JPH06229981A - Measuring method utilizing enzyme reaction - Google Patents

Abstract

PURPOSE:To enable measurement of various substances handily at a high accuracy by using a dehydrogenase or the like for catalyzing a reaction to form a substrate oxidation body and a coenzyme reduction body from a substrate reduction body and a coenzyme oxidation body in a sample. CONSTITUTION:A reaction is catalyzed by a first dehydrogenase E1 to form a substrate oxidation body Xox and a coenzyme reduction body Cred from a substrate reduction body Xred and a coenzyme oxidation body Cox in a sample. Then, a second substrate reduction body Yred and the coenzyme oxidation body Cox are formed with the second dehydrogenase E2 from the coenzyme reduction body Cred and a second substrate oxidation body Yox. Then, the second reduction body is formed by a reaction system containing the second substrate oxidation body Yox. The substrate reduction body Yred formed is brought into contact with an immobilizing body of an oxidation enzyme E3 for catalyzing an oxidizing reaction to detect a variation of an electrode active substance, namely, oxygen or hydrogen peroxide decreasing or increasing electrochemically. Thus, the amount of the substrate reduction body Xred in the sample is determined.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring method using an enzymatic reaction, and more particularly to a method for measuring various substances such as glutamic acid.

[0002]

2. Description of the Related Art Compared to a detection method using a chemical reaction, an assay method using an enzyme reagent does not use a dangerous reagent such as an acid, a base or an organic solvent, and a complicated reaction operation such as heating or extraction. It is a safe and environmentally friendly analysis method because it does not need to be performed and there is almost no need to consider the treatment of waste liquid.
In addition, the enzyme has the advantage that it has a high substrate specificity, excellent selectivity, and mild reaction conditions.

Therefore, various analytical methods utilizing enzyme reactions have been known. For example, a method of detecting reduced oxygen or increased hydrogen peroxide by the electrode method using the reaction of the substrate oxidant and hydrogen peroxide generated by the action of the oxidase from the substrate and oxygen, or the generated hydrogen peroxide Generally, the colorimetric method is used for quantification. In addition, in the measurement method using dehydrogenase, the coenzyme is added to the substrate and the redox reaction between the substrate and the coenzyme occurs due to the action of the dehydrogenase, which is used to increase or decrease the coenzyme by the colorimetric method. The method of detection is common. If the final detection method is a colorimetric method among these methods,
There have been problems such as turbidity and coloring of the sample hindering accurate measurement, and complicated pretreatment is required to remove these.

Further, the measuring method of detecting with an electrode using an oxidase is not affected by turbidity or a coloring substance, but the kinds of oxidases having a sufficient activity suitable for practical use are limited, and thus various methods are used. We were unable to meet the measurement request. Regarding the dehydrogenase, for example, alcohol dehydrogenase does not act much on methanol as compared with ethanol, and thus is used when measuring ethanol in a system in which methanol is mixed. However, it is difficult to reproducibly measure nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) used as a coenzyme of dehydrogenase by the electrode method, and dehydrogenase cannot be simply used. .

Further, L-glutamic acid is an amino acid well known as an umami component of foods, and is added to various foods, and among many measurement objects, there is a high demand for measurement of L-glutamic acid. Glutamic acid is measured by an amino acid analyzer, a method of converting it to pyroglutamic acid and then chemically quantifying it by extracting with an organic solvent, and a colorimetric method has been often used, but pretreatment is troublesome. Etc. still remains a problem.

Further, L-glutamic acid oxidase is known as an oxidase which acts on L-glutamic acid. This enzyme produces α-ketoglutaric acid, ammonia, and hydrogen peroxide from L-glutamic acid and oxygen. The combination of this oxidase and the electrode should allow the measurement of L-glutamic acid. However, the specific activity of L-glutamate oxidase is low and it is difficult to obtain sufficient sensitivity. Further, there is a problem that the range in which the proportional relationship is established between the substrate concentration and the amount of hydrogen peroxide produced or the amount of oxygen reduced is narrow, and the dilution rate of the actual sample becomes large, which is not suitable for practical use.

On the other hand, L-lactic acid is abundant in foods produced by fermentation processes such as miso, soy sauce and dairy products, and it is highly necessary to measure it at the same time as glutamic acid. But,
Conventionally, L-glutamic acid and L-lactic acid were measured independently, and there was no method for continuous and simple measurement.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a small number of types of enzymes having sufficient activity in conventional electrochemical measurements using only oxidases, and the measurement target is limited.
It is an object of the present invention to solve the above problems and provide a method capable of measuring various substances. Further, it provides a method capable of accurately and easily measuring various measurement substances.

[0009]

The present invention can be summarized in the following constitutions, but is not limited thereto.

(1) a. Sample that may include substrate reductant Xred, first dehydrogenase E1, coenzyme oxidant Cox, coenzyme that catalyzes reaction of forming substrate oxidant Xox and coenzyme reductant Cred from Xred and coenzyme oxidant Cox Reductant Cred
And the second substrate reductant Yox and the second substrate reductant Yred and the second dehydrogenase E2 that produces Cox, and the second substrate oxidant Yox. Generating b. Measuring the generated Yred,
A measuring method for determining the amount of Xred in a sample having.

(2) b. The step of measuring the produced Yred is by contacting Yred with an immobilized form of oxidase E3 which catalyzes the oxidation reaction of Yred, and electrochemically detecting the amount of change in the electrode active substance that decreases or increases ( 1) The measuring method described.

(3) Oxygen or hydrogen peroxide is the substance that decreases or increases due to the reaction of oxidase E3 (2)
Measuring method.

(4) c. The method further comprises the step of measuring the amount of Yred in the sample, and the step b. The measured value of Yred obtained in the process is referred to as c. Yred originally existed in the sample obtained in the process
The measuring method according to (1), which is corrected by the amount.

(5) In (1), Xred is L-glutamic acid, E1 is L-glutamate dehydrogenase, Cox is nicotinamide adenine dinucleotide oxidase, and E2 is L-lactate dehydrogenase. And Yox
Is pyruvic acid and Yred is L-lactic acid.

(6) The method for measuring L-glutamic acid according to (2), wherein Yred is L-lactic acid and E3 is L-lactate oxidase.

[0016]

ACTION FIG. 2 shows the enzymatic reaction of the present invention. A.
In the process, the first dehydrogenase E1 reaction and the second dehydrogenase E2 reaction are combined to produce an amount of Yred corresponding to the amount of Xred in the sample. First, the dehydrogenase E1 is the measurement target. The reaction of oxidizing the substrate reductant Xred is described. The general reaction formula of dehydrogenase E1 is shown below. However, the substrate reductant is Xred, the coenzyme oxidant is Cox, the substrate oxidant is Xox, and the coenzyme reductant is Cred. E1: Xred + Cox → Xox + Cred

Although this reaction is reversible, the equilibrium of the reaction is almost always biased toward the production of Xred and Cox. Therefore, even if E1 is used alone and Xred and Cox act, Xred hardly changes to Xox, so it is necessary to convert the produced Xox or Cred into another substance and shift the equilibrium to proceed the reaction. Further, since this Xox or Cred consumption reaction has no effect unless it is carried out at the same time as the E1 reaction, a reaction using an enzyme capable of reacting under the same conditions is conceivable. The present invention is characterized by utilizing the second dehydrogenase E2 which shares the coenzyme C utilized in the reaction of E1. The analysis cost can be reduced by combining the reaction of reoxidizing the reduced coenzyme Cred, recycling the coenzyme, and conducting the reaction with a small amount of the expensive coenzyme used.

In the case of a dehydrogenase, the equilibrium state of the reaction is almost always the substrate reductant Yred and the coenzyme oxidant Co.
It can be used because x is biased in the direction of generation. A second dehydrogenase E2 and a second substrate oxidant Yox are further added to the above E1 reaction system to combine the following E2 reactions. E2: Cred + Yox.fwdarw.Cox + Yred In the present invention, Xred and Yox of the present mole or more are added, and 1 mole of Yred can be produced from 1 mole of Xred. Then b. As a step, the amount of Xred in the sample can be obtained by measuring the produced Yred.

For the quantification of Yred, a method using oxidase E3 which acts specifically on Yred is preferable. A representative reaction of E3 is illustrated below. E3: Yred + O ₂ → Zox + H ₂ O ₂ In the example of this oxidase, it is a one-way reaction that produces hydrogen peroxide from oxygen, and even if Zox coexists, it does not affect the reaction, and NAD (P) , NAD (P) H coexist, there is no effect. The E1 and E2 enzymes do not need to be inactivated because they do not inhibit the E3 reaction or the E3 reaction. The detection method of the E3 reaction includes electrochemical detection and the like, which will be described later.

In the present invention, the coenzyme oxidant Cox is reduced by dehydrogenase E1 to Cred, and further oxidized by E2 to return to Cox. For this reason, Cox apparently does not seem to be involved in the reaction, but it is recycled as an electron carrier. Therefore, in the present invention, Cox is added to the E1 reaction system.
However, even if the amount of Cox is less than the amount of Xred in the sample, Xred can be completely converted to Yred. In the measurement, the conversion coefficient between Xred and Yred does not necessarily have to be 100%, and can be calculated according to a predetermined coefficient. In other words, the reaction can be stopped before the 100% reaction is completed, and the Xred concentration can be determined kinetically. However, since the reaction is completed, it becomes unnecessary to precisely control the reaction time, which is practically preferable.

In order to achieve such reaction conditions,
All Xred cannot be oxidized unless the amount of Yox added to the reaction system is larger than the amount of sample Xred. If the sample contains Yred in addition to Xred, then step c. As a result, the amount of Yred in the sample is first measured, and then Xre in the sample is measured.
It is necessary to measure the amount of Xred by measuring the amount of Yred after the conversion reaction from d to Yred and measuring the difference between the two.

The reactions of the dehydrogenases E1 and E2 proceed at the same time, but the reaction of the oxidase E3 is performed in a separate step.
Zox generated by the oxidation of d may be the same as Yox. Further, Xred does not react with Zox produced by the oxidase E3 because it adds the coenzyme oxidant Cox to the reaction system. Therefore, the recycling of Xred does not occur. Tables 1 to 3 show examples of combinations of the substrate reductant Xred that can be measured in the present invention and the enzyme used for the measurement.

Table 1 exemplifies a reaction in which the substrate reductant Xred is oxidized by the dehydrogenase E1 in the presence of Cox to produce the substrate oxidant Xox.

[0024]

[Table 1]

Table 2 shows that the second substrate oxidant Yox is added in the presence of the generated Cred, and the second dehydrogenase E2 is allowed to act thereon to produce the second substrate reductant Yred a.
The process is shown. It also exemplifies a combination of reactions in which oxidase E3 that oxidizes Yred is caused to act to produce Zox. The reaction of E3 can be utilized to quantify Yred (step b.).

[0026]

[Table 2]

Further, in the present invention, a substance which produces a substrate reductant Xred by the enzyme E0 can also be measured.
For example, the substance to be measured can be expanded by performing a step of decomposing the substance to be measured into a reduced substance Xred by the enzyme E0 and then quantifying Xred.

[0028]

[Table 3]

The substances to be measured are dehydrogenases E1 and E.
It can be changed freely by combining the two. As shown in Table 1, various dehydrogenases can be used as E1.

For example, measurement of glutamate using glutamate dehydrogenase, measurement of glycerol using glycerol dehydrogenase, measurement of L-lactic acid using L-lactate dehydrogenase, alcohol using alcohol dehydrogenase. The measurement of glucose, the measurement of glucose using glucose dehydrogenase, the measurement of D-lactic acid using D-lactate dehydrogenase, and the like.

As shown in Table 2, L produced by adding pyruvate and L-lactate dehydrogenase to the E1 reaction system.
-Lactic acid can be measured using L-lactate oxidase. In addition, acetaldehyde and alcohol dehydrogenase can be added to the reaction system of E1 and the produced ethanol can be measured using alcohol oxidase. Further, D-glucono-δ-lactone and D-glucose dehydrogenase are added to the E1 reaction system, and the produced D-glucose can be measured using D-glucose oxidase.

The E2 reaction combined with the E1 reaction is
Select the one with the same coenzyme. The coenzyme required by E2 may be NAD or NADP (nicotinamide adenine dinucleotide phosphate) as long as it is common to E1. That is, when the coenzyme added to the sample is NAD, E2 and E1
Must be NAD-responsive dehydrogenase, and NADP-responsive dehydrogenase when the coenzyme is NADP.

Next, the Yred produced by the E2 reaction is converted to E
The step of quantifying using 3 reactions will be specifically described (b. Step). Of course, measure Yred originally present in the sample c. The process is the same as the method. The amount of hydrogen peroxide produced by the reaction of oxidase E3 is determined, for example, by using peroxidase and 2,2′-azino-di (3-ethylbenzthiazoline) -6-sulfonic acid or 4-aminoantipyrine ( (Trinder method)
It can be measured by measuring the absorbance in the visible region. The visible light absorbance measurement is less susceptible to turbidity than the ultraviolet light absorbance measurement used to quantify coenzymes. However, the measurement using a spectrophotometer is difficult to measure accurately when the sample is turbid or contains a coloring substance, and therefore pretreatment is required.

Compared to this method, the electrochemical measuring method in which reduced oxygen or generated hydrogen peroxide is converted into an electric current value by an electrode and measured is easy to operate and less susceptible to turbidity and coloring substances. It is an excellent detection means. As the oxygen electrode for measuring the consumed oxygen, various known electrodes such as galvanic type and Clark type can be used.

As a hydrogen peroxide electrode for measuring the hydrogen peroxide produced, carbon, platinum, nickel,
It is possible to use a known one using palladium or the like and silver or the like on the cathode side. 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 a polysiloxane film, an acrylic resin film, a protein film, an acetyl cellulose film, an albumin film on the surface of the electrode is desirable from the viewpoint of removing obstacles. Also, dichloroindophenol,
It is also possible to measure with an electrode in which an electron carrier such as potassium ferricyanide or benzoxone, a so-called mediator or the like is interposed. As the electrode system, a two-electrode hydrogen peroxide electrode or an oxygen electrode composed of a working electrode and a counter electrode can be used, but a three-electrode system composed of a working electrode, a reference electrode and a counter electrode is more preferable in terms of stability and accuracy. desirable.

Although oxidase E3 in the form of a solution can be used, the use of oxidase E3 immobilized makes it possible to repeatedly use the enzyme, which is advantageous in that the reaction conditions of the enzyme can be easily defined. is there. Further, it is preferable to use the oxidase E3 immobilized body in combination with an electrode for detecting the changing amount of increasing or decreasing oxygen or hydrogen peroxide, because Yred can be detected in one step in a short time.

The method for immobilizing the enzyme is not particularly limited, and an adsorption method, a chemical bonding method, an encapsulation method, etc. can be used. Above all, a chemical bonding method that can form a strong immobilized body is preferable.
Carriers used for immobilization include diatomaceous earth, silica gel, glass beads, alumina, ceramics, carbon, activated carbon, molecular sieves, silicone rubber, cellulose,
Agarose, amino acid polymers, etc. can be used. As the chemical bonding method, a method in which an amino group is introduced on the surface of a carrier with an aminosilanization reagent and further formylation is performed using a polyfunctional aldehyde such as glutaraldehyde, and then an enzyme is brought into contact to immobilize it is preferable. Take as an example. As a form of the immobilized enzyme, a method of immobilizing it on a carrier and packing it in a reactor such as a column is considered.

Further, a membrane in which L-lactate oxidase is fixed with a cross-linking agent such as glutaraldehyde, formaldehyde, succinyl aldehyde can be attached to the electrode and used. When immobilizing in a membrane form, albumin, globulin,
Other proteins such as gelatin can be added to crosslink the L-lactate oxidase. The buffer solution used for the measurement may be any buffer solution having a pH suitable for the oxidase E3 and having no buffering effect on the electrode electrochemically.

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 L-glutamic acid sample and a mixture sample of L-glutamic acid and L-lactic acid were used as samples, and NA was used for each sample.
After contacting D, glutamate dehydrogenase, L-lactate dehydrogenase, and pyruvate and reacting for a certain period of time (a. Step), the L-lactic acid concentration was measured (b. Step).

L-lactic acid concentration was measured by a method in which hydrogen peroxide produced when L-lactic acid was oxidized was measured with a platinum electrode using an immobilized L-lactic acid oxidase. (1) Production of L-lactate oxidase-immobilized column 150 mg of fire-resistant brick (30 to 60 mesh), which is calcined diatomaceous earth, is well dried and immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour. After that, wash well with toluene and dry. After the aminosilane-treated carrier was immersed in 5% glutaraldehyde for 1 hour, it was washed thoroughly with distilled water and finally pH adjusted.
Replace with 7.0, 100 mM sodium phosphate buffer and remove this buffer as much as possible. To this formylated refractory brick, 200 μl of a solution prepared by dissolving 100 mM sodium phosphate buffer of pH 7.0 at a concentration of 50 units / ml of L-lactate oxidase (manufactured by Sigma, derived from Pediococcus) was contacted, and 0- Let stand for 1 day at 4 ° C to immobilize. This enzyme-immobilized carrier has an inner diameter of 3.5 mm and a length of 3
It was packed in a 0 mm acrylic column to give an L-lactate oxidase-immobilized column.

(2) Production of hydrogen peroxide electrode A side surface of a platinum wire having a diameter of 2 mm was 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 +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 selectively 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.

(3) L-lactic acid measuring device A flow type L-lactic acid measuring device is shown in FIG. The buffer solution is sent from the buffer solution tank (1) by the pump (2), and 5 μl of the sample is injected from the sampler (3). When L-lactic acid in the sample is sent to the L-lactate oxidase-immobilized column (5) in the thermostat (4), hydrogen peroxide is produced, and the hydrogen peroxide electrode (6) produces a current value. Is detected and detected by the detector (7). Further, the signal can be sent to the personal computer (10). The composition of the buffer solution is 100 mM sodium phosphate, 50 mM potassium chloride,
It contains 1 mM sodium azide and has a pH of 7.0.

(4) Measuring method For the L-glutamic acid sample and the mixed solution sample of L-lactic acid and L-glutamic acid, the [A] sample was left as it was (step c.) And the [B] L-glutamic acid oxidation was completed. The L-lactic acid concentration was measured using the above-mentioned L-lactic acid measuring device for two points of the reaction liquid in which pyruvic acid was reduced.
In the reaction system of [B], the concentration was NAD 5 mM, pyruvic acid 10 mM, glutamate dehydrogenase (Boehringer, bovine liver) 100 units / ml, L-lactate dehydrogenase (Boehringer, pig muscle) 100 A solution containing unit / ml of sodium phosphate and having a pH of 7.5 was reacted at room temperature of 25 ° C. for about 10 minutes.

As a sample, 1 m of L-glutamic acid was used.
M, L-glutamic acid 2 mM, L-lactic acid 1 mM, L
-Glutamic acid 1 mM, L-lactic acid 1 mM, L-glutamic acid 2 mM were used. (5) Results The amount of L-lactic acid produced after 10 minutes was as shown in Table 4.

The measurement result before the reaction [A] is L of each sample.
-Lactic acid measurement value is shown (step c). After the reaction [B] is L-
Oxidation of glutamic acid was completed, and pyruvic acid was reduced to produce L-lactic acid, and L that had been present in the sample before the reaction.
-Shows the total value of lactic acid (b. Step). L in the sample
The amount of glutamic acid was determined by subtracting the L-lactic acid measurement value before the reaction from the L-lactic acid measurement value after the reaction.

[0046]

[Table 4]

Example 2 L-lactic acid and L-glutamic acid in food samples were measured using the same L-lactate oxidase-immobilized column and L-lactic acid measuring device having a hydrogen peroxide electrode as in Example 1.

(1) Method of measurement For samples (seasoned vinegar store: "Asasuke-san" shiso flavor), which are seasonings for pickles, and (seasoned vinegar store: "Asatsuki-san" ginger flavor), 250 times each It diluted and L-lactic acid density | concentration was measured (c. Process). Furthermore, using a 250-fold diluted solution of the sample, the concentration of the reaction system was NAD 5 mM, pyruvic acid 10 mM, glutamate dehydrogenase (Boehringer, bovine liver-derived) 20 units / ml, L-lactate dehydrogenase (Boehringer). , (From pig muscle), 20 units / ml, containing sodium phosphate and having a pH of 7.5 were reacted at 25 ° C. for 1 hour (step a.), And then L-lactic acid concentration was measured (step b). The L-glutamic acid concentration and L-lactic acid concentration in the sample are% (W /
W). (2) Results L-lactic acid and L-glutamic acid concentrations of each sample are shown in Table 5.

[0049]

[Table 5]

[0050]

By using the enzyme reaction of the present invention,
It has become possible to easily and accurately quantify a substance that can be the substrate reduced form Xred of the dehydrogenase E1. Also,
The second substrate reductant Yred can also be continuously measured. Further, according to the present invention, it is possible to measure an object to be measured whose substrate specificity of an oxidase, Km value (measurement range), heat resistance, specific activity, pH and the like are not practical. For example, L-glutamic acid, glycerol, etc. can be easily measured. Further, it becomes possible to measure the two components of L-glutamic acid and L-lactic acid continuously and easily, and it is possible to measure representative amino acids and organic acids in foods. As shown in the embodiment, there is an advantage that the two components of Xred and Yred can be measured by using only the measuring device of Yred.

[Brief description of drawings]

FIG. 1 shows a flow-type L-lactic acid measuring apparatus used in Examples.

FIG. 2 shows the enzymatic reaction of the present invention.

[Explanation of symbols]

 1 Buffer Tank 2 Pump 3 Sampler 4 Constant Temperature Tank 5 L-Lactate Oxidase Immobilization Column 6 Hydrogen Peroxide Electrode 7 Detector 8 Single Board Computer 9 RS232C Code 10 Personal Computer 11 Sampler Control Signal 12 Liquid Transfer Pump Control Signal 13 Waste Liquid

Claims (6)

[Claims] 1. A. A sample that may contain the substrate reductant Xred,
The first dehydrogenase E1, the coenzyme oxidant Cox, the coenzyme reductant Cred, and the second dehydrogenase E1 which catalyze the reaction of forming the substrate oxidant Xox and the coenzyme reductant Cred from Xred and the coenzyme oxidant Cox
The second substrate reductant Yred is produced in a reaction system containing a second substrate reductant Yred and a second dehydrogenase E2 that produces Cox from the substrate oxidant Yox of Process, b. A method of measuring the amount of Xred in a sample, comprising the step of measuring the generated Yred. 2. b. The process of measuring the generated Yred
2. The measuring method according to claim 1, wherein Yred is brought into contact with an immobilized form of oxidase E3 which catalyzes the oxidation reaction of Yred, and the amount of change in the electrode active substance that decreases or increases is detected electrochemically. 3. The method according to claim 2, wherein the substance that decreases or increases due to the reaction of oxidase E3 is oxygen or hydrogen peroxide. 4. c. The method further comprises the step of measuring the amount of Yred in the sample, and the step b. The measured value of Yred obtained in the process is
c. The measuring method according to claim 1, wherein the amount of Yred originally present in the sample obtained in the step is corrected. 5. The method according to claim 1, wherein Xred is L-glutamic acid, E1 is L-glutamate dehydrogenase, Cox is nicotinamide adenine dinucleotide oxidase, and E2 is L-lactate dehydrogenase. Yes, Yox is pyruvic acid, and Yred is L-lactic acid. A method for measuring L-glutamic acid. 6. The method for measuring L-glutamic acid according to claim 2, wherein Yred is L-lactic acid and E3 is L-lactate oxidase.