JPH0798295A - Measuring device and activation method therefor - Google Patents

Abstract

PURPOSE:To provide the measuring device utilizing immobilized enzyme and the activation method thereof. CONSTITUTION:This measuring device has an enzyme fixed object 6, wherein L-lactic-acid dehydrogenase and other dehydrogenase are fixed, an L-lactic-acid oxidizing-enzyme fixed object 6 and an electrode 7, which detects the electrode activity material that is increased or decreased by the L-lactic-acid oxidizing- enzyme fixed object reaction. At least, other dehydrogenase is fixed by a physisorption method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a rapid and simple measuring apparatus for a substrate reductant of dehydrogenase using an immobilized enzyme and a method for activating the same.

[0002]

2. Description of the Related Art A measuring method using an enzymatic reaction is a safe method as compared with a detecting method using a chemical reaction without using dangerous reagents such as acids, bases and organic solvents. Further, it has advantages that it is not easily affected by coexisting substances because of its high substrate specificity, that reaction conditions are mild and controllable. Therefore, there is an increasing demand for establishment of an analytical method utilizing an enzymatic reaction in food industry, fermentation control, clinical tests, etc., such as quantification of specific components in foods, fermented liquors, and body fluids.

There are various analytical methods utilizing enzyme reactions. For example, a method of detecting reduced oxygen and hydrogen peroxide produced by the electrode method using the reaction of the substrate oxidant and hydrogen peroxide produced from the substrate and oxygen by the oxidase, and comparing the produced hydrogen peroxide A method of quantifying by a color method can be mentioned.
In addition, the dehydrogenase causes a redox reaction between the substrate and the coenzyme by causing a coenzyme such as nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP) to act on the substrate. A method of quantifying the amount of coenzyme that increases or decreases by using a colorimetric method can be mentioned.

However, when the final detection means is a colorimetric method, the sensitivity is low depending on the substance to be detected, and it is necessary to measure the blank of the sample. There is a problem that processing is required. In addition, when quantifying a substrate for dehydrogenase by a solution reaction, there is a problem that the direction of the reaction is often limited. In other words, for example, a dehydrogenase reaction utilizing NAD is a nicotinamide adenine dinucleotide reductant (hereinafter referred to as N
There is a marked equilibrium in the direction of producing NAD and a substrate reductant from the substrate oxidant and ADH). Therefore, when measuring the substrate reductant, the generated NADH
Is coupled with another dehydrogenase reaction to regenerate NAD to proceed the reaction. Also in this method, the compounds other than NADH produced by the dehydrogenase reaction often feedback-inhibit the reaction system, and the reaction often does not proceed completely. A typical example thereof is a glutamate dehydrogenase system, and ammonia and α-ketoglutarate produced when NAD coexists and oxidizes glutamic acid, which is a substrate reductant, inhibits the reaction. Therefore, if these compounds are not removed, the concentration of glutamic acid that can be oxidized is limited, resulting in a narrow measurement range of glutamic acid.

On the other hand, the measuring method in which the oxidase is used for detection by the electrode method is not affected by turbidity or coloring substances, but the number of oxidases suitable for practical use is small.

Therefore, a method combining two kinds of dehydrogenase and oxidase has been devised. That is, in addition to the dehydrogenase using the substance to be measured as a substrate, a dehydrogenase and an oxidase acting on a specific substrate are caused to act. For example, in measuring L-glutamic acid, L-lactate is used as a substrate in addition to L-glutamate dehydrogenase (hereinafter abbreviated as GLDH).
-Acting lactate dehydrogenase (hereinafter abbreviated as L-LDH) and L-lactate oxidase (hereinafter abbreviated as LOD). This method will be described by taking L-glutamic acid as an example.
Oxidized coenzymes NAD and GLDH are allowed to act on glutamic acid, and L-lactic acid is produced by allowing the produced NADH to act on L-LDH and pyruvate, which is a substrate oxidant of L-LDH. Hydrogen peroxide or reduced oxygen produced by the action of LOD on this L-lactic acid is detected by the electrode method (Japanese Patent Application No. 5-157547).

Further, immobilization of an enzyme is carried out in that an expensive enzyme capable of reacting a large number of samples under the same conditions can be continuously used.

However, some of the enzymes used are not suitable for practical use because their activity immediately decreases even after immobilization, and it is difficult to carry out stable measurement for a long period of time.

[0009]

The object of the present invention is to provide a measuring device capable of stable measurement over a long period of time, and also to provide a method for recovering the activity of an immobilized form having a reduced enzyme activity.

[0010]

The present invention is illustrated by the embodiments shown below, but the invention is not limited thereto.

(1) Enzyme-immobilized product in which L-lactate dehydrogenase and other dehydrogenase are immobilized; L-lactate oxidase-immobilized product; and electrode activity increased or decreased by L-lactate oxidase reaction A measuring device for a substrate reductant of the other dehydrogenase, comprising an electrode for detecting a substance, wherein the measuring device comprises at least the other dehydrogenase immobilized by a physical adsorption method. (2) An enzyme-immobilized body in which the two enzyme-immobilized bodies are encapsulated in the same reactor and which contains L-lactate dehydrogenase, another dehydrogenase, and L-lactate oxidase in a mixed state. The measuring device according to (1). (3) The measuring device according to (1) or (2), further comprising an L-lactate oxidase-immobilized body and an electrode for detecting an electrode active substance that increases or decreases due to the L-lactate oxidase reaction. (4) L-lactate oxidase is immobilized on a carrier by a covalent bond, and L-lactate dehydrogenase and another dehydrogenase are immobilized on the carrier by physical adsorption (1) or (2 ) The measuring device described. (5) L-lactate oxidase and L-lactate dehydrogenase are immobilized on a carrier by a covalent bond, and another dehydrogenase is immobilized on the carrier by physical adsorption. (1) or (2) measuring device. (6) The measuring device according to (1) or (2), wherein the other dehydrogenase is L-glutamate dehydrogenase. (7) By passing a solution containing other dehydrogenase through an enzyme-immobilized body in which the other dehydrogenase is immobilized in the measuring apparatus according to (1) or (2), the other dehydrogenase is added. A method for activating a measuring device by immobilizing by physical adsorption.

[0012]

The enzymatic reaction of L-LDH and LOD used in the present invention is shown as follows.

L-LDH reaction: pyruvate + NADH +
H ⁺ → L-lactic acid + NAD ⁺ LOD reaction: L-lactic acid + O ₂ → pyruvic acid + H ₂ O ₂

Pyruvic acid or NADH can be measured by detecting the decreasing oxygen or the produced hydrogen peroxide by using the reaction in which these two kinds of enzymes are combined. Various substances can be measured by further carrying out the following dehydrogenase reaction before this enzymatic reaction.

Dehydrogenase reaction: Substrate + NAD ⁺ → Substrate oxidant + NADH + H ⁺ That is, the substance to be measured (substrate reductant of another dehydrogenase) is oxidized and reduced by another dehydrogenase in the presence of NAD. It receives the action of the reaction and produces an oxidized substrate and NADH.
Next, the produced NADH was L-LD in the presence of pyruvic acid.
H produces L-lactic acid and regenerates NAD. The produced L-lactic acid is oxidized by LOD under dissolved oxygen to regenerate pyruvic acid and produce hydrogen peroxide.

In the present invention, dehydrogenases that can be used include various dehydrogenases having NAD as a coenzyme, L-glutamate dehydrogenase, glycerol dehydrogenase, sorbitol dehydrogenase and the like.

Electrochemical methods are used for the quantification of the electrode active substances such as hydrogen peroxide finally produced by these reactions or reduced oxygen. The method of quantifying hydrogen peroxide by a spectroscopic method involves a chemical reaction and is detected by a colorimetric method, so that the coloring and turbidity of the sample affect. In comparison, the electrochemical method is simple and accurate because the reduced oxygen is detected at the oxygen electrode and the increased hydrogen peroxide is detected at the hydrogen peroxide electrode. Further, the measurement can be performed by interposing an electron carrier such as dichloroindophenol, potassium ferricyanide, or benzoquinone, a so-called mediator.

As the oxygen electrode for measuring the consumed oxygen, various known ones such as a carbani type and a Clark type can be used, and it may have a permselective membrane. As the hydrogen peroxide electrode for measuring the hydrogen peroxide produced, a known 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 against hydrogen peroxide and high sensitivity. An electrode having a selective permeation film such as a polysiloxane film, an acrylic resin film, a protein film, and an acetyl cellulose film on the electrode surface is desirable from the viewpoint of removing obstacles.

The electrode system is composed of a working electrode and a counter electrode 2
A hydrogen peroxide electrode or an oxygen electrode can be used as the electrode. From the viewpoint of stability and accuracy, a three-electrode structure including a working electrode, a reference electrode and a counter electrode is preferable.

The enzyme used in the present invention is used after being immobilized. Of course, the same reaction proceeds even when used in a solution, but when immobilized and used, there are advantages that the enzyme can be repeatedly used and that the reaction conditions of the enzyme can be easily adjusted.

L-lactate dehydrogenase and other dehydrogenases can be mixed and fixed, and L-lactate oxidase can be separately fixed and encapsulated in different reactors, or three enzymes can be mixed and fixed. It is also possible to incorporate them in the same reactor.

The form of the immobilized enzyme may be a method of immobilizing it on the membrane on the surface of the electrode, a method of immobilizing it on a carrier and filling it in a reactor such as a column, or a reactor using a membrane or hollow fiber. Among them, the method of packing the carrier immobilized on the column can increase the amount of enzyme that can be immobilized as compared with the case where it is immobilized on the electrode surface, and can easily immobilize a sufficient amount of enzyme for the reaction. it can. The material of the column is
Acrylic resins, fluororesins, vinyl chloride resins, metals such as glass and stainless steel, or combinations thereof can be used.

Examples of the enzyme immobilization method include an adsorption method, a chemical bonding method, and an entrapping method. Each immobilization method has advantages and disadvantages. The adsorption method is an immobilization method that utilizes the fact that an enzyme is physically adsorbed on a carrier depending on the nature of the carrier. The advantage is that it can be immobilized under relatively mild conditions. In addition, it can be easily immobilized, and the carrier can be regenerated so that it can be immobilized many times. However, the disadvantage is that the bond is weak and the enzyme is easily released.

On the other hand, the chemical bonding method is an immobilization method in which a carrier and an enzyme are formed into a strong covalent bond with a crosslinking reagent such as glutaraldehyde. The advantage is that the bond is strong and the immobilized enzyme is difficult to release, but the disadvantage is that the activity of the immobilized form may be low because the conditions of the covalent bond reaction are severe, and the enzyme activity cannot be regenerated. Is.

Immobilization of all enzymes by the same method is operationally simple, but depending on the nature of the enzyme it may not be suitable. For example, it is desirable to firmly immobilize an enzyme that is relatively stable even if immobilized and can withstand the treatment by the chemical bond method by a chemical bond. For an enzyme that is unstable when immobilized, and whose activity decreases rapidly, an adsorption method that can easily immobilize the enzyme and does not cause a chemical reaction is suitable.

LOD and L-LDH are stable even when chemically bound, and L-LDH is also relatively stable when immobilized by a chemical bond. Therefore, LOD and L-LDH are chemically bound to each other by another dehydrogenase. Is immobilized by an adsorption method, or LOD is immobilized by a chemical bonding method, and L-LDH and other dehydrogenases are immobilized by an adsorption method. Taking advantage of each immobilization method, in the present invention, LOD is first immobilized on a carrier by a chemical bonding method.
At this time, L-LDH may also be bonded by a chemical bonding method.
After packing the LOD-immobilized carrier in a column reactor, if L-LDH is not immobilized, a solution containing L-LDH and other dehydrogenase is passed through the column reactor to physically adsorb and immobilize it. You can

When other dehydrogenases are immobilized by the chemical bonding method, the activity of other dehydrogenases, which have poor stability due to immobilization, may be observed even though the activities of LOD and L-LDH are not lowered. The problem of having to replace the column reactor with a new immobilized enzyme column reactor in a short period of time is eliminated because it drops in a short period of time.

In the present invention, when the activity of another dehydrogenase having poor stability is reduced, the adsorption of the dehydrogenase again causes the adsorption of the dehydrogenase, thereby extending the life of the column. be able to. As the carrier used for immobilization, diatomaceous earth, calcined diatomaceous earth, silica gel, glass beads, alumina, ceramics, carbon, activated carbon, molecular sieves, silicone rubber, cellulose, agarose, amino acid polymers and the like can be used. Particularly, diatomaceous earth, calcined diatomaceous earth, and silica gel are preferable.

A suitable method is to add NAD and pyruvate required for the reaction of L-LDH and another dehydrogenase to a carrier. At this time, the concentration of NAD is 0.5 mM to 5
About 0.5 mM to 5 mM of mM and pyruvic acid are preferable. L-LDH, LOD as carriers to be sent
Another phosphate-based buffer solution or the like that has a buffering capacity near pH 7 which is suitable for another dehydrogenase and does not have an electrochemical effect on the electrode is used. Although the substrate reductant measurement of other dehydrogenases has been described above, when measuring a sample in which L-lactic acid may be contained in the sample, the amount of L-lactic acid is measured, and thereby the substrate reduction is performed. Body measurements need to be corrected.

For that purpose, it is necessary to provide, for example, two types of detection units. That is, one is an L-lactic acid detection unit equipped with an LOD-immobilized body and an electrode, and a substrate reduced body of another dehydrogenase equipped with an immobilized body of L-LDH, LOD and another dehydrogenase and an electrode. And a detection unit for detecting L-lactic acid. Based on the current values obtained by these two detectors, the substrate reductant and L
-The concentration of lactic acid is determined. At this time, the two detectors may be arranged in series or in parallel.

Specifically, GLD is used as another dehydrogenase.
The flow type apparatus using H shown in FIGS. 1 and 2 can be exemplified.

FIG. 1 shows an in-line type L-glutamic acid and L-lactic acid measuring apparatus. First, the buffer solution is sent from the buffer solution tank (1) by the pump (2), and 5 μl of the sample is injected by the sampler (3). The injected sample passes through the LOD column (4), hydrogen peroxide is produced from L-lactic acid, and the change in current value is detected by the hydrogen peroxide electrode (5). This column and the electrode are used as the first component detecting section. Next GLD
H, L-LDH, and LOD were passed through the column (6), and NADH and L were extracted from L-glutamic acid.
-Hydrogen peroxide is generated through lactic acid and hydrogen peroxide electrode (7)
The change in current value is detected at. This is the second component detector. Keep these columns and electrodes in a constant temperature bath at 30 ° C (8)
Install inside. The change in the current value at each electrode is detected by the detector (9). Further, the signal can be sent to the personal computer (11).

FIG. 2 shows a parallel type measuring apparatus for L-glutamic acid and L-lactic acid. First, the buffer solution is sent from the buffer solution tank (21) by the pump (22), and 5 μl of the sample is injected by the sampler (23). The injected sample is divided into two by a three-way joint (24). One passes through the LOD column (25), hydrogen peroxide is produced from L-lactic acid, and the change in current value is detected by the hydrogen peroxide electrode (26). This column and the electrode are used as the first component detecting section. One passed through a column (27) on which GLDH, L-LDH, and LOD were immobilized and passed from L-glutamic acid to NADH,
Hydrogen peroxide is produced via L-lactic acid, and a change in current value is detected at the hydrogen peroxide electrode (28). This is the second component detector. These columns and electrodes are installed in a constant temperature bath (29) at 30 ° C. The change in current value at each electrode is detected by the detector (30). Further, the signal can be sent to the personal computer (33).

In these cases, the L-
A standard curve is prepared from the current values obtained by measuring the standard solutions of glutamic acid and L-lactic acid. Calibration curve 1 is a calibration curve of L-lactic acid prepared from the current value obtained at the first electrode when L-lactic acid is injected. Calibration curve 2 is L created from the current value obtained at the second electrode when L-lactic acid is injected.
-A calibration curve for lactic acid. The calibration curve 3 is a calibration curve of L-glutamic acid created from the current value obtained at the second electrode when L-glutamic acid is injected. At this time, of course, a current value cannot be obtained even if L-glutamic acid is injected into the first electrode. In the case of measuring an actual sample, a current value 1 obtained from the first electrode and a current value 2 obtained from the second electrode are obtained.

The amounts of L-glutamic acid and L-lactic acid are calculated, for example, by the following methods. The L-lactic acid amount is calculated by substituting the current value 1 into the calibration curve 1. The calculated L-lactic acid concentration is substituted into the calibration curve 2 to calculate the current value of L-lactic acid contributing to the current value 2. By subtracting the L-lactic acid current value that contributes to the current value 2 from the current value 2, the current value based on L-glutamic acid is obtained. The amount of L-glutamic acid can be calculated by substituting the current value of this L-glutamic acid contribution to the calibration curve 3.

[0036]

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 (1) Manufacture of LOD-immobilized column 150 mg of fire-resistant brick (30 to 60 mesh), which was fired diatomaceous earth, was well dried and then added to an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane. After soaking for a long time, it is thoroughly washed with toluene and dried. The aminosilane-treated carrier is immersed in 5% glutaraldehyde for 1 hour to formylate. Then, it is washed thoroughly with distilled water and finally replaced with 100 mM sodium phosphate buffer of pH 7.0 to remove this buffer as much as possible. This formylated refractory brick has a pH of 7.0,
Solution 200μ dissolved in 100 mM sodium phosphate buffer at a concentration of 50 units / ml of LOD (manufactured by Sigma)
1 is contacted and left at 0 to 4 ° C. for 1 day for immobilization. The enzyme-immobilized carrier was packed in an acrylic resin column having an inner diameter of 3.5 mm and a length of 30 mm to obtain a LOD-immobilized column.

(2) Production of GLDH, L-LDH, and LOD-immobilized column In the same LOD-immobilized column prepared in (1),
GL in 100 mM sodium phosphate buffer, pH 7.0
DH (Boehringer) 50 units / ml, LL
5 ml of a solution having a concentration of 10 units / ml of DH (Boehringer) was contacted at a flow rate of 0.6 ml / min,
It was fixed.

(3) Manufacture of hydrogen peroxide electrode A side surface of a platinum wire having a diameter of 2 mm is coated with heat-shrinkable Teflon,
One end of the line is smoothed with a file and 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, 0.1 M
Electrolyte in sulfuric acid at +2.0 V for 10 minutes. After that, the platinum wire was washed well with water and then dried at 40 ° C for 10 minutes.
It is immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour and then washed. 20 mg of bovine serum albumin (Fraction V, manufactured by Sigma) is dissolved in 1 ml of distilled water, and glutaraldehyde is added to the solution to make the concentration 0.2%. 5 μl of this mixed solution was quickly put on a platinum wire prepared in advance, and dried and cured at 40 ° C. for 15 minutes to form a hydrogen peroxide selective permeable membrane, which was used as a hydrogen peroxide electrode.

An Ag / AgCl reference electrode was used as the reference electrode, and a conductive pipe was used as the counter electrode.

(4) Measuring apparatus The flow type measuring apparatus shown in FIG. 1 was used. The composition of the buffer solution is 100 mM sodium phosphate, 50 mM potassium chloride,
It contains 1 mM sodium azide, 1 mM NAD, 1 mM pyruvic acid and has a pH of 7.0. Pump flow velocity is 0.6m
It was 1 / min.

(5) Measurement of standard solution Distilled water, 1,2,3 mM L-lactic acid, 1,2,3 mM L-glutamic acid was injected into the measuring device of (4), and
The current values obtained at the electrodes and the second electrode are as shown in Table 1,
The following calibration curve was obtained. However, Y is the current value (nA),
X is the L-lactic acid concentration (mM) in the sample.

[0043]

[Table 1]

Calibration curve 1 First electrode L-lactic acid calibration curve Y = 4
5.06X + 0.81 calibration curve 2 second electrode L-lactic acid calibration curve Y = 3
3.21X + 0.31 calibration curve 3 2nd electrode L-glutamic acid calibration curve Y =
9.40X + 0.35

Example 2 (1) Production of LOD-immobilized column An LOD-immobilized column was produced in the same manner as in Example 1 (1). (2) Manufacture of GLDH, L-LDH, and LOD-immobilized column In the formylated carrier as in (1) of Example 1, pH was added.
LOD in 7.0, 100 mM sodium phosphate buffer
A solution (200 μl) dissolved at a concentration of 50 units / ml (manufactured by Sigma) and 250 units / ml L-LDH (manufactured by Boehringer) is brought into contact and left standing at 0 to 4 ° C. for 1 day to be immobilized. This enzyme-immobilized carrier has an inner diameter of 3.5 mm and a length of 3
It is packed in a 0 mm acrylic resin column. GLDH (manufactured by Boehringer) 50 units / ml in 100 mM sodium phosphate buffer, pH 7.0.
5 ml of a solution dissolved at a concentration of 0 unit / ml is contacted and immobilized at a flow rate of 0.6 ml / min.

(3) Production of Hydrogen Peroxide Electrode A hydrogen peroxide electrode was produced in the same manner as in (3) of Example 1. (4) Measuring apparatus The flow type measuring apparatus shown in FIG. 1 was used. The composition of the buffer solution is 100 mM sodium phosphate, 50 mM potassium chloride,
It contains 1 mM sodium azide, 1 mM NAD, 1 mM pyruvic acid and has a pH of 7.0. Pump flow velocity is 1.0m
It was 1 / min.

(5) Measurement of standard solution Distilled water, 1, 2, 3 mM L-lactic acid, 1, 2, 3 mM L-glutamic acid was injected into the measuring device of (4), and
The current values obtained at the electrode and the second electrode are as shown in Table 2,
The following calibration curve was obtained. However, Y is the current value (nA),
X is the L-lactic acid concentration (mM) in the sample.

[0048]

[Table 2]

Calibration curve 1 First electrode L-lactic acid calibration curve Y = 3
4.26X + 0.74 calibration curve 2 2nd electrode L-lactic acid calibration curve Y = 2
7.91X + 0.24 calibration curve 3 second electrode L-glutamic acid calibration curve Y =
3.88X + 0.13

[Brief description of drawings]

FIG. 1 is a flow-type L used in Examples 1 and 2.
-Glutamic acid L-lactic acid measuring device.

FIG. 2 illustrates another aspect of the invention.

FIG. 3 shows the enzymatic reaction of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Buffer tank 2 Pump 3 Sampler 4 LOD fixed column 5 Hydrogen peroxide electrode (1st electrode) 6 GLDH, L-LDH, LOD fixed column 7 Hydrogen peroxide electrode (2nd electrode) 8 Constant temperature bath 9 Detector 10 Single Board Computer 11 Personal Computer 12 RS232C Code 13 Sampler Control Signal 14 Liquid Sending Pump Control Signal 15 Waste Liquid 21 Buffer Liquid Tank 22 Pump 23 Sampler 24 Three-way Joint 25 LOD Fixing Column 26 Hydrogen Peroxide Electrode (First Electrode) 27 GLDH , L-LDH, LOD-immobilized column 28 Hydrogen peroxide electrode (second electrode) 29 Constant temperature bath 30 Detector 31 Single board computer 32 RS232C code 33 Personal computer 34 Sampler control signal 35 Liquid feed pump control signal 36 Waste liquid

Claims (7)

[Claims] 1. An enzyme-immobilized body in which L-lactate dehydrogenase and another dehydrogenase are immobilized; L-lactate oxidase-immobilized body;
And an electrode for detecting an electrode active substance that increases or decreases due to L-lactate oxidase reaction, and is a device for measuring a substrate reductant of the other dehydrogenase, which is a physical adsorption method for at least the other dehydrogenase. Measuring device fixed by. 2. An enzyme immobilization in which the two enzyme-immobilized bodies are encapsulated in the same reactor and which contains L-lactate dehydrogenase, another dehydrogenase, and L-lactate oxidase in a mixed state. The measuring device according to claim 1, which is a compound. 3. An L-lactate oxidase-immobilized product and L-
The measuring device according to claim 1, further comprising an electrode that detects an electrode active substance that increases or decreases due to a lactate oxidase reaction. 4. The L-lactate oxidase is immobilized on a carrier by a covalent bond, and the L-lactate dehydrogenase and other dehydrogenases are immobilized on the carrier by physical adsorption.
Or the measuring device according to 2. 5. The L-lactate oxidase and the L-lactate dehydrogenase are immobilized on the carrier by a covalent bond, and the other dehydrogenase is immobilized on the carrier by physical adsorption.
The measuring device described. 6. The measuring device according to claim 1, wherein the other dehydrogenase is L-glutamate dehydrogenase. 7. The other dehydrogenase is fixed by passing a solution containing the other dehydrogenase through the enzyme-immobilized body having the other dehydrogenase immobilized in the measuring apparatus according to claim 1 or 2. A method for activating a measuring device by immobilizing by physical adsorption.