Amino acid quantitative test paper, method for quantitatively determining amino acid concentration in a test sample, and method for producing amino acid quantitative test paper

Abstract

To provide an amino acid quantification test strip which enables quick and easy quantification of amino acid concentration in a test sample, a method of quantifying amino acid concentration in a test sample, and a method of manufacturing the amino acid quantification test strip.SOLUTION: A test strip of the present invention is for quantifying amino acid concentration in a test sample by a colorimetric method, and comprises an L-glutamate oxidase, coloring reagent, and surfactant carried by a liquid-permeable carrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a test paper for quantitatively determining an amino acid concentration, a method for quantitatively determining an amino acid concentration in a test sample, and a method for producing the test paper for quantitatively determining an amino acid concentration. [Background technology]

[0002] Measurement of glutamic acid, one of the umami components in food, requires a large, expensive dedicated device. A method and a measurement device for measuring umami components in food samples using a flow analysis system have been proposed (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 3-151894 Summary of the Invention [Problem to be solved by the invention]

[0004] Conventionally, expensive amino acid analyzers and liquid measurement kits have been used to quantify amino acids such as glutamic acid. In addition, the measurement requires time and skill, so there has been a demand for test strips that can quickly and easily measure amino acid concentrations. Therefore, an object of the present invention is to provide an amino acid quantification test paper that can easily and quickly quantitate the amino acid concentration in a test sample, a method for quantifying the amino acid concentration in a test sample, and a method for manufacturing the amino acid quantification test paper. [Means for solving the problem]

[0005] In order to achieve the above object, the present inventors have conducted extensive research and have discovered that L-glutamic acid oxyesterase is a hydroxylase inhibitor of L-glutamic acid. D We have found that by supporting a surfactant, together with an enzyme and a color-developing reagent, on a liquid-permeable carrier, it is possible to obtain a test paper that can quantitatively determine amino acids simply and inexpensively.

[0006] In other words, the present invention is a test paper for quantifying the amino acid concentration in a test sample by a colorimetric method, characterized in that L-glutamate oxidase, a color-developing reagent, and a surfactant are carried on a liquid-permeable carrier.

[0007] The present invention also provides a method for quantifying the concentration of an amino acid in a test sample, which comprises contacting the test sample with the above-mentioned test paper and quantifying the concentration by colorimetry.

[0008] Furthermore, the present invention relates to a method for producing the above-mentioned test paper, in which the surfactant is polyoxyethylene (20) sorbitan monolaurate, D A staining solution containing ELISA kit, a color developing reagent, and polyoxyethylene (20) sorbitan monolaurate was prepared. Penetration Inclusion in a carrier Soaking The manufacturing method includes the step of: Effect of the Invention

[0009] According to the present invention, it is possible to provide an amino acid quantification test paper that can easily and quickly quantitate the amino acid concentration in a test sample, a method for quantifying the amino acid concentration in a test sample, and a method for manufacturing the amino acid quantification test paper. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described in detail below. The test paper of the present invention is used to quantify the amino acid concentration in a test sample by colorimetry. The test sample is not particularly limited as long as it is a sample suspected of containing an amino acid, and examples thereof include biological samples (blood, urine, saliva, tears, etc.) and food and beverage products (nutritional drinks, amino acid drinks, etc.). The L-glutamic acid in the test sample may be at a low concentration of less than 1 mM or at a high concentration of 1 mM or more. Examples of amino acids in such test samples include glutamic acid, aspartic acid, glycine, tryptophan, and lysine, and the test paper of the present invention is particularly effectively used for quantifying glutamic acid. Glutamic acid is specifically exemplified by L-glutamic acid.

[0011] The amino acid quantitative test paper of the present invention is a test paper for determining the amount of L-glutamic acid hydroxylase which catalyzes the oxidative deamination of amino acids. D The surfactant, together with the enzyme and color-developing reagent, is supported on a liquid-permeable carrier. As the liquid-permeable carrier, a filter paper or a porous membrane made of cellulose, glass fiber, or the like can be used. The shape and size of the carrier are not particularly limited, and it can be of any shape and size, for example, a small piece of cellulose fiber filter paper measuring 5 mm x 5 mm x 1 mm can be mentioned.

[0012] L-Glutamic Acid Oxygen D As the enzyme, any enzyme can be used. In particular, the following (a) L-glutamic acid oxyenzyme can be used: D (b) L-glutamate oxytocin mutants, and (b) D The L-glutamate oxytocin mutant of the present invention is D It is preferred as a lysine. (a) an L-glutamic acid oxidizing agent comprising an amino acid sequence having an identity of 90% or more to the amino acid sequence of SEQ ID NO: 3 and having an L-glutamic acid oxidizing activity; D L-glutamate oxytocin mutants (provided that the L-glutamate oxytocin mutants have the amino acid sequence of SEQ ID NO: 1) D (Excluding -ze) (b) the L-glutamic acid oxyester of (a) Dand (3) a region near the boundary between the γ region and the β region in the mutant L-glutamate oxidizing enzyme, the mutant having an L-glutamate oxidizing activity, and a peptide linker consisting of 1 to 20 amino acid residues inserted at one or more sites selected from the group consisting of (1) a site in the region near the boundary between the α1 region and the α2 region, (2) a site in the region near the boundary between the α2 region and the γ region, and (3) a site in the region near the boundary between the γ region and the β region. D A mutant of the enzyme the site in the region near the boundary between the α1 region and the α2 region is a site in a region consisting of amino acid residues at positions 349 to 363 in SEQ ID NO: 3; the site in the region near the boundary between the α2 region and the γ region is a site in the region consisting of amino acid residues at positions 372 to 377 in SEQ ID NO: 3; and The site in the region near the boundary between the γ region and the β region is a site in the region consisting of amino acid residues 466 to 469 in SEQ ID NO: 3. D -ase mutant. The above (a) L-glutamic acid oxy D (b) L-glutamate oxytocin mutants, and (b) D Either one of the enzyme mutants may be used, but a more excellent effect is exhibited when both are used.

[0013] The above-mentioned L-glutamic acid oxy D The enzyme variants can be prepared and confirmed in accordance with WO 2021 / 193598.

[0014] Examples of color-developing reagents include a combination of 4-aminoantipyrine and Trinder's reagent, benzidine, o-tolidine, o-dianisidine, 3,3'-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine (TMBZ), and the like, with 3,3',5,5'-tetramethylbenzidine (TMBZ) being particularly preferred.

[0015] The surfactant may be an ionic surfactant selected from an anionic surfactant, a cationic surfactant, and an amphoteric surfactant, or a nonionic surfactant, with the nonionic surfactant being preferred. Examples of nonionic surfactants include polyethylene glycol mono-p-isooctylphenyl ether, polyoxyethylene lauryl ether, polyoxyethylene (20) sorbitan monolaurate (Tween 20), etc., with polyoxyethylene (20) sorbitan monolaurate (Tween 20) being particularly preferred. The surfactant acts as a reaction promoter that promotes the oxidative deamination reaction of glutamic acid.

[0016] The test paper of the present invention is D The staining solution, which contains enzyme, coloring reagent, and surfactant, is contained in the carrier. Soaking The dye can be produced by drying the dye. The solvent for the dye solution can be a mixed solution of alcohol and ketone. The volume ratio of alcohol to ketone (alcohol:ketone) is preferably 10:90 to 90:10, more preferably 40:60 to 60:40. The alcohol can be selected from methanol, ethanol, isopropyl alcohol, etc., and the ketone can be selected from acetone, methyl ethyl ketone, cyclohexanoate, etc.

[0017] L-glutamic acid oxy in staining solution D The concentration of the enzyme in the staining solution is preferably about 500 to 5000 U / L, and more preferably about 1000 to 2000 U / L. The concentration of the color-developing reagent in the staining solution is preferably about 1 to 10 mM, and more preferably about 3 to 5 mM. The concentration of the surfactant in the staining solution is preferably about 0.01 to 0.5%, and more preferably about 0.01 to 0.1%.

[0018] In addition to the above-mentioned components, the staining solution may contain a hydrogen peroxide detection reagent, a buffer solution, etc. The hydrogen peroxide detection reagent is used when hydrogen peroxide is detected by, for example, color development or fluorescence. D Specifically, horseradish peroxylase and / or a coloring agent that can be a substrate thereof are included. DExamples of suitable combinations include, but are not limited to, a combination of 4-aminoantipyrine and Trinder's reagent TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline). The concentration of 4-aminoantipyrine in the staining solution is preferably about 0.1 to 10 mM, and more preferably about 0.5 to 3 mM. The concentration of Trinder's reagent in the staining solution is preferably about 0.1 to 10 mM, and more preferably about 2 to 5 mM.

[0019] The buffer solution is used to maintain the pH in the reaction solution at a value suitable for the target enzyme reaction. Examples of the buffer solution include TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphoric acid, etc., and 0.05 to 0.5 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is particularly preferred. For example, 0.2 M HEPES is a buffer solution with a pH of 7.0. In this case, it is preferable to mix the buffer solution so that the concentration in the staining solution is about 40 to 60%.

[0020] Furthermore, the staining solution may contain one or more selected from the group consisting of an ammonia detection reagent and a 2-oxoglutaric acid detection reagent. Examples of the ammonia detection reagent include the indophenol method, which combines phenol and hypochlorous acid. Examples of the 2-oxoglutaric acid detection reagent include 2-oxoacid reductase.

[0021] The staining solution in the present invention contains, for example, 1000 to 2000 U / L of L-glutamic acid oxytocin. D peroxidase, 20,000-40,000 U / L D The solution can be obtained by adding 0.07 to 1.2 g of 3,3',5,5'-tetramethylbenzidine, and 40 to 60 mL of 0.2 M HEPES (pH 7.0) to a solvent consisting of a mixture of 20 to 30 mL of methanol and 20 to 30 mL of acetone and mixing them uniformly.

[0022] Furthermore, the staining solution may contain an interference reducer that reduces the influence of interference substances in the oxidative deamination reaction. Interfering substances in the oxidative deamination reaction include reducing substances and oxidizing substances. Fruits and vegetables such as tomatoes contain ascorbic acid, which is a type of reducing substance. Processed foods such as processed meat products and processed fruit products contain trace amounts of ascorbic acid for the purpose of promoting color development and preventing fading. For this reason, the color reaction of the color-developing reagent is interfered with by the influence of ascorbic acid. Therefore, the amino acid concentration, particularly the glutamic acid concentration, in fruit and vegetables and processed foods is determined by the glutamic acid oxyconcentration. D It was difficult to measure the amount of acetylcholine using test paper carrying the enzyme.

[0023] Ascorbic acid oxyacetate is a method for quantifying amino acid concentrations while reducing the effects of ascorbic acid. D However, the method of adding ascorbic acid oxylase is known. D However, the use of ascorbic acid as an inhibitor of the oxidative deamination reaction is not practical because it is expensive and requires a complicated pretreatment procedure. By using an interference-reducing agent that reduces the effects of interferences in the oxidative deamination reaction, it is possible to obtain a test paper that can quantitatively measure the amino acid concentration in a test sample while avoiding such inconveniences and reducing the effects of ascorbic acid.

[0024] Examples of interference reducers that reduce the effect of interference substances in the oxidative deamination reaction include 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical, 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl free radical, 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl free radical, 4-carboxy-2,2,6,6-tetramethylpiperidine, 4-hydroxy-2,2,6,6-tetramethylpiperidine, 2,2,6,6-tetramethylpiperidine-1-oxyl free radical, 4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl free radical, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical, and the like. carbamide, 3-cyano-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical, 4-(4-nitrobenzoyloxy)-2,2,6,6-tetramethyl-1-piperidinyloxy free radical, 3-aminomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical, 2-butyl-4,4-dimethyl-2-pentyl-3-oxazolidinyloxy free radical, 2-(4-methoxy-4-oxobutyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy free radical, tetramethylene sulfoxide, 3,4-dibromotetrahydrothiopene-1,1-dioxide, 5,5-dimethyl-1-pyrroline-N-oxide, and pyridine sulfur trioxide.

[0025] These compounds may be used alone or in combination of two or more. Free radicals, especially 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radicals, are particularly preferred as interference reducers, because they can sufficiently reduce the interference reaction caused by ascorbic acid.

[0026] The interference reducing agent is added to the amino acid quantitative test paper of the present invention at a concentration of 680 to 2270 g / m 3If the amount of the interference reducer supported is about 3 to 10 g / L, the effect of reducing the influence of ascorbic acid is exhibited. Therefore, the concentration of the interference reducer in the staining solution may be appropriately set so that the test paper has a predetermined content. For example, when a staining solution containing 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical at a concentration of about 3 to 10 g / L is used, the interference reducer is effective in reducing the influence of ascorbic acid. 3 Therefore, a test paper carrying a certain amount of 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical can be obtained. 680~2270g / m 3 By using a test paper carrying an interference-reducing agent of about 0.001 to 0.1 mM, the amino acid concentration can be measured without interference from ascorbic acid even in a test sample in which about 0.001 to 0.1 mM ascorbic acid is coexistent.

[0027] In preparing the test paper of the present invention, the dye solution is placed in a glass beaker or a stainless steel tank, and a carrier such as paper is immersed in the dye solution for about 0.01 to 1 minute to allow the carrier to absorb the dye solution. Soaking Thereafter, the mixture is dried at 35 to 55° C. for 5 to 25 minutes using an air blower, drum dryer, or the like, to produce the test paper of the present invention carrying the predetermined component.

[0028] The amino acid concentration in a test sample can be quantified using the test paper of the present invention by the following procedure. First, the test paper of the present invention is immersed in the test sample for about 1 to 30 seconds and then removed. The temperature of the test sample is not particularly limited and can be about 20 to 30°C. The immersion time in the test sample is preferably about 1 to 10 seconds, more preferably about 1 to 2 seconds. Excess test sample is shaken off, and after 10 to 600 seconds, preferably 60 to 180 seconds, the color chart is compared visually. The color chart is prepared in advance using a solution with a known amino acid concentration. Specifically, the color chart can be prepared using L-glutamic acid, sodium L-glutamate, and their hydrates.

[0029] Examples of test samples include biological samples, food and beverage products, seasonings, culture media, wastewater, river water, groundwater, seawater, soil, and fertilizer. As representative foods, tomatoes contain about 9 to 14 mg / 100 g of ascorbic acid, and potatoes contain about 13 to 34 mg / 100 g of ascorbic acid. Examples of foods to which ascorbic acid is added for the purpose of promoting color development or preventing fading include the foods and beverages listed in Table 1 below.

[0030] [Table 1] *The data in the above table was transcribed from the "Report of the Additives Subcommittee on the Designation of Calcium L-Ascorbate as a Food Additive" (Ministry of Health, Labour and Welfare).

[0031] The test paper of the present invention contains L-glutamic acid hydroxylase. D Since the gel contains a surfactant as well as the enzyme and coloring reagent, strong coloring can be confirmed 120 seconds after immersion in the test sample. Even when the test sample contains a low concentration of L-glutamic acid, for example 0.05 mM, a predetermined coloring is observed in 120 seconds. In this way, by using the test paper of the present invention, it is possible to quickly and easily determine the concentration of amino acids such as glutamic acid.

[0032] L-Glutamic Acid Oxygen D In the case of test papers that contain an interference reducer such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical in addition to enzymes, coloring dyes, and surfactants, the concentration of glutamic acid can be quantified without the influence of interfering substances such as ascorbic acid. Such test papers are effective for quantifying glutamic acid in fruits and vegetables and processed foods. EXAMPLES

[0033] The present invention will be specifically described below with reference to examples. The materials, contents, mixing ratios, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

[0034] <Example 1> A staining solution was prepared according to the following formulation. L-Glutamic Acid Oxygen D -Ze 160U Peroxy D -Ze (Toyobo PEO-301) 3000U 3,3',5,5'-Tetramethylbenzidine (TMBZ) 0.1g (4.16mM) 0.2M HEPES (pH 7.0) 50mL Methanol 25mL 25mL acetone Tween20 0.05g (0.05%)

[0035] The L-glutamic acid oxyester used here D The enzyme is a GluOX mutant prepared according to Example 1 of WO 2021 / 193598.

[0036] The carrier was a qualitative filter paper No. 2 (300 mm x 300 mm) made by Toyo Roshi Co., Ltd. The staining solution was placed in a metal tray, and the carrier was immersed in the staining solution for 5 seconds to allow the carrier to absorb the staining solution. Soaking Then, the staining solution was added. Soaking The carrier was taken out and dried at 45° C. for 15 minutes using a Toyo Seisakusho blower dryer to remove the solvent. Thus, the L-glutamic acid oxyester was added to the qualitative filter paper as a carrier. D The test paper of Example 1 carrying the enzyme, the coloring reagent (TMBZ), and the nonionic surfactant (Tween 20) was obtained.

[0037] The concentration of L-glutamic acid in the test sample was quantified using the test paper of Example 1. Five types of liquids with different concentrations of L-glutamic acid were prepared as the test samples. The concentrations of L-glutamic acid were 0 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM. For quantitative determination, one end of the test paper was dipped in each test sample for 2 seconds, then removed. Excess test sample was shaken off, and the color after 120 seconds was visually compared with the color chart.

[0038] For all test samples with L-glutamic acid concentrations between 0.05 and 1 mM, the color tone of the test paper after 120 seconds was comparable to that of the color chart. The color was maintained even after storage at room temperature for five months, demonstrating excellent stability.

[0039] In the case of a test paper prepared using the same staining solution as described above except that the concentration of the nonionic surfactant was changed to 0.5%, coloration was observed in 180 seconds when the L-glutamic acid concentration was 0 to 1 mM. Thus, it was confirmed that the test paper of Example 1 reacted very quickly, and color developed in 30 to 120 seconds at L-glutamic acid concentrations of 0.05 to 5 mM.

[0040] <Comparative Example> Using the same components as above, a primary staining solution and a secondary staining solution were prepared according to the following recipe. Using the primary staining solution and the secondary staining solution thus obtained, a test paper of a comparative example was produced. <Primary staining solution> Glutamic Acid Oxygen D -Ze 160U Peroxy D -Ze (Toyobo PEO-301) 3000U 0.1M HEPES (pH 7.0) 100mL <Secondary staining solution> 3,3',5,5'-Tetramethylbenzidine (TMBZ) 0.1g (4.16mM) Methanol 50mL 50mL acetone

[0041] The same carrier as above is used with the primary staining solution. Soaking The fabric was then dried in a fan dryer at 45°C for 30 minutes. Soaking The carrier is then further treated with a secondary staining solution. Soaking The test paper of the comparative example was obtained by subjecting the test paper to a drying process at 35° C. for 6 minutes in a blower dryer. The test paper of the comparative example did not carry a nonionic surfactant.

[0042] The concentration of L-glutamic acid in the test sample was quantified using the test paper of the comparative example. Five types of liquids with different concentrations of L-glutamic acid were prepared as the test samples. The concentrations of L-glutamic acid were 0 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM, and the color was observed using the same method as described above. As a result, in the case of the test paper of the comparative example, it took a long time for color to appear, and even after 120 seconds, the color was very weak.

[0043] The color development in the test papers of Example 1 and Comparative Example described above was evaluated according to the following criteria, and is summarized in the table below. ○: Color appears as per the color chart. △: Color is produced but different from the color chart. ×: Almost no coloring.

[0044] [Table 2]

[0045] By using the test paper of the present invention, it is possible to easily determine the concentration of L-glutamic acid in a test sample in a short period of time.

[0046] <Example 2> A dyeing solution of Example 2 was prepared in the same manner as in Example 1, except that 0.45 g of 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical was added as a hindrance reducing agent. Using the obtained dyeing solution, a test paper of Example 2 was prepared in the same manner as in Example 1. The test paper of Example 2 had a dyeing density of 1021 g / m 3A degree of 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical is carried.

[0047] The concentration of L-glutamic acid in a test sample containing ascorbic acid was quantified using the test paper of Example 2. The concentrations of ascorbic acid were set to five levels: 0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, and 0.1 mM, and the concentrations of L-glutamic acid were set to five levels: 0 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM. For quantitative determination, one end of the test paper was dipped in each test sample for 2 seconds, then removed. Excess test sample was shaken off, and the color after 120 seconds was visually compared with the color chart.

[0048] The color development in the test paper of Example 2 was evaluated according to the same criteria as above, and the results are summarized in the table below.

[0049] [Table 3]

[0050] In all test samples with ascorbic acid concentrations between 0.02 and 0.1 mM, the color tone of the test paper after 120 seconds was similar to that of the color chart. It was confirmed that the color reaction was not hindered even in the presence of ascorbic acid.

[0051] For reference, the concentration of L-glutamic acid in a test sample containing ascorbic acid was quantified in the same manner as in Example 2 using the test paper obtained in the same manner as in Example 1. The color of the test paper was evaluated according to the same criteria as above, and the results are summarized in the table below.

[0052] [Table 4]

[0053] In the case of test papers that do not contain an interference-reducing agent, the coexistence of ascorbic acid may interfere with color development. It is expected that test papers containing the interference-reducing agent will be effectively used for measuring glutamic acid in fruits and vegetables and processed foods that contain ascorbic acid.

Claims

1. A test paper for quantifying the concentration of L-glutamic acid in a test sample by a colorimetric method, comprising: A liquid-permeable carrier; L-glutamate oxidase, a color-developing reagent selected from 3,3'-diaminobenzidine and 3,3',5,5'-tetramethylbenzidine, and a surfactant, all contained in the liquid-permeable carrier. A test paper comprising:

2. 2. The test paper according to claim 1, wherein the surfactant is a non-ionic surfactant.

3. 3. The test paper according to claim 2, wherein the non-ionic surfactant is polyoxyethylene (20) sorbitan monolaurate.

4. 4. The test paper according to claim 1, wherein the L-glutamate oxidase comprises the following L-glutamate oxidase mutant (a) and / or the L-glutamate oxidase mutant (b): (a) an L-glutamate oxidase variant that contains an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 3 and has an L-glutamic acid oxidizing activity (excluding L-glutamate oxidase that contains the amino acid sequence of SEQ ID NO: 1); (b) an L-glutamate oxidase mutant of (a) above, in which a peptide linker consisting of 1 to 20 amino acid residues is inserted at one or more sites selected from the group consisting of (1) a site in the region near the boundary between the α1 region and the α2 region, (2) a site in the region near the boundary between the α2 region and the γ region, and (3) a site in the region near the boundary between the γ region and the β region, and which has an L-glutamate oxidizing activity, the site in the region near the boundary between the α1 region and the α2 region is a site in a region consisting of amino acid residues at positions 349 to 363 in SEQ ID NO: 3; The site in the region near the boundary between the α2 region and the γ region is a site in the region consisting of amino acid residues 372 to 377 in SEQ ID NO: 3, and An L-glutamate oxidase mutant, wherein the site in the region near the boundary between the γ region and the β region is a site in the region consisting of amino acid residues 466 to 469 in SEQ ID NO:

3.

5. 4. The test paper according to claim 1, further comprising an interference-reducing agent for reducing the effect of an interfering substance on the oxidative deamination reaction, which is supported on the carrier.

6. 5. The test paper according to claim 4, further comprising an interference-reducing agent for reducing the effect of an interfering substance on the oxidative deamination reaction, which is supported on the carrier.

7. 6. The test strip of claim 5, wherein said interference-reducing agent comprises a free radical.

8. 8. The test paper according to claim 7, wherein the free radical is a 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical.

9. 13. A method for quantifying the concentration of L-glutamic acid in a test sample, comprising contacting the test paper according to claim 1 with the test sample and quantifying the concentration by colorimetry.

10. 10. The quantitative method according to claim 9, wherein the test sample is a biological sample or a food or beverage product.

11. A method for quantifying the concentration of L-glutamic acid in a test sample containing ascorbic acid, comprising contacting the test sample with the test paper described in claim 6 and quantifying the concentration by a colorimetric method.

12. A method for producing the test paper described in claim 3, comprising the step of impregnating a liquid-permeable carrier with a staining solution containing L-glutamate oxidase, a color-developing reagent selected from 3,3'-diaminobenzidine and 3,3',5,5'-tetramethylbenzidine, and polyoxyethylene (20) sorbitan monolaurate.

13. The method according to claim 12, wherein the concentration of the polyoxyethylene (20) sorbitan monolaurate in the staining solution is 0.01% or more and 0.5% or less.

14. The method according to claim 12, wherein the concentration of L-glutamate oxidase in the staining solution is 500 to 5000 U / L.

15. The method according to claim 12 or 13, wherein the staining solution further contains one or more selected from the group consisting of a hydrogen peroxide detection reagent, a buffer solution, an ammonia detection reagent, and a 2-oxoglutaric acid detection reagent.

16. A method for producing the test paper described in claim 6, comprising the step of impregnating a liquid-permeable carrier with a staining solution containing L-glutamate oxidase, a color-developing reagent selected from 3,3'-diaminobenzidine and 3,3',5,5'-tetramethylbenzidine, polyoxyethylene (20) sorbitan monolaurate, and an interference-reducing agent which reduces the effect of interfering substances in the oxidative deamination reaction.

17. The method of claim 16, wherein the interference reducer comprises 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical.

18. The method for producing a dye according to claim 17, wherein the concentration of the 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical in the staining solution is 3 to 10 g / L.

19. The method according to claim 16, wherein the concentration of the polyoxyethylene (20) sorbitan monolaurate in the staining solution is 0.01% or more and 0.5% or less.

20. 17. The method according to claim 16, wherein the concentration of the L-glutamate oxidase in the staining solution is 500 to 5000 U / L.

21. The method according to claim 16, wherein the staining solution further contains one or more selected from the group consisting of a hydrogen peroxide detection reagent, a buffer solution, an ammonia detection reagent, and a 2-oxoglutarate detection reagent.

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