesulfonic acid, 2
-(N-morpholino)ethanesulfone "li, 3-(N
-morpholino) furopanesulfonic acid, pipezine-N,
N'-bis(2-ethanesulfonic acid), N-+-lith(
Examples include hydroxymethyl)methyl-3-aminopropanesulfonic acid, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and N-tris(hydroxymethyl)methylglycine.゛Also, examples of aliphatic amines include 2,2-bis(hydroxymethyl)-2,2',
Examples include 2'-nitrilotriethanol, 1,3-bis[tris(hydroxymethyl)meglutami7no]propane, and glycinamide. Also tris(hydroxymethyl)
Aminomethane and the like can also be used.

These can be used alone or in combination of two or more.

Also, M, 2+ ions and Mn2+ ions are ]'v
Preferably it is used in the form of lyCt2 and MnC15.

The immobilized enzyme of the present invention is described in the specification of the patent filed on December 1, 1982 (Japanese Patent Application No. 1982-211.117).
．． It can also be coated with a cold-curable blocked isocyanate compound by contacting it with an aqueous solution of the compound.

(Uses of the immobilized enzyme of the present invention) The uses of the immobilized enzyme of the present invention are, for example, as follows. A membrane-shaped immobilized enzyme can be used as an enzyme sensor by being attached to an electrode such as an enzyme electrode, a hydrogen peroxide electrode, or a carbon dioxide electrode. The immobilized enzyme in the form of a tube can be used as an immobilized enzyme reaction device as a pretreatment reaction section for an analyzer in the field of clinical testing, and the immobilized enzyme in the form of beads can be used as a packed reaction device.

The sample system to be measured may be pyruvate itself or a sample system that has a system that directly or indirectly releases pyruvate, such as analysis of pyruvate present in serum, urine, etc. Analysis of transaminases (GOT, GPT), lactic acid and lactate dehydrogenase (hereinafter abbreviated as LDH), azone syndiphosphate (hereinafter abbreviated as ADP) and pyruvate kinase (hereinafter abbreviated as PK) , triglyceride analysis, etc.

The following are representative examples of the use of ``2.''

(b) For the purpose of measuring pyruvate in a sample containing pyruvate Niacetyl phosphate - + CO2 + HzO2 The enzyme reaction to be measured in the presence of a substrate directly converts pyruvate to 1ζ [L For example, ←) A system for measuring GPT activity: PT alanine + α-ketoglutar P --- → pyruvate +
Although the purpose is to measure glutamate(III) lactic acid or LDH activity, the enzyme to be measured also involves an enzymatic reaction in the presence of a substrate, and the production of pyruvate from the product of this reaction, the required substrate, and the enzyme. As a system that indirectly releases pyruvate by coupling with an enzyme reaction, for example, one for the purpose of measuring GOT activity: OT aspartic acid + α-ketoglutaric acid --- one ---
-→Oxaloacetate + Glutamic acid AC Oxaloacetate--→Pyruvate+CO20→Those aimed at measuring ADP or PK activity: I) K ADP+Phosphoenolpyruvate---11-→Adenosine triposphene) ( For the purpose of measuring ATP) + pyruvate triglyceride: 3-
Phosphoric acid + ADP Pyruvic acid + ATP Examples Next, the present invention will be explained in more detail with reference to Examples and Comparative Examples. Similarly, "parts" used in the examples are based on weight. In addition, in the measurement of the enzyme activity of immobilized POP fermentation in the example, POP activity was measured using L and P.

Hager and Li', Lipmann et al.
, Vol. 1.482 (1955)), 1 unit (IJ) is 1 unit (IJ) of 1 min per minute from pyruvate, phosphoric acid and oxygen at 37°C and pI (, 6,7).
This is the amount of enzyme activity that produces 7ztnol of hydrogen peroxide.

Examples 1 to 8, Comparative Example 1 (1) Production of phase material PVC (manufactured by Kanebuchi Kagaku Co., Ltd., polymerization degree 1000) 10 parts, DM
A homogeneous solution was prepared from 90 parts of F solvent, a portion of this solution was cast onto a 15×15 cr++ glass plate, and the glass plate was immediately immersed in a methanol bath. After 30 minutes, this glass plate was immersed in distilled water to obtain a PVC membrane (II''r 740 pm) carrier in which methanol had been replaced with water.

(n) Production of immobilized composite enzyme membrane Punch out the above PVC membrane into a circle with a diameter of 8 gates, and insert it into a po
p (manufactured by Toyo Jozo Co., Ltd., concentration 500 rq/de) and coenzyme FAD (manufactured by Gyudon Kagaku Co., Ltd., 2 mmol/1).
, coenzyme TPP (manufactured by Gyudon Kagaku Co., Ltd., 2 mmol/1),
Buffer solution containing MgCl2 (20 mmol//-) (manufactured by Gyudon Kagaku Co., Ltd., 50 mmol/t, pif7.0)
Immobilized f'i by immersing it in 0.6m of
7: Get the raw film. The POP activity of the obtained immobilized enzyme membrane was measured depending on the buffer solution used at this time, and the activity was as shown in Table 1.

Table-1 MES: 2-(N-(,Ikeris)ethanesulfonic acid))I))ES: Piperazine-N, N'-bis(2-ethanesulfonic acid) B,
, N, N-bis(2-hydroxyethyl)-2-
Tricine (aminoethanesulfonic acid) Tricine:
) lis(hydroxymethyl)aminomethane phosph
ate: KF12PO4-NaOH Comparative Examples 2 to 4 The PVC membrane obtained in (i) of Examples 1 to 8 was punched out into a circle with a diameter of 8, and the coenzyme TPP in the production of the immobilized enzyme membrane was
A sand-immobilized enzyme membrane was obtained in the same manner as in Example 3.6 and Comparative Example 1, except that MgC62 and MgC62 were removed. The POP activity of the obtained immobilized enzyme membrane was as shown in Table 2.

Table-2 Comparative Examples 5 to 7 Examples 1 to 8 t7) (1) f obtained p v CI+! ,
Coenzyme FAD, coenzyme T P P and MyC in the production of immobilized enzyme membrane.
f1! Immobilized enzyme membranes were obtained by the same operations as in Example 3.6 and Comparative Example 1 except for the following. The POP activity of the obtained immobilized enzyme membrane was as shown in Table 3.

Table 3 Example 9 (1) Production of room temperature curable blocked isocyanate compound Urethane prepolymer containing 5.6 parts of unreacted tolylene diisocyanate by reacting 70 parts of polyethylene glycol with molecular weight 1000 and 30 parts of tolylene diisocyanate. I got it. Free NCO in this urethane prepolymer
The base content was 8.6%.

Next, 13 parts of imidazole was added to 87 parts of this urethane prepolymer, and the mixture was reacted with stirring at 90° C. for 3 hours to obtain a room temperature curable blocked isocyanate compound.

[11) Production of immobilized enzyme membrane 10 parts of PVC (manufactured by Kanebuchi Kagaku Co., Ltd., Polymerization 1i1000), D
A homogeneous solution was made from 90 parts of MF solvent to P]%, a portion of this solution was cast onto a 15 x 15 crn glass plate covered with a polyester nonwoven fabric (manufactured by Nippon Vilene ■, thickness approximately 38 pm), and immediately poured with methanol. After immersing the glass plate together in a bath for 30 minutes, it was placed in water for water replacement to obtain a porous polymer membrane.

Next, punch out this film into a circle with a diameter of 8 Wm (film thickness: 70 mm).
p m ), immobilized enzyme membrane 30 in the same manner as in Example 6.
aqueous solution (50 mmol/1 MES buffer, pH 7.0
) for 1 minute, and then left for 30 minutes under room Fl to obtain an immobilized enzyme membrane coated with the urethane prepolymer. The activity of the obtained immobilized enzyme membrane was 0 for pop activity 2.
．． 45 pm, thickness t4opm) was punched out into a circle with a diameter of 8WrR to obtain an immobilized enzyme membrane in the same manner as in Example 6. Further, a 30% aqueous solution (50 mmol, MES laxative solution, pH 7.0) of the cold-curable blocked isocyanate compound obtained in Example 9 (1) was applied to this layer, and the mixture was allowed to stand at room temperature for 30 minutes. An immobilized enzyme membrane coated with urethane prepolymer was obtained.

The activity of the obtained immobilized enzyme membrane was POP activity 75m[J
/cJ.

Reference Example 1m Preparation of a composite enzyme sensor A homogeneous solution was prepared from 6 parts of acetylcellulose, 87 parts of cyclohexanone, and 7 parts of isopropanol 1. A part of this solution was cast onto a 15 x 15 cm glass plate, and the mixture was heated at room temperature. After air drying for 48 hours, a transparent acetylcellulose film with a thickness of 10/1 m was obtained. This acetylcellulose membrane was then immobilized in Example 9. 'P=
R' and 'S were bonded together, and the acetylcellulose membrane was attached to the electrode side, and the immobilized enzyme membrane was attached to the hydrogen peroxide electrode so as to be in contact with the sample solution, to produce an enzyme sensor.

(II) Determination of pyruvate Adjust the above enzyme sensor to 37℃ and attach it to the cell: coenzyme FAD (0.1 mmol//), coenzyme TPP
(1,0 mmol/l), 8~C/-11 (10 mmol//,), Tricine buffer (50 mmol/LXpH
7,0) 0.68 m/ into the cell, then 0-20
While pressing 1 apt of mmol/l lithium pyruvate standard solution, each was injected into the cell using a microdispenser, the enzyme reaction was performed for 15 seconds, and the oxidation current of hydrogen peroxide generated was measured by polarographic method. I measured it.

The measurement mounting used for this measurement is shown in Figure 1. Further, the measurement results are shown in FIG. 2. As is clear from FIG. 2, there is a good linear relationship between the pyruvate concentration and the current value, and if the immobilized enzyme membrane of the present invention is used, θ ~ 2
Potassium pyruvate with a composition of 0 mmol/l can be easily expressed.

Next, this enzyme sensor was subjected to 1500 repeated measurements at 37° C. in the same manner as the above-mentioned pyruvic acid constant m·, and no decrease in electrode activity was observed, indicating good stability in use.

Reference Example 2 (1) Quantification of GPT The enzyme sensor obtained in (1) of Reference Example 1 was placed in a cell adjusted to 37°C.
α-ketoglutal F2 (10 mmol/l), coenzyme F
AD (o, t mmol/'t), coenzyme TPp (1,
0 mmol/l), MgC12 (16 mmol/l)
, and I(I(zPO4(1,0 mmol/l) J
Tricine 1lFj solution (so millimorph t,
1pIL7.0) 0.68 m/l into the cell17, then a G of known activity between 0 and 850 international units (1,U)/l.
50 pt of each sample containing PT (manufactured by Boehringer Manno Im Yamanouchi Co., Ltd.) was placed into a cell under stirring using a microdispenser, and an enzyme reaction was allowed to occur for 1 minute. The oxidation current of hydrogen peroxide generated by this reaction was measured by polarographic method.

The measurement results are shown in Figure 3. As is clear from Figure 3, KGPT
A good linear relationship was observed between the activity and the rate of change in current value, and when the immobilized enzyme membrane of the present invention is used, a small amount of sample can be adjusted to θ~8501. GPT in a wide range of concentrations (U/l) can be measured quickly and with good accuracy using a simple method. Note that 1 international year (1, U) is α- at pH 7, 5, and 30℃.
This is the amount of enzyme activity that produces 1 micromole of L-glutamic acid per minute from ketoglutaric acid.

Reference Examples 3 to 9 The immobilized enzyme membranes obtained in Comparative Examples 1 to 7 were attached to a hydrogen peroxide electrode in the same manner as in Reference Example 1, and pyruvic acid and GPT were quantified in the same manner as in Reference Example 1.2. As a result of our attempts, the oxidation current caused by the enzymatic reaction was small, making it difficult to measure GPT in biological samples.

[Brief explanation of drawings]

FIG. 1 is a conceptual diagram showing an example of an apparatus for measuring pyruvic acid and GPT using an enzyme sensor in which the immobilized enzyme membrane of the present invention is attached to a hydrogen peroxide electrode. Figures 2 and 3 show changes in lithium pyruvate concentration-current value and GPT activity current value, respectively, measured using a 907 element sensor with the immobilized enzyme membrane of the present invention attached to a hydrogen peroxide electrode. FIG. 3 is a correlation diagram showing the relationship between speeds. 1. Sample injection port 2, Reaction and detection cell 3, Immobilized enzyme membrane 4, Hydrogen peroxide electrode 5, Amplifier 6, Recorder 7,...・Digital multimeter 8, S), 10. ... Ponfu" 11, ... Substrate and buffer supply 12, ... Waste liquid tank Patent applicant Mitsubishi Yuka Co., Ltd. Patent attorney Hidetoshi Furukawa Agent Patent attorney Tadashi Hase Admission 1 Figure 2 Pirubishi Lithium oxide) Agricultural products (rrll-q/yu) Figure 3 0 200 400 EiOO800+00
0GPT activity'I case value (river/su

Claims (5)

[Claims] (1) Pyruvate oxidase (
POP), coenzyme flavin adenine dinucleotide (F
AD), coenzyme thiamine pyrophosphate) (TPP)
and an immobilized enzyme obtained by immersion in a solution containing Mn2+ and/or Mn2+ ions. (2) 411 porous 1-silica molecules dissolve vinyl chloride resin in one or more solvents, and the resin Y small holes 2
A solution obtained by making a solution of ~25 sinter and immersing the solution in two or more types of molten I & Q 3) which is a poor solvent for vinyl chloride resin and a good solvent for solvent (5). It is/l! j. The immobilized enzyme according to claim 1. (3) Pyruvate oxidase (
POP), coenzyme flavin adenine cylophosphate (
A method for producing immobilized light red, which comprises immersing it in a solution containing TPP) and Rh2+ and U/or Mn2+ ions. (4) A porous polymer carrier is prepared by dissolving vinyl chloride resin in one or more solvents (5) to obtain a resin concentration of 2 to 25%.
weight% solution, and the solution is immersed in one or more solvents that are a poor solvent for vinyl chloride resin and a good solvent for the solution ff(A)... A method for producing the immobilized enzyme according to scope 3. (5) Enzyme, coenzyme and Δ2+ and/or Mn2+
Claim 3 or 4, wherein the ion-containing solution is a buffer solution containing one or more of N-substituted dipolar amino acids with a PKa of 6 to 8.5, aliphatic amines, and tris(hydroxymethyl)aminomethane. Method for producing the described immobilized enzyme.