JPH0530973A - Immobilized enzyme substance and apparatus for measurement using the same - Google Patents

Abstract

PURPOSE:To obtain an immobilized pyruvate oxidase used for measuring pyruvic acid concentration of living body humors or fermentation liquors, etc., by immobilizing the pyruvate oxidase and proteins other than the pyruvate oxidase in a specific state on a carrier. CONSTITUTION:Pyruvate oxidase and proteins other than the pyruvate oxidase are chemically immobilized on a carrier to coat >=50% carrier surface. A reagent (glutaraldehyde, etc.) reactive with carbonyl, amino, imino groups, etc., in the proteins is used for immobilization.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme-immobilized body and a measuring apparatus using the same, and more particularly to a stable enzyme used for measuring the concentration of pyruvic acid or its salt contained in biological fluids, fermentation broths, etc. Immobilized pyruvate oxidase,
The present invention relates to a simple and accurate measuring device using the same.

[0002]

BACKGROUND OF THE INVENTION Pyruvate is a compound that is produced in vivo as a product of glycolysis and amino acid metabolism, and occupies an important position as an intermediate in the tricarboxylic acid cycle and fatty acid metabolism pathway. Pyruvate is not only involved in intracellular metabolism, but is also secreted into animal body fluids, culture fluids such as microorganisms, and fermentation fluids. This measurement of pyruvic acid concentration is an essential item in a wide range of fields such as clinical tests, substance metabolism research, and fermentation control.

For example, it is known that when yeast is starved at a high alcohol concentration, the concentration of pyruvic acid increases, although the reason is not clear. In recent years, in the sake brewing process, there is a tendency to utilize the increase / decrease in the pyruvic acid concentration for fermentation process control. In addition, a method of measuring blood enzyme activity using the amount of pyruvic acid as an index has also been developed. Enzymes such as glutamate oxaloacetate transaminase (GOT) or glutamate pyruvate transaminase (GPT) contained in animal hepatocytes are released into the blood due to liver dysfunction, and the blood enzyme activity of these enzymes should be measured. Is an important item in clinical examination. Since these enzymes consume or generate pyruvic acid as a substrate or a reaction product, development of a method for measuring pyruvic acid concentration quickly and accurately is an important issue.

However, since pyruvic acid is a labile, low-molecular weight, water-soluble compound, its analysis is not easy.
Due to instability, analysis of pyruvate requires rapidity. In addition, in order to quantitatively analyze only low-concentration pyruvic acid from among many kinds of organic acids coexisting in biological fluid,
High selectivity and high sensitivity are required. In order to meet these demands, analytical methods utilizing enzyme reactions have been developed. Conventionally, lactate dehydrogenase (L-lactate dehydrogenase, EC 1.1.1.27 or D-lactate dehydrogenase, EC 1.1.1.28) has been used for this purpose. Lactate dehydrogenase reduces pyruvate to lactate in the presence of reduced nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) which is a coenzyme. At this time, NADH is converted into an oxidized form (NAD). With this change,
The absorbance of NADH in the near ultraviolet region decreases. Therefore,
Pyruvate can be quantified by following the specific absorption of NADH. However, in this method, a small amount of sample and an enzyme reaction solution are mixed and a spectrophotometer is used, so that there is a drawback that the operation is complicated and a large amount of expensive enzyme is consumed.
At the same time, in order to trace the decrease in ultraviolet absorption of coenzymes, the pyruvic acid concentration in the sample was higher than that of the added NADH,
There is a problem in that no apparent change occurs when NADH is completely consumed, and it is also difficult to apply it to a system containing a coloring component.

Therefore, in recent years, pyruvate oxidase (E
Analytical methods using C 1.2.3.3) are drawing attention. Pyruvate oxidase is an enzyme isolated from bacteria such as Escherichia, Pediococcus, Aerobacterium, Lactobacillus and the like. This enzyme catalyzes the oxidative decarboxylation of pyruvate in the presence of inorganic phosphate and dissolved oxygen. That is, pyruvate oxidase participates in the reaction of [Chemical Formula 1].

[0006]

[Chemical formula 1] pyruvate + inorganic phosphate + O ₂ → acetyl phosphate + CO ₂ + H ₂ O ₂ This enzyme requires thiamine pyrophosphate (hereinafter abbreviated as TPP) and flavin adenine dinucleotide (hereinafter abbreviated as FAD) as coenzymes. However, it is known that divalent ions such as magnesium, calcium, cobalt and manganese act as an activator. It is necessary to add TPP and the divalent ion at the time of reaction, and unlike glucose oxidase, alcohol oxidase, and other oxidases, FAD is relatively loosely bound to the enzyme in this enzyme. Probably, FAD is added to the reaction solution. It is known that this stabilizes the enzyme.

[0007] A measuring method for constructing a measuring kit using the present enzyme for colorimetric reaction is already known (Japanese Patent Publication No. 59-15637). However, when the colorimetric method is used in the solution system, it is difficult to recover and utilize the enzyme, which causes a problem of increasing the analysis cost. In addition, in a color development reaction in which a compound such as peroxidase (EC 1.11.1.7) and 4-aminoantipyrine, which are generally used for the determination of hydrogen peroxide, and a phenol compound are combined, the color of pyruvic acid itself, which is an α-keto acid, becomes a color. It had a demeritizing effect on the drug substance, lacked color development stability, and adversely affected quantification accuracy.

Therefore, it has been attempted to immobilize the present enzyme to reuse the enzyme and to quantify oxygen consumed or hydrogen peroxide produced in the reaction.
For example, a so-called enzyme electrode type detector has been developed in which a membrane-shaped immobilization product in which an enzyme is immobilized on a polymer membrane is used and a membrane is arranged on the surface of an oxygen electrode or a hydrogen peroxide electrode (JP-A-56-56). No. 122947). However, the life span of these immobilized enzymes was not sufficient.

Therefore, a method of adding a high concentration of FAD, which is a stabilizer, was also developed (Japanese Patent Publication No. 2-48059).
issue). However, in this method, the expensive FAD is made disposable, and the increase in analysis cost cannot be avoided. Also,
A system in which the effect of FAD is not recognized has also been reported (JP-A-55-111786). As described above, a sufficiently practical immobilized pyruvate oxidase has not been obtained by the conventional measurement. High concentration of FAD in these technologies
However, the increase in analysis cost cannot be avoided, and a sufficiently stable immobilization product has not been obtained, and the sensitivity decreases when a large number of samples are continuously analyzed, that is, the enzyme is deactivated. There was a flaw.

[0010]

Generally, there are several known methods for stabilizing an enzyme when the enzyme is used in a solution state. For example, in an enzyme containing a thiol residue in the active center involved in the enzymatic reaction, unnecessary oxidative reaction may be prevented and stabilized by adding a thiol reagent such as cysteine or glutathione to the reaction system. Further, when the enzyme is susceptible to denaturation due to foaming due to stirring of the solution, etc., there is known a method of promoting stabilization by adding a protein other than the enzyme to the solution to reduce the ratio of enzyme molecules involved in foaming. There is. However, according to the experiments conducted by the present inventors, the stabilizing effect of pyruvate oxidase could not be found even when a thiol reagent was added in a solution system or a protein other than an enzyme was added.

Further, by immobilizing the enzyme, the storage stability is generally increased in many cases, but in the case of pyruvate oxidase, no remarkable effect was observed. The present invention particularly provides a stable pyruvate oxidase-immobilized body used when measuring the concentration of pyruvic acid contained in a biological fluid, a fermentation broth, and the like, and a simple and accurate measuring device using the same. With the goal.

[0012]

The present invention provides an enzyme-immobilized body in which pyruvate oxidase and other proteins are chemically immobilized on a carrier, and the pyruvate oxidase and other proteins are the carrier surface. The enzyme-immobilized body covers 50% or more of the enzyme. The present invention also discloses the above-mentioned enzyme-immobilized body in which the carrier is aminosilanized, then formylated with a polyfunctional aldehyde, and further coated with pyruvate oxidase and other proteins.

Further, the present invention provides a mechanism for bringing a sample in a buffer into contact with the above-mentioned enzyme-immobilized body, and a mechanism for electrochemically detecting the reaction between pyruvic acid or its salt in the sample and the enzyme-immobilized body. A pyruvic acid measuring device having the same is disclosed.

[0014]

[Function] As a result of investigating various immobilization conditions, the enzyme is chemically immobilized on the carrier, and 50% or more of the carrier surface is coated with pyruvate oxidase and at least one protein other than pyruvate oxidase. It was found that an extremely stable immobilized pyruvate oxidase was obtained. These findings are surprising facts that cannot be inferred from the behavior of solution enzymes.

The surface area of the carrier in the present invention is the amount of surface area of the carrier on which pyruvate oxidase and other proteins can be chemically bound. The amount of pyruvate oxidase and other proteins that can be bound should be measured. Can be obtained by The coverage of the carrier can be obtained, for example, by the following method. Actually changing the concentration of protein etc. to bind to the carrier, measure the binding amount by the Kjeldahl method etc., and consider the equilibrium binding amount when the concentration of protein etc. is extrapolated to infinity as the amount proportional to the carrier surface area. be able to.

Next, the amounts of pyruvate oxidase and other proteins actually bound to the enzyme-immobilized body used in the column of the measuring device are determined by the Kjeldahl method or the like, and this value is determined as the amount proportional to the chemically bound area. I can see it.
Then, the coverage of the carrier can be obtained by obtaining the ratio of the latter to the former.

In the present invention, pyruvate oxidase and other proteins are immobilized on the surface of the carrier by a chemical bond, and molecules of pyruvate oxidase and protein are almost monolayered and immobilized on the carrier. Conceivable. Therefore, it is considered that the amount of pyruvate oxidase or protein bound is almost directly proportional to the coated area on the carrier.

To actually determine the immobilized amount of pyruvate oxidase or protein, the amount of enzyme activity or protein remaining in the solution used for the immobilization of pyruvate oxidase or protein is determined, and the original enzyme activity or concentration is determined. It can also be calculated by finding the difference between The amount of the enzyme or protein remaining in the solution can be determined by, for example, a solution-based color reaction using a phosphotungstic acid-phenol reagent or the like.

The specific surface area of the carrier can be determined by the BET method, but in many carriers, the specific surface area by BET is the same as the specific surface area of the carrier to which the above-mentioned enzyme or protein can be chemically bound. If the molecular cross-section of a protein is known, the specific surface area can be directly calculated from the amount of the above-mentioned bound enzyme or protein, and even if the hydrodynamic radius is unknown, the carrier is based on that protein. It is possible to calculate what percentage of the total surface area is covered.

Examples of usable carriers include silica gel, diatomaceous silicic acid, silicic acid-based carriers such as porous glass, sand, alumina, zirconia, ceramics, activated carbon,
Examples thereof include molecular sieves, titanium oxide, acidic clay, crystalline cellulose, dextran gel, chitin, collagen, amino acid resins, polystyrene resins, polyacrylamide gels, polyvinyl alcohol crosslinked products, nylon fibers, ion exchange resins and the like. Among these, inorganic carriers such as silica gel, diatomaceous earth silicic acid, and porous glass are advantageous in that they have high strength and there is no fear of collapsing the carriers in actual use. The carrier preferably has a specific surface area of 0.5 to 200 square meters per 1 g of the carrier. If the specific surface area is too small, the enzyme activity cannot be sufficiently supported, and if it is too large, a large amount of protein needs to be immobilized, which is not economically desirable.

Although it is possible to immobilize pyruvate oxidase and other proteins on these carriers by physical adsorption, in the present invention, in order to obtain an immobilization product that can be used continuously and stably, It fixes pyruvate oxidase and other proteins. As a method for chemically immobilizing a protein containing an enzyme on a carrier, a reagent that reacts with a carbonyl group, an amino group, an imino group or the like in the protein, for example, glutaraldehyde, a carbodiimide reagent, an isocyanate, a diazonium compound or the like is used. Various crosslinking immobilization methods can be used.

For example, an aminoalkylalkoxysilane such as γ-aminopropyltriethoxysilane is brought into contact with the surface of the carrier in an anhydrous solvent, and the surface of the carrier is aminosilanized by heating. The surface-treated support is contacted with an aqueous solution of an aldehyde having two or more functional groups (eg, glutaraldehyde) to formylate. Thereafter, the protein dissolved in the buffer is brought into contact with the amino group in the protein molecule to react with the formyl group to immobilize the protein.
The advantage of this method is that there is no direct contact between excess polyfunctional aldehyde and protein due to the separate steps of formylation and protein binding, which prevents unnecessary deactivation when the protein is an enzyme. It is a point. Silica-based carriers are preferred for this method. Of course, other immobilization methods can be used by adjusting the reaction conditions.

In order to obtain a high coverage, it is preferable to increase the concentration of the enzyme solution and the protein solution and allow them to react for a long time. When immobilizing pyruvate oxidase and a protein other than pyruvate oxidase, a mixture of both may be prepared and immobilized from the beginning, or either one may be immobilized first and another protein or Immobilization may be performed by post-adding the enzyme. This can be used properly when the reactivity with the cross-linking reagent used for chemical bonding differs depending on the enzyme or protein used.

When pyruvate oxidase is first immobilized and then another protein solution having a high concentration is immobilized, there is an advantage that a high coverage can be easily obtained. For example, when bovine serum albumin is immobilized as pyruvate oxidase and another protein, bovine serum albumin differs from pyruvate oxidase in reactivity with a cross-linking reagent, protein size, and shape. Easy to fix. Therefore, by immobilizing pyruvate oxidase first, it is possible to prevent immobilization of only a large amount of bovine serum albumin.

As the pyruvate oxidase used for immobilization, various enzymes isolated from bacteria such as Escherichia, Pediococcus, Aerobacterium, Lactobacillus and the like can be targeted. Examples of proteins other than pyruvate oxidase immobilized on a carrier include structural proteins such as collagen, elastin and fibroin, and proteins such as gelatin which is a partial degradation product thereof, serum proteins such as albumin and globulin, casein, ovo. Proteins such as albumin,
And various enzyme molecules and modified products thereof can be used. When an enzyme molecule is used, it may be positively used for its enzyme activity or may simply exist as a protein. In the latter case, a modified product with no enzyme activity can be used. Of course, two or more kinds of proteins may be used as the protein other than pyruvate oxidase.

The reason why stabilization of pyruvate oxidase is realized by co-immobilizing pyruvate oxidase and a protein other than pyruvate oxidase is not clear, but is presumed as follows. Pyruvate oxidase has low thermostability and its molecular structure is maintained by a relatively weak intramolecular hydrogen bond, and it is considered that hydrogen bond between enzyme molecules has no effect of improving stability. Therefore, the molecular structure is easily destroyed by heating etc., but by immobilizing it on the surface of the carrier, protein molecules other than pyruvate oxidase come close to the enzyme molecule, and the interaction between this protein molecule and the enzyme molecule. Would promote stabilization. On the other hand, in the solution, a large amount of water molecules exist between the pyruvate oxidase and the other protein, and it is considered that the stabilizing effect is not recognized. For this reason, it is desirable that pyruvate oxidase and other proteins are sufficiently close to each other on the surface of the carrier, and it is necessary to cover 50% or more, and more preferably 70% or more of the total specific surface area of the carrier. The weight ratio of pyruvate oxidase to proteins other than pyruvate oxidase is preferably about 1: 100 to 10: 1, and more preferably 1:10 to 5: 1 from the viewpoint of sensitivity.

The pyruvate oxidase-immobilized body prepared as described above can be stably used. Pyruvate oxidase and at least one kind of protein other than pyruvate oxidase are chemically bound to a carrier and 50% or more of the carrier surface is coated with an enzyme immobilization body of an analyzer using this immobilized body and mixed with a buffer solution. As a mechanism for bringing the sample into contact, a batch type analysis system may be configured, or a carrier may be packed in a column to form a flow type analysis device.

Further, as a mechanism for electrochemically detecting the reaction between the pyruvic acid or its salt in the sample and the enzyme-immobilized body, for example, it is consumed by contacting the pyruvic acid or the like contained in the sample with the enzyme-immobilized body. A mechanism for electrochemically detecting increase / decrease in oxygen, hydrogen peroxide produced, or an electron acceptor that exchanges electrons with pyruvate oxidase can be exemplified.

As the oxygen electrode for measuring the consumed oxygen, various known electrodes such as galvanic type and Clark type can be used.
As the hydrogen peroxide electrode for measuring the hydrogen peroxide produced, one using carbon, platinum, nickel, palladium or the like for the anode substrate and silver or the like for the cathode side can be used. Generally, platinum is often used as the anode because of its low overvoltage and high sensitivity. An electrode having a selective permeation film such as polysiloxane, acrylic resin, protein film, and acetylcellulose film on the electrode surface is desirable from the viewpoint of removing measurement obstacles.

Further, in the method using these oxygen electrode and hydrogen peroxide electrode in which the dissolved oxygen in the buffer solution is used as the electron acceptor of the reduced pyruvate oxidase, according to the Michaelis constant of pyruvate oxidase with respect to the dissolved oxygen,
The measurement range is limited. In order to eliminate this, various electron acceptors can be made to coexist in the buffer solution to eliminate the effect of oxygen concentration. For example, it can be carried out using a so-called mediator such as potassium ferricyanide, phenazine methosulfate, dichloroindophenol, and ferrocenecarboxylic acid. In this case, a carbon electrode, a platinum electrode, or the like is used to detect the reduced mediator. Of course, the mediator can be used in either a batch type or flow type measuring device using the above-mentioned enzyme-immobilized body.

The buffer used for the measurement is 0.01 to 10
mM phosphate, 0.1-50 μM FAD, 0.00
A buffer containing 1 to 1 mM TPP and 0.1 to 10 mM magnesium ion is preferable.
Although pyruvate oxidase catalyzes a reaction containing inorganic phosphate, it tends to be inhibited by a high concentration of phosphate, and if the concentration is too low, a sufficient reaction rate cannot be obtained. Therefore, the concentration of phosphate contained in the buffer solution is preferably 0.01 to 10 mM. The pH range in which the immobilized pyruvate oxidase stably acts is 6 to 8, and the phosphate may be used to give the solution a buffering ability, or other compounds may be allowed to coexist with the buffering ability. You may let me.

Although it has been conventionally considered that the stability of pyruvate oxidase is improved by increasing the FAD concentration, the immobilization product of the present invention is destabilized if the FAD concentration is too high. A trend is recognized,
If the concentration is too low, the reaction rate will decrease, so
It is desirable to add in the range of 1 to 50 μM. TPP
Also, for low concentration, the reaction did not proceed at all, and 1 mM
Since there is no change in the reaction rate or stability even if a concentration exceeding the above is added, it is preferably 0.001 to 1 mM.

Pyruvate oxidase is activated by various divalent ions as described above, but when divalent ions form a precipitate with phosphoric acid when calcium ions, manganese ions, cobalt ions, etc., and interfere with the analytical operation. Because there is
It is desirable to use magnesium ions. At a concentration of 0.1 mM or more, a remarkable activating effect is recognized, and when it exceeds 10 mM, the reaction is inhibited.
If it exceeds 10 mM at the same time, precipitation of magnesium phosphate may occur depending on the pH and the temperature of the buffer solution.
It is preferable to add it at a concentration of 10 mM. The thus-obtained measuring apparatus of the present invention can stably analyze pyruvic acid (including pyruvic acid salts) for a long time with high accuracy.

[0034]

The contents of the present invention will be described in more detail with reference to the following examples, but of course the present invention is not limited thereto.

Example 1 The stability of the pyruvate oxidase-immobilized product of the present invention was confirmed by constructing a flow type measuring apparatus. (1) Manufacturing method of carrier for immobilization 150 mg of refractory brick (30 to 60 mesh) is thoroughly dried. Next, after soaking in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, it is washed with toluene and methanol and dried at 120 ° C. for 2 hours. This was used as a carrier used in the following experiments. (2) Production of Pyruvate Oxidase-Immobilized Body and Column The carrier thus treated with aminosilane was immersed in a 5% glutaraldehyde aqueous solution for 1 hour and then washed with distilled water. Then, it is replaced with a 10 mM sodium phosphate buffer containing 10 μM FAD having a pH of 7.0, and this buffer is removed as much as possible.

10 m containing 10 μM FAD in the above carrier
20 units of pediococcus pyruvate oxidase (corresponding to 0.63 mg as protein) dissolved in M sodium phosphate buffer is added, and the mixture is allowed to stand at 4 ° C for 16 hours. Then, a part of the supernatant was taken and the amount of enzyme protein was quantified by a color reaction (Lowry improved method) using a phosphotungstic acid-phenol reagent. Almost no added pyruvate oxidase remained in the supernatant, and 99% was immobilized on the carrier. 10 μMFA
Bovine serum albumin (Sigma, fraction V) 1 m dissolved in 10 mM sodium phosphate buffer containing D
g was added to the above-mentioned carrier on which pyruvate oxidase had already been immobilized, and this state was allowed to stand at 4 ° C. for 16 hours to chemically immobilize pyruvate oxidase and bovine serum albumin on the carrier. The carrier is 10 μM
10 mM sodium phosphate buffer containing FAD (pH
It was thoroughly washed with 7.0), and a part of the washing solution was taken to quantify the amount of protein that was not immobilized again.

A column having an inner diameter of 3.5 mm and a length of 30 mm was packed with the enzyme-immobilized carrier to obtain a pyruvate oxidase-immobilized column. (3) Measurement of carrier coverage Using the same aminosilanized carrier obtained in (1), glutaraldehyde was allowed to act in the same manner as in the case of enzyme immobilization to prepare a formylated carrier. 10 mM at pH 7.0
0.1-10 mg / bovine serum albumin in phosphate buffer
It is dissolved at various concentrations of ml, and 1 ml of this solution is brought into contact with the formylated carrier. After leaving it at 4 ° C. for 16 hours, it is washed with a large amount of distilled water. After the amount of protein bound to the carrier is decomposed with sulfuric acid, the amount of ammonia is measured to calculate the total amount of chemically bound protein (Kjeldahl method). Then, the equilibrium binding amount was calculated when the bovine serum albumin concentration was extrapolated to infinity. A similar experiment was conducted by changing albumin to pyruvate oxidase.

The equilibrium binding amounts of bovine serum albumin and pyruvate oxidase to the carrier were almost equal, and both were 7.2 mg / g to this carrier. In this way, a value proportional to the surface area per 1 g of the carrier was obtained. Next, as described in (2) above, after leaving the pyruvate oxidase alone in contact with the formylated carrier, the amount of residual enzyme in the supernatant was quantified, and 99% or more of the added enzyme was bound to the carrier. . It is considered that substantially all of the enzyme protein is bound to the carrier. That is, 0.63 mg / 0.15 g = 4.2 mg / g of enzyme protein was bound to the carrier.

The amount of residual albumin in the supernatant after contact with bovine serum albumin was 0.7 mg. Since the added amount of bovine serum albumin was 1 mg, the immobilized amount was 0.3 mg, and the amount proportional to the coated area per 1 g of the carrier was 0.3 mg / 0.15 g = 2 mg / g.

[0040]

## EQU1 ## (4.2 + 2) /7.2=0.86 Therefore, in Example 1, a carrier in which 86% of the carrier surface was coated was obtained. (4) Method for manufacturing hydrogen peroxide electrode The side surface of a platinum wire having a diameter of 2 mm is coated with heat-shrinkable Teflon,
One end of the wire is smoothed with a file and a No. 1500 emery paper. Using this platinum wire as a working electrode, a 1 cm square platinum plate as a counter electrode, and a saturated calomel electrode as a reference electrode, 0.1M
Electrolyte in sulfuric acid at +1.4 V for 10 minutes. After that, the platinum wire is thoroughly washed and dried at 40 ° C. for 10 minutes. Next, it is immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour and then washed. 20 mg of bovine serum albumin is dissolved in 1 ml of distilled water, and glutaraldehyde is added to the solution in an amount of 0.2%. Place 5 μl of this solution on the platinum wire prepared earlier, and keep it at 40 ° C.
Dry cure for 15 minutes. This is used as a hydrogen peroxide electrode. (5) Measuring apparatus using enzyme-immobilized body The flow type measuring apparatus shown in FIG. 1 was used for the measurement. First, 10 μM FA was added to the buffer reservoir (1).
D, 0.1 mM TPP, 1 mM magnesium chloride,
And a 10 mM sodium phosphate buffer (pH 7.0) containing 50 mM potassium chloride is stored. This buffer solution is continuously sent by the solution sending pump (2). The liquid feeding rate is 1.0 ml / min. Autosampler (3)
The sample containing pyruvic acid injected from the
Sent to. The constant temperature bath is controlled at 25 ± 0.2 ° C. When the sample passes through the column (5) filled with the immobilized pyruvate oxidase in the thermostat, pyruvic acid is oxidized and hydrogen peroxide is produced. The produced hydrogen peroxide is detected by the hydrogen peroxide electrode (6). +0.6 for the silver / silver chloride reference electrode (7) for the hydrogen peroxide electrode (6)
The voltage of V is applied. The piping up to the measurement cell (8) equipped with a hydrogen peroxide electrode was a fluororesin tube (9) with an inner diameter of 0.5 mm, and a stainless steel pipe with an inner diameter of 0.5 mm and a length of 5 mm was installed on the outlet side of the measurement cell. It is used as a counter electrode and takes a three-electrode type. This stainless steel pipe is further connected to a fluororesin tube (10).

The change in the current value depends on the potentiostat (1
The voltage is converted by 1), digitized by the A / D converter (12), and then analyzed by the microcomputer (13). Using the current value in the absence of the sample as a reference, the difference from the peak current due to hydrogen peroxide detected when the sample passes is calculated as the detected value. This value is output to the printer (14). In addition, (15) is a waste liquid reservoir. (6) Stability Experimental Method Using the pyruvate measuring apparatus described above, 5 μl of a sample containing 10 mM lithium pyruvate was injected at 2-minute intervals, and changes in current value when 300 times of injection were recorded. (7) Results FIG. 2 shows the current value every 10 times in order to clarify the change (marked with a circle). As a result, no decrease in current value was observed over a long period of time, indicating that the immobilized body of the present invention can be stably used.

Comparative Example 1 (1) Experimental Method The carrier for immobilization, the measuring device and the like are the same as in Example 1. However, when the pyruvate oxidase-immobilized product was produced, only pyruvate oxidase was immobilized without adding bovine serum albumin. The stability was confirmed using this immobilized body. (2) Results After the pyruvate oxidase alone was left in contact with the formylated carrier, the amount of residual enzyme in the supernatant was quantified. As a result, in the same manner as in Example 1, 99% or more of the added enzyme was bound to the carrier. That is, 4.2 mg / g of enzyme protein was bound to the carrier. This means that 58% of the surface of the carrier was coated with the enzyme.

FIG. 2 shows the current value every 10 times in order to make the change clear (marked by Δ). 300 from this result
By performing the injections once, a gradual decrease in the current value is recognized, and finally a sensitivity decrease of about 30% occurs. Example 1
It is clear from Comparative Example 1 that the immobilized body of the present invention is stable.

Example 2 (1) Experimental method The carrier for immobilization, the measuring device and the like are the same as in Example 1. However, the method for producing a pyruvate oxidase-immobilized product has been changed to that described below. The method for confirming the stability and the like are the same as in Example 1. (2) Method for producing immobilized pyruvate oxidase and column A formylated carrier was prepared in the same manner as in Example 1, and 10 units of pyruvate oxidase (0% as protein) were dissolved in a 10 mM sodium phosphate buffer containing 10 μM FAD. (Corresponding to .32 mg) and 0.5 mg of bovine serum albumin are added at the same time, and the mixture is allowed to stand at 4 ° C. for 16 hours. afterwards,
A part of the supernatant was taken and the amount of protein was quantified by a color reaction using a phosphotungstic acid-phenol reagent.

A column having an inner diameter of 3.5 mm and a length of 30 mm is packed with this enzyme-immobilized carrier to obtain a pyruvate oxidase-immobilized column. (3) Results First, when the mixture of pyruvate oxidase and albumin was left in contact with the formylated carrier and the residual amount of the supernatant was quantified, 77% of the added protein was bound to the carrier. 0.63 mg / 0.15 g = 4.2 mg /
That is, 4.2 mg / g of the enzyme protein was bound to the carrier, that is, to the carrier.

4.2 / 7.2 = 0.58, and in Example 2, 58% of the surface of the carrier was coated. FIG. 3 shows the current value every 10 times in order to make the change clear (circle mark).
As a result, no decrease in current value was observed over a long period of time, indicating that the immobilized body of the present invention can be stably used.

Comparative Example 2 (1) Experimental Method The carrier for immobilization, the measuring device and the like are the same as in Example 2. However, the time for contacting the mixture of pyruvate oxidase and bovine serum albumin with the formylated carrier at the time of producing the immobilized pyruvate oxidase was set to 0.5 hours. (2) Results Pyruvate oxidase and bovine serum albumin were left in contact with the formylated carrier for 0.5 hour, and the amount of residual total protein in the supernatant was quantified. Since the contact time was short, only 40% of the added protein was bound. However, 2.1 mg / g of enzyme and protein were bound per 1 g of the carrier.
This means that 30% of the surface of the carrier was covered.

The results are shown in FIG. 3 for comparison with Example 2 (marked with Δ). The initial sensitivity was naturally low because the amount of binding was small, but a decrease in current value was observed by further performing 300 times of injection, and finally a sensitivity decrease of about 50% occurred. It is clear from comparison with Example 2 that there is a significant difference in stability depending on the coverage of the carrier surface.

[0049]

INDUSTRIAL APPLICABILITY According to the present invention, a stable pyruvate oxidase-immobilized product can be obtained, and an apparatus for stably and easily quantifying the concentration of pyruvate can be constructed.

[Brief description of drawings]

FIG. 1 is an example of a measuring device using the immobilized pyruvate oxidase of the present invention used in Examples.

FIG. 2 shows the results of Example 1 (◯ mark) and Comparative Example 1 (Δ mark). The horizontal axis represents the number of injections, and the vertical axis represents 10.
It is an output current value (μA) when 5 μl of a mM lithium pyruvate solution is injected.

FIG. 3 shows the results of Example 2 (marked with ◯) and Comparative Example 2 (marked with Δ). The horizontal axis represents the number of injections, and the vertical axis represents 10.
It is an output current value (μA) when 5 μl of a mM lithium pyruvate solution is injected.

[Explanation of symbols]

1 buffer reservoir 2 Liquid feed pump 3 Autosampler 4 constant temperature bath 5 Pyruvate oxidase-immobilized column 6 Hydrogen peroxide electrode 7 Silver / silver chloride electrode 8 measuring cells 9 Fluororesin tube 10 Fluororesin tube 11 Potentiostat 12 A / D converter 13 Microcomputer 14 Printer 15 Waste liquid reservoir

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical indication location G01N 27/26 7235-2J (72) Inventor Ryuzo Hayashi 4-3-1, Jokoji, Amagasaki City, Hyogo Prefecture Kanzaki Paper Co., Ltd. Kanzaki Mill

Claims (3)

[Claims] 1. An enzyme-immobilized body in which pyruvate oxidase and other proteins are chemically immobilized on a carrier, and 50% or more of the carrier surface is covered with pyruvate oxidase and other proteins. Immobilized enzyme. 2. The carrier is aminosilanized, then formylated with a polyfunctional aldehyde, and further coated with pyruvate oxidase and other proteins.
The immobilized enzyme described. 3. A mechanism for bringing a sample in a buffer into contact with the enzyme-immobilized body according to claim 1 or 2 and a reaction between pyruvic acid or a salt thereof in the sample and the enzyme-immobilized body is electrochemically performed. A pyruvic acid measuring device having a detecting mechanism.