L-glutamate oxidase mutant

Abstract

The present invention provides an alternative L-glutamic acid oxidase which enables the measurement of L-glutamic acid. More specifically, the present invention provides an L-glutamic acid oxidase mutant (a) or (b) described below, and the like. (a) an L-glutamic acid oxidase mutant which comprises an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 3, while having oxidation activity of L-glutamic acid (excluding an L-glutamic acid oxidase which comprises the amino acid sequence set forth in SEQ ID NO: 1 (b) an L-glutamic acid oxidase mutant which is obtained by inserting a peptide linker consisting of 1-20 amino acid residues into one or more sites that are selected from the group consisting of (1) a site in a region near the boundary between an α1 region and an α2 region, (2) a site in a region near the boundary between the α2 region and a γ region and (3) a site in a region near the boundary between the γ region and a β region in the L-glutamic acid oxidase mutant (a), and which has oxidation activity of L-glutamic acid

Description

Technical Field

[0001] The present invention relates to L-glutamate oxidase mutants and the like.

Background Art

[0002] L-glutamate oxidase (GluOX) is an enzyme that catalyzes the following reaction (EC1.4.3.11). L-glutamate + O2 + H2O → 2-oxoglutaric acid + H2O2 + NH3

[0003] Conventionally, GluOX has been known to exhibit activity as a hetero-oligomer (α2β2γ2) having different subunits through analysis based on protein isolation and purification. However, the gene encoding GluOX had not been identified. Subsequently, it was reported that the GluOX gene encodes an immature protein (molecular weight of about 70 kDa) containing an αβγ-containing chain (single chain), and that GluOX also retains activity as a homo-dimer ((αβγ-containing single chain)2) having two subunits consisting of αβγ-containing single chains (Patent Document 1). As a result of subsequent detailed analysis, currently, native GluOX is not only cleaved into three segments of αβγ by protease, but also cleaved by protease within the α region into two α fragments (referred to as α1 and α2 in this case as necessary), a β unit, and a γ unit, and is considered to exhibit its activity by taking a unique three-dimensional structure of a hetero-octamer (α12α22β2γ2).

[0004] L-glutamate is known as a major umami component in foods and is also an important component present in biological samples such as blood. L-glutamate can be easily measured using GluOX. Therefore, GluOX is used for measuring L-glutamate in samples.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] International Publication No. 2001 / 079503 [Non-patent literature]

[0006] [Non-Patent Document 1] Arima J et al.,FEBS J. 2009 Jul;276(14):3894-903 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide an L-glutamate oxidase that enables the measurement of L-glutamate.

[0008] Unexpectedly, the present inventors have discovered that a single-chain polypeptide (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3) lacking both the processing removal region between the γ and β regions (the region consisting of amino acid residues at positions 467-506 in the amino acid sequence of SEQ ID NO: 1) and the processing removal region downstream of the β region (the region consisting of amino acid residues at positions 670-687 in the amino acid sequence of SEQ ID NO: 1) in the αβγ-containing single-chain GluOX described in Patent Document 1 (amino acid sequence of SEQ ID NO: 1) can exhibit higher L-glutamic acid oxidative activity than the αβγ-containing single-chain GluOX described in Patent Document 1 (Figure 1, Example). The inventors have also found that a single-chain polypeptide in which a peptide linker is inserted into at least one of the near-boundary regions of the α1-α2 region, the α2-γ region, and the γ-β region (particularly the near-boundary regions of the α1-α2 region and the α2-γ region) of the above single-chain polypeptide can exhibit higher L-glutamic acid oxidation activity than the above immature protein (Figure 1, Examples). The inventors have further succeeded in discovering several variants that can exhibit higher L-glutamic acid oxidation activity than the αβγ-containing single-chain GluOX described in Patent Document 1, thereby completing the present invention.

[0009] In other words, the present invention is as follows: [1] L-glutamate oxidase mutants of (a) or (b) below: (a) L-glutamate oxidase variants that contain an amino acid sequence showing 90% or more identity with the amino acid sequence of SEQ ID NO: 3 and that have L-glutamate oxidative activity (excluding L-glutamate oxidases containing the amino acid sequence of SEQ ID NO: 1); or (b) An L-glutamate oxidase mutant having L-glutamate oxidative activity, wherein a peptide linker consisting of 1 to 20 amino acid residues is inserted in one or more sites selected from the group consisting of (1) a site in the vicinity of the boundary between the α1 and α2 regions, (2) a site in the vicinity of the boundary between the α2 and γ regions, and (3) a site in the vicinity of the boundary between the γ and β regions in the L-glutamate oxidase mutant of (a) above, The region near the boundary between the α1 and α2 regions is the region consisting of amino acid residues from positions 349 to 363 in SEQ ID NO: 3. The region near the boundary between the α2 and γ regions is the region consisting of amino acid residues from positions 372 to 377 in SEQ ID NO: 3, and An L-glutamate oxidase mutant in which the region near the boundary between the γ and β regions corresponds to the region consisting of amino acid residues at positions 466-469 in SEQ ID NO: 3. [2] The L-glutamate oxidase mutant of [1], wherein the L-glutamate oxidase mutant is a mutant of L-glutamate oxidase derived from a microorganism belonging to the genus Streptomyces. [3] An L-glutamate oxidase mutant of [2], wherein the microorganism belonging to the genus Streptomyces is Streptomyces SP X-119-6. [4] An L-glutamate oxidase mutant of any of [1] to [3], wherein the peptide linker is inserted in either (1) a site in the region near the boundary between the α1 and α2 regions, or (2) a site in the region near the boundary between the α2 and γ regions, or both thereof. [5] The region near the boundary between the α1 and α2 regions is the region between amino acid residues at positions 356 and 357 in SEQ ID NO: 3. The region near the boundary between the α2 and γ regions is the region between amino acid residues at positions 376 and 377 in SEQ ID NO: 3, or The region near the boundary between the γ and β regions is the L-glutamate oxidase mutant [1] to [4], which is the region between amino acid residues at positions 466 and 467 in SEQ ID NO: 3. [6] An L-glutamate oxidase mutant of any of [1] to [5], wherein the L-glutamate oxidase mutant has a mutation in one or more amino acid residues selected from the group consisting of A106, C210, Q235, D236, D237, P244, T311, W313, Q333, I334, M336, Q338, R339, T416, A438, K441, Y455, Q456, Q457, L505, P558, C561, and P569 in SEQ ID NO: 3. [7] An L-glutamate oxidase mutant of any of [1] to [6], wherein the L-glutamate oxidase mutant has a mutation in one or more amino acid residues selected from the group consisting of A106S, C210S, Q235E, D236E, D237E, P244H, T311S, W313F, Q333E, I334V, I334L, M336L, Q338E, R339K, T416S, A438P, K441E, Y455F, Q456R, Q457E, Q457K, L505I, P558A, C561S, and P569A in SEQ ID NO: 3. A method for analyzing L-glutamic acid, comprising measuring the L-glutamic acid contained in a test sample using one of the L-glutamic acid oxidase mutants of [8] [1] to [7]. [9] The method of [8], wherein L-glutamic acid is measured using N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (TOOS) and 4-aminoantipyrine, as well as peroxidase, in addition to any of the L-glutamic acid oxidase variants of [1] to [7]. A method for producing 2-oxoglutaric acid, comprising producing 2-oxoglutaric acid from L-glutamic acid in the presence of any of the L-glutamic acid oxidase mutants of

[10] [1] to [7]. A polynucleotide encoding one of the L-glutamate oxidase variants

[11] , [1], to [7]. An expression vector containing polynucleotides

[12] and

[11] . A transformed microorganism comprising an expression unit comprising a polynucleotide encoding one of the L-glutamate oxidase variants of [1] to [7], and a promoter operably linked thereto. A method for producing L-glutamate oxidase mutants, comprising generating one of the L-glutamate oxidase mutants from

[14] [1] to [7] using the transformed microorganism from

[13] . A reagent or kit for detecting L-glutamate containing any of the L-glutamate oxidase variants listed in

[15] , [1], to [7].

[16] The L-glutamic acid detection reagent or kit of

[15] , further comprising one or more selected from the group consisting of reaction buffer or buffer salt, hydrogen peroxide detection reagent, ammonia detection reagent and 2-oxoglutaric acid detection reagent.

[17] (a) a device, and (b) a detection system for L-glutamate analysis comprising one of the L-glutamate oxidase variants of [1] to [7].

[18] (c) A detection system for L-glutamic acid analysis according to

[17] , further comprising one or more selected from the group consisting of reaction buffer or buffer salt, hydrogen peroxide detection reagent, ammonia detection reagent and 2-oxoglutaric acid detection reagent.

[19] An enzyme sensor for L-glutamate analysis comprising (a) a detection electrode and (b) an L-glutamate oxidase variant of any of [1] to [7] fixed to or positioned on the detection electrode. [Effects of the Invention]

[0010] The L-glutamate oxidase variant of the present invention has high activity against L-glutamate and is therefore useful for rapid and highly sensitive measurement of L-glutamate and / or production of 2-oxoglutaric acid. The L-glutamate oxidase variant of the present invention is also easy to prepare because it does not require protease treatment or subsequent purification of the protease-treated product. The analytical method of the present invention is useful in a wide range of fields, such as biological research, health and nutrition, medicine, and food manufacturing. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the relationship between L-glutamate oxidase (GluOX) processing and the L-glutamate oxidase mutant of the present invention. The GluOX gene naturally expresses a single-chain polypeptide encoding the α region (α1 region, α2 region), β region, and γ region. In nature, this single-chain polypeptide is known to be cleaved by a protease expressed by microorganisms that naturally produce this single-chain polypeptide (microorganisms belonging to the genus Streptomyces) to form a heterooctamer (α12α22β2γ2). The GluOX variants of the present invention relate to (A) a shortened single-chain polypeptide lacking both the processing removal region between the γ and β regions and the processing removal region downstream of the β region, and (B) an inserted single-chain polypeptide in which a peptide linker is inserted at one or more sites selected from the group consisting of (1) a site in the vicinity of the boundary between the α1 and α2 regions, (2) a site in the vicinity of the boundary between the α2 and γ regions, and (3) a site in the vicinity of the boundary between the γ and β regions in the shortened single-chain polypeptide of (A). [Figure 2]Figure 2 shows the amino acid sequence (SEQ ID NO: 1) of wild-type GluOX from Streptomyces sp. X-119-6. This wild-type GluOX amino acid sequence (SEQ ID NO: 1) is encoded by the nucleotide sequence of SEQ ID NO: 2. Each region corresponds to the amino acid sequence of SEQ ID NO: 1 as follows: α region: region consisting of amino acid residues from positions 1 to 376 (α1 region: region consisting of amino acid residues from positions 1 to 352; α2 region: region consisting of amino acid residues from positions 361 to 376); γ region: region consisting of amino acid residues from positions 377 to 466; β region: region consisting of amino acid residues from positions 507 to 669. The protease cleavage sites (black triangles) correspond to the amino acid sequence of SEQ ID NO: 1 as follows. Protease cleavage region within the α-domain: between amino acid residues at positions 353-360 (underlined); boundary region between the α- and γ-domains: between amino acid residues at positions 376 and 377; downstream region of the γ-domain: between amino acid residues at positions 466 and 467; upstream region of the β-domain: between amino acid residues at positions 506 and 507; downstream region of the β-domain: between amino acid residues at positions 664 and 669 (underlined). Preferred point mutation sites that can be introduced into L-glutamate oxidase mutants are A106, C210, Q235, D236, D237, P244, T311, W313, Q333, I334, M336, Q338, R339, T416, A438, K441, Y455, Q456, Q457, L545, L598, C601, and P609. [Figure 3]Figure 3 shows a shortened single-chain polypeptide (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3) lacking both the processing and removal region (the region consisting of amino acid residues at positions 467 to 506 in the amino acid sequence of SEQ ID NO: 1) between the γ region and the β region and the processing and removal region downstream of the β region (the region consisting of amino acid residues at positions 670 to 687 in the amino acid sequence of SEQ ID NO: 1) in the amino acid sequence of wild-type GluOX of Streptomyces sp. X-119-6 (SEQ ID NO: 1). Each region corresponds as follows in the amino acid sequence of SEQ ID NO: 3. α region: the region consisting of amino acid residues at positions 1 to 376 (α1 region: the region consisting of amino acid residues at positions 1 to 352; α2 region: the region consisting of amino acid residues at positions 361 to 376); γ region: the region consisting of amino acid residues at positions 377 to 466; β region: the region consisting of amino acid residues at positions 467 to 629. Each neighboring region corresponds as follows in the amino acid sequence of SEQ ID NO: 3. Region near the boundary between the α1 region and the α2 region: the region consisting of amino acid residues at positions 349 to 363; Region near the boundary between the α2 region and the γ region: the region consisting of amino acid residues at positions 372 to 377; Region near the boundary between the γ region and the β region: the region consisting of amino acid residues at positions 466 to 469. The underlined part corresponds to the protease cleavage region within the α region and the downstream region of the β region described in Figure 2. The positions of preferred point mutations that can be introduced into the L-glutamate oxidase mutant are A106, C210, Q235, D236, D237, P244, T311, W313, Q333, I334, M336, Q338, R339, T416, A438, K441, Y455, Q456, Q457, L505 (corresponding to L545 in SEQ ID NO: 1), P558 (corresponding to L598 in SEQ ID NO: 1), C561 (corresponding to C601 in SEQ ID NO: 1), and P569 (corresponding to P609 in SEQ ID NO: 1).

Mode for Carrying Out the Invention

[0012] The present invention provides an L-glutamate oxidase mutant of the following (a) or (b): (a) An L-glutamate oxidase variant that contains an amino acid sequence showing 90% or more identity to the amino acid sequence of SEQ ID NO: 3 and has the oxidizing activity of L-glutamic acid (however, excluding the L-glutamate oxidase containing the amino acid sequence of SEQ ID NO: 1); or (b) An L-glutamate oxidase variant 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 vicinity of the boundary between the α1 region and the α2 region, (2) a site in the vicinity of the boundary between the α2 region and the γ region, and (3) a site in the vicinity of the boundary between the γ region and the β region in the L-glutamate oxidase variant of (a), and which has the oxidizing activity of L-glutamic acid, where the site in the vicinity of the boundary between the α1 region and the α2 region is a site in the region consisting of the amino acid residues at positions 349 to 363 in SEQ ID NO: 3, the site in the vicinity of the boundary between the α2 region and the γ region is a site in the region consisting of the amino acid residues at positions 372 to 377 in SEQ ID NO: 3, and the site in the vicinity of the boundary between the γ region and the β region is a site in the region consisting of the amino acid residues at positions 466 to 469 in SEQ ID NO: 3. An L-glutamate oxidase variant.

[0013] The L-glutamate oxidase variant of (a) corresponds to a shortened single-chain polypeptide (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3) lacking both the processing removal region between the γ region and the β region (the region consisting of the amino acid residues at positions 467 to 506 in the amino acid sequence of SEQ ID NO: 1) and the processing removal region downstream of the β region (the region consisting of the amino acid residues at positions 670 to 687 in the amino acid sequence of SEQ ID NO: 1) naturally expressed by the GluOX gene. Therefore, the L-glutamate oxidase variant of (a) does not contain the regions consisting of the amino acid residues at positions 467 to 506 and 670 to 687 in the amino acid sequence of SEQ ID NO: 1.

[0014] The degree of amino acid sequence identity percentage in the L-glutamate oxidase mutant of (a) with respect to the amino acid sequence of SEQ ID NO: 3 is preferably 92% or higher, more preferably 95% or higher, even more preferably 97% or higher, and most preferably 98% or higher or 99% or higher. The amino acid sequence identity percentage can be calculated using GENETYX Ver13.1.1 software from Genetics Co., Ltd., using the full length of the polypeptide portion encoded in the ORF, and the value obtained after performing Muscle alignment, ClustalW alignment, or Multiple sequence alignment with the setting "Gaps are taken into account".

[0015] In the L-glutamate oxidase mutant of (b), the region in which the peptide linker is inserted is one or more sites selected from the group consisting of (1) a site in the region near the boundary between the α1 and α2 regions (the region consisting of amino acid residues from positions 349 to 363 in SEQ ID NO: 3), (2) a site in the region near the boundary between the α2 and γ regions (the region consisting of amino acid residues from positions 372 to 377 in SEQ ID NO: 3), and (3) a site in the region near the boundary between the γ and β regions (the region consisting of amino acid residues from positions 466 to 469 in SEQ ID NO: 3) (e.g., 1, 2, or 3). Preferably, the region in which the peptide linker is inserted may be either (1) a site in the region near the boundary between the α1 and α2 regions, or (2) a site in the region near the boundary between the α2 and γ regions, or both.

[0016] The region near the boundary between the α1 and α2 regions is preferably a region within the boundary region between the α1 and α2 regions (the region consisting of amino acid residues from positions 349 to 363 in SEQ ID NO: 3), and more preferably a region between amino acid residues at positions 356 and 357 in SEQ ID NO: 3.

[0017] The region in the vicinity of the boundary between the α2 and γ regions is the region in the vicinity of the boundary between the α2 and γ regions (the region consisting of amino acid residues from positions 372 to 377 in SEQ ID NO: 3), and is preferably the region between amino acid residues at positions 376 and 377 in SEQ ID NO: 3.

[0018] The region in the vicinity of the boundary between the γ and β regions is the region in the vicinity of the boundary between the γ and β regions (the region consisting of amino acid residues at positions 466 to 469 in SEQ ID NO: 3), and is preferably the region between amino acid residues at positions 466 and 467 in SEQ ID NO: 3.

[0019] The peptide linker is a peptide linker consisting of 1 to 20 amino acid residues. The peptide linker may have an amino acid sequence different from the partial amino acid sequence of L-glutamate oxidase. The peptide linker may consist of 2 or more, 3 or more, 4 or more, or 5 or more amino acid residues. The peptide linker may also consist of 18 or fewer, 16 or fewer, 14 or fewer, 12 or fewer, or 10 or fewer amino acid residues. More specifically, the peptide linker may consist of 2 to 18, 3 to 16, 4 to 14, 5 to 12, or 5 to 10 amino acid residues.

[0020] The types of amino acid residues that make up the peptide linker are natural L-α-amino acids and glycine (G) residues that make up normal proteins. More specifically, such natural L-α-amino acid residues include L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), and L-lysine (K). Preferably, the types of amino acid residues that make up the peptide linker are amino acid residues that readily form highly flexible peptide linkers. Examples of amino acid residues that readily form highly flexible peptide linkers include glycine (G), L-alanine (A), L-serine (S), and L-threonine (T).

[0021] Preferably, the peptide linker is GGGGS (SEQ ID NO: 4) or a repeating sequence thereof. The number of repetitions in the repeating sequence is 2 to 4, preferably 2 or 3, and more preferably 2.

[0022] The oxidative activity of L-glutamic acid is its ability to catalyze the following reaction: L-glutamic acid + O2 + H2O → 2-oxoglutaric acid + H2O2 + NH3

[0023] The L-glutamic acid oxidation activity of the L-glutamic acid oxidase variant of the present invention is not particularly limited as long as it is equal to or greater than that of the L-glutamic acid oxidase consisting of the amino acid sequence of SEQ ID NO: 1. Preferably, it may have 1.1 times or more L-glutamic acid oxidation activity (more preferably 1.2 times or more L-glutamic acid oxidation activity) compared to the L-glutamic acid oxidase consisting of the amino acid sequence of SEQ ID NO: 1. Such L-glutamic acid oxidation activity can be measured using the oxidation reaction of L-glutamic acid (10 mM) and its conjugate reaction, as described in the examples. (Oxidation reaction of L-glutamic acid) A reaction catalyzed by L-glutamic acid oxidase. L-glutamic acid + O2 + H2O → 2-oxoglutaric acid + H2O2 + NH3 (Conjugated reaction) Reaction catalyzed by peroxidase H2O2+TOOS+4-AA → Pigment compound (absorbance approximately 555nm) TOOS: N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline 4-AA:4-aminoantipyrine

[0024] The L-glutamate oxidation activity of the L-glutamate oxidase variant of the present invention is not particularly limited, as long as it exhibits higher L-glutamate oxidation activity than the αβγ-containing single-chain L-glutamate oxidase described in Patent Document 1. Preferably, the L-glutamate oxidation activity of the L-glutamate oxidase variant of the present invention may be equal to or greater than that of the L-glutamate oxidase consisting of the amino acid sequence of SEQ ID NO: 3. The L-glutamic acid oxidation activity of the L-glutamic acid oxidase variant of the present invention may be more preferably 1.1 times or more, even more preferably 1.2 times or more, and particularly preferably 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times or more, or 1.8 times or more, compared to the αβγ-containing single-chain L-glutamic acid oxidase described in Patent Document 1, or the L-glutamic acid oxidase consisting of the amino acid sequence of SEQ ID NO: 3 (preferably the L-glutamic acid oxidase consisting of the amino acid sequence of SEQ ID NO: 3). Such L-glutamic acid oxidation activity can be measured using the oxidation reaction of L-glutamic acid (10 mM) and its conjugate reaction, as described above.

[0025] The L-glutamate oxidase mutant of the present invention may have one or more mutations that can enhance the oxidative activity of the L-glutamate oxidase mutant. Examples of such mutations include mutations in one or more amino acid residues (e.g., 1, 2, or 3) selected from the group consisting of A106, C210, Q235, D236, D237, P244, T311, W313, Q333, I334, M336, Q338, R339, T416, A438, K441, Y455, Q456, Q457, L505, P558, C561, and P569 in SEQ ID NO: 3. The mutated amino acid residues at these sites are desired native L-α-amino acid residues that are different from the pre-mutated amino acid residues. Such desired natural L-α-amino acid residues are L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), or L-glutamic acid (G). If the L-glutamate oxidase variant of the present invention has two or more mutations, such mutations may include two or more combinations of D236 with other mutations (e.g., D236 and Q338, D236 and R339, D236 and T416), combinations of T311 with other mutations (e.g., T311 and D236, T311 and Q457), and combinations of Q457 with other mutations (e.g., Q457 and D236, Q457 and T311, Q457 and Q338, Q457 and R339).

[0026] Preferably, one or more mutations that can enhance the oxidative activity of the L-glutamate oxidase mutant are one or more amino acid residue mutations (e.g., 1, 2, or 3) selected from the group consisting of A106S, C210S, Q235E, D236E, D237E, P244H, T311S, W313F, Q333E, I334V, I334L, M336L, Q338E, R339K, T416S, A438P, K441E, Y455F, Q456R, Q457E, Q457K, L505I, P558A, C561S, and P569A in SEQ ID NO: 3. If the L-glutamate oxidase variant of the present invention has two or more mutations, such mutations may include two or more combinations of D236E with other mutations (e.g., D236E and Q338E, D236E and R339K, D236E and T416S), combinations of T311S with other mutations (e.g., T311S and D236E, T311S and Q457E), and combinations of Q457E with other mutations (e.g., Q457E and D236E, Q457E and T311S, Q457E and Q338E, Q457E and R339K).

[0027] The L-glutamate oxidase derived from the L-glutamate oxidase variant of the present invention can be an enzyme from any organism (e.g., microorganisms, animals, and plants), but it is preferable to use an enzyme from a microorganism belonging to the genus Streptomyces. Among microorganisms belonging to the genus Streptomyces, Streptomyces SP X-119-6 is preferred.

[0028] The L-glutamate oxidase variant of the present invention may also contain other peptide components (e.g., tag portions) at its C-terminus or N-terminus. Other peptide components that may be included in the L-glutamate oxidase variant of the present invention include, for example, peptide components that facilitate the purification of the target protein (e.g., tag portions such as histidine tag and Strep-tag II; proteins commonly used for the purification of target proteins such as glutathione-S-transferase and maltose-binding protein), peptide components that improve the solubility of the target protein (e.g., Nus-tag), peptide components that act as chaperones (e.g., trigger factors), and peptide components that have other functions, such as proteins or protein domains or linkers that connect them to the L-glutamate oxidase variant.

[0029] The L-glutamate oxidase variant of the present invention can be prepared using the transformed microorganism of the present invention that expresses the L-glutamate oxidase variant of the present invention, or using a cell-free system or the like. The transformed microorganism of the present invention can be prepared, for example, by preparing the expression vector of the present invention and then introducing this expression vector into a host.

[0030] The expression vector of the present invention comprises the polynucleotide (e.g., DNA, RNA) of the present invention encoding the L-glutamate oxidase variant of the present invention. The expression vector of the present invention may further include, in addition to the polynucleotide of the present invention, regions such as a promoter, a terminator, and a region encoding a drug (e.g., tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin) resistance gene. The expression vector of the present invention may be a plasmid or an integrative vector. The expression vector of the present invention may also be a viral vector or a cell-free vector. The expression vector of the present invention may further include, at the 3' or 5' end of the polynucleotide of the present invention, a polynucleotide encoding another peptide component that can be added to the L-glutamate oxidase variant of the present invention. Examples of polynucleotides encoding other peptide components include, for example, a polynucleotide encoding a peptide component that facilitates the purification of the target protein as described above, a polynucleotide encoding a peptide component that improves the solubility of the target protein as described above, a polynucleotide encoding a peptide component that acts as a chaperone, or a polynucleotide encoding a protein or protein domain with other functions or a peptide component that acts as a linker connecting them to the L-glutamate oxidase variant. Various expression vectors containing polynucleotides encoding other peptide components are available. Therefore, such expression vectors may be used to prepare the expression vector of the present invention.For example, expression vectors can be used that contain polynucleotides encoding peptide components that facilitate the purification of the target protein (e.g., pET-15b, pET-51b, pET-41a, pMAL-p5G), expression vectors that contain polynucleotides encoding peptide components that improve the solubility of the target protein (e.g., pET-50b), expression vectors that contain polynucleotides encoding peptide components that act as chaperones (e.g., pCold TF), or expression vectors that contain polynucleotides encoding proteins or protein domains with other functions, or peptide components that act as linkers connecting them to the L-glutamate oxidase variant. To enable cleavage of the L-glutamate oxidase variant of the present invention and other peptide components attached thereto after protein expression, the expression vector of the present invention may include a region encoding a protease cleavage site between the polynucleotide encoding the L-glutamate oxidase variant of the present invention and the polynucleotide encoding the other peptide component.

[0031] As hosts for expressing the L-glutamate oxidase mutant of the present invention, various prokaryotic cells can be used, such as Escherichia coli and other Escherichia species, Corynebacterium species (e.g., Corynebacterium glutamicum), and Bacillus species (e.g., Bacillus subtilis), as well as various eukaryotic cells, such as Saccharomyces species (e.g., Saccharomyces cerevisiae), Pichia species (e.g., Pichia stipitis), and Aspergillus species (e.g., Aspergillus oryzae). Furthermore, as a host for expressing the L-glutamate oxidase mutant of the present invention, microorganisms that do not express a protease capable of cleaving the L-glutamate oxidase mutant (e.g., microorganisms belonging to the genus Streptomyces) can also be suitably used. As a host, a strain lacking a predetermined gene may also be used. Examples of transformed microorganisms include transformed microorganisms that possess an expression vector in the cytoplasm, and transformed microorganisms in which the target gene has been introduced into the genome.

[0032] The transformed microorganisms of the present invention can be cultured in a culture medium having, for example, the composition described below, using a predetermined culture apparatus (e.g., test tubes, flasks, jar fermenters). Culture conditions can be set as appropriate. Specifically, the culture temperature may be 10°C to 37°C, the pH may be 6.5 to 7.5, and the culture time may be 1h to 100h. Culture may also be carried out while controlling the dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture medium may be used as a control indicator. The aeration and stirring conditions can be controlled so that the relative dissolved oxygen concentration (DO value), assuming an atmospheric oxygen concentration of 21%, does not fall below, for example, 1 to 10%, preferably 3% to 8%. Furthermore, the culture may be performed by batch culture or fed-batch culture. In the case of fed-batch culture, the culture can be continued by sequentially or discontinuously adding a sugar source solution or a solution containing phosphate to the culture medium.

[0033] As mentioned above, the host organism to be transformed is Escherichia coli. Specifically, for Escherichia coli, it can be selected from the Escherichia coli K12 subspecies, including strains JM109, DH5α, HB101, and BL21(DE3). Methods for performing transformation and selecting transformed microorganisms are described in publications such as Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001 / 01 / 15). Below, a more detailed example will be provided of how to prepare transformed Escherichia coli and use it to produce a specific enzyme.

[0034] As promoters for expressing the polynucleotides of the present invention, promoters commonly used for heterologous protein production in E. coli can be used. Examples of strong promoters include PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, and T5 promoter, with PhoA, PhoC, and lac being preferred. As vectors, for example, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and their derivatives may be used. Other vectors may include phage DNA vectors. Furthermore, expression vectors containing a promoter and capable of expressing the inserted DNA sequence may be used. Preferably, the vectors may be pUC, pSTV, or pMW.

[0035] Furthermore, a terminator, which is a transcription termination sequence, may be ligated downstream of the polynucleotide of the present invention. Examples of such terminators include the T7 terminator, the fd phage terminator, the T4 terminator, the tetracycline resistance gene terminator, and the E. coli trpA gene terminator.

[0036] The vector for introducing the polynucleotide of the present invention into E. coli is preferably a so-called multicopy type, and examples include plasmids having a replication origin site derived from ColE1, such as pUC plasmids or pBR322 plasmids or their derivatives. Here, "derivative" means a plasmid that has been modified by base substitution, deletion, insertion and / or addition.

[0037] Furthermore, to select transformed microorganisms, it is preferable that the vector contains markers such as ampicillin resistance genes. Such plasmids, expression vectors with strong promoters, are commercially available [e.g., pUC series (Takara Bio Inc.), pPROK series (Clonetec), pKK233-2 (Clonetec)].

[0038] By transforming Escherichia coli using the obtained expression vector of the present invention and culturing this Escherichia coli, the L-glutamate oxidase mutant of the present invention can be obtained.

[0039] As the culture medium, you may use a medium commonly used for culturing E. coli, such as M9-casamino acid medium or LB medium. The medium may contain a specified carbon source, nitrogen source, and coenzyme (e.g., pyridoxine hydrochloride). Specifically, you may use peptone, yeast extract, NaCl, glucose, MgSO4, ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, etc. Furthermore, the culture conditions and production induction conditions should be appropriately selected according to the type of vector marker, promoter, host bacteria, etc. used.

[0040] The L-glutamate oxidase variant of the present invention can be recovered by the following methods. The L-glutamate oxidase variant of the present invention can be obtained as a lysate and lysate by recovering the transformed microorganism of the present invention and then disrupting (e.g., sonication, homogenization) or lysing (e.g., lysozyme treatment) the microbial cells. The L-glutamate oxidase variant of the present invention can be obtained by subjecting such lysates and lysates to methods such as extraction, precipitation, filtration, and column chromatography.

[0041] The present invention provides a method for analyzing L-glutamic acid. The analytical method of the present invention may include measuring the amount of L-glutamic acid contained in a test sample using the L-glutamic acid oxidase variant of the present invention.

[0042] The test sample is not particularly limited as long as it is a sample suspected to contain L-glutamic acid, and examples include biological samples (e.g., blood, urine, saliva, tears, etc.) and food and beverages (e.g., nutritional drinks, amino acid drinks, etc.). The L-glutamic acid in the test sample may be at a low concentration (e.g., less than 1 mM, such as 1 μM or more and less than 1 mM) or a high concentration (e.g., 1 mM or more and less than 1 M, such as 1 mM or more and less than 1 M).

[0043] The analytical method of the present invention is not particularly limited as long as L-glutamic acid can be measured using the L-glutamic acid oxidase mutant of the present invention. The generated 2-oxoglutaric acid may be detected, or NH3 or H2O2 produced as a by-product in conjunction with the generation of 2-oxoglutaric acid may be detected. Alternatively, the product of a conjugated reaction may be detected by coupling it with another reaction. Examples of such conjugated reactions include the following.

[0044] (Oxidation reaction of L-glutamic acid) A reaction catalyzed by L-glutamic acid oxidase. L-glutamic acid + O2 + H2O → 2-oxoglutaric acid + H2O2 + NH3 (Conjugated reaction) Reaction catalyzed by peroxidase H2O2+TOOS+4-AA → Pigment compound (absorbance approximately 555nm) TOOS and 4-AA are the same as above.

[0045] When utilizing the above conjugated reaction, L-glutamic acid can be measured using TOOS and 4-AA, as well as peroxidase, in addition to the L-glutamic acid oxidase variant of the present invention. Specifically, the test sample is mixed with TOOS and 4-AA, as well as peroxidase, in an aqueous solution (e.g., buffer solution). The mixed sample is then subjected to the above enzymatic reaction, and finally, L-glutamic acid is measured by detecting the absorbance (approximately 555 nm) of the resulting pigment compound. The measurement can be performed qualitatively or quantitatively. The measurement may be performed, for example, based on an endpoint method in which the measurement is carried out until all substrates have reacted, or based on a rate method (initial rate method). Since the amount of oxygen required in the oxidation reaction is small, the amount of oxygen needed can be supplied by the dissolved oxygen in the reaction system, so it is usually not necessary to forcibly supply oxygen or oxygen-containing gas to the reaction system.

[0046] The L-glutamate oxidase variant of the present invention does not react with amino acids other than L-glutamate (e.g., L-α-amino acids), or its reactivity to them is extremely low. Therefore, even when the test sample contains not only L-glutamate but also other amino acids, the amount of L-glutamate in the test sample can be specifically evaluated by using the L-glutamate oxidase variant of the present invention.

[0047] Furthermore, by using a hydrogen peroxide electrode utilizing the L-glutamate oxidase variant of the present invention, the amount of L-glutamate in the test sample can be specifically evaluated.

[0048] Furthermore, the present invention includes (A) a kit for L-glutamate analysis comprising the L-glutamate oxidase variant of the present invention. The kit of the present invention may further comprise at least one of (B) a reaction buffer or buffer salt, (C) a hydrogen peroxide detection reagent, (D) an ammonia detection reagent, and (E) a 2-oxoglutaric acid detection reagent.

[0049] (B) Reaction buffer or buffer salt is used to maintain the pH of the reaction mixture at a value suitable for the desired enzymatic reaction.

[0050] (C) Reagents for detecting hydrogen peroxide are used when detecting hydrogen peroxide by means of color development or fluorescence, for example. Examples include combinations of peroxidase and a color developer that can serve as its substrate. Specifically, examples include, but are not limited to, combinations of horseradish peroxidase, 4-aminoantipyrine, and phenol.

[0051] (D) Examples of ammonia detection reagents include the indophenol method, which combines phenol and hypochlorous acid.

[0052] (E) Examples of 2-oxoglutaric acid detection reagents include 2-oxoacid reductase.

[0053] The present invention also provides (a) a device and (b) a detection system for L-glutamate analysis comprising the L-glutamate oxidase variant of the present invention.

[0054] The L-glutamate oxidase variant of the present invention may exist as a unit independent of the microdevice that can be supplied into the device at the time of use, or it may be pre-injected, immobilized, or positioned in the device. Preferably, the L-glutamate oxidase variant of the present invention is provided in a form pre-injected, immobilized, or positioned in the device. Immobilization or positioning of the L-glutamate oxidase variant of the present invention to the device is performed directly or indirectly. As the device, a microdevice such as a microfluidic chip with a channel can be suitably used, for example.

[0055] The L-glutamic acid detection system of the present invention may further include one or more components selected from the group consisting of (c) a reaction buffer or buffer salt, a hydrogen peroxide detection reagent, an ammonia detection reagent, and a 2-oxoglutaric acid detection reagent. In the L-glutamic acid detection system of the present invention, all of the components of (c) may be provided in a form in which they are housed in a device. Alternatively, some of the components of (c) may be provided in a form in which they are housed in a device, and the remainder may be provided not in a form in which they are housed in a device (e.g., in a form in which they are housed in a different container). In this case, the components of (c) not housed in a device may be used by being injected into the device when measuring the target substance.

[0056] Examples of devices include: 1) a device comprising a first area for mixing a sample and the components of (c) to prepare a mixture, and a second area for contacting the prepared mixture with the L-glutamate oxidase variant of the present invention to detect L-glutamate (a device in which the mixing and detection steps are performed in different areas); 2) a device comprising an area for mixing a sample, the components of (c) and the L-glutamate oxidase variant of the present invention to detect L-glutamate using the L-glutamate oxidase variant of the present invention (a device in which the mixing and detection steps are performed in the same area); and 3) a device comprising a channel that enables mixing of a sample and the components of (c) (and optionally with the L-glutamate oxidase variant of the present invention), and an area for detecting L-glutamate using the L-glutamate oxidase variant of the present invention (a device in which, when a sample is injected into the device's inlet, the sample is automatically mixed via the channel, and L-glutamate in the resulting mixture is automatically detected in the detection area). From an automation standpoint, device 3), particularly device 3) in the form of a microfluidic device, is preferred. In device 3), the L-glutamate oxidase variant of the present invention may be provided in the fluid flowing through the channel, or in a form fixed or positioned in the detection area, but preferably in a form fixed or positioned in the detection area.

[0057] The present invention also provides an enzyme sensor for L-glutamate analysis, comprising (a) a detection electrode and (b) an L-glutamate oxidase variant of the present invention immobilized or positioned on the detection electrode. The L-glutamate oxidase variant of the present invention is immobilized or positioned directly or indirectly on the electrode.

[0058] As the aforementioned detection electrode, for example, a hydrogen peroxide detection electrode can be used, and more specifically, examples include an enzymatic hydrogen peroxide detection electrode and a diaphragm-type hydrogen peroxide detection electrode. In this case, L-glutamic acid can be analyzed by detecting the hydrogen peroxide produced when L-glutamic acid is oxidized by L-glutamic acid oxidation activity. Other components can be used as they are, or modified as appropriate, from known sensors. [Examples]

[0059] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0060] [Example 1] Preparation of GluOX variants Each GluOX variant was prepared as follows. First, the GluOX gene (SEQ ID NO: 3) was chemically synthesized and cloned into the NdeI-HindIII cloning site of pET-16b (Merck KGaA). Hereafter, the plasmid containing the GluOX sequence (SEQ ID NO: 3) with a His-tag added to the N-terminus will be referred to as pET-16b-GluOX. The standard GluOX (SEQ ID NO: 1) was prepared in the same manner. For the preparation of GluOX variants, mutations were introduced into the GluOX gene using the KAPA HiFi HotStart ReadyMix PCR kit (Nippon Genetics Co., Ltd.) with pET-16b-GluOX as a template, following a general protocol for site-directed mutagenesis. In cases of single mutations, insertion of specific amino acid residues, or deletion of specific amino acid residues, the mutant plasmids were prepared using this method.

[0061] [Example 2] GluOX expression and purification Recombinant expression systems for reference GluOX and GluOX mutants were constructed using Escherichia coli, and all were prepared using the same method. Here, the expression and purification method for GluOX of SEQ ID NO: 3 is described. Transformants of E. coli BL21(DE3) were obtained using pET-16b-GluOX according to a standard method. Hereafter, the BL21(DE3) transformants produced by pET-16b-GluOX will be referred to as pET-16b-GluOX-BL21(DE3).

[0062] GluOX was prepared as follows: First, glycerol stock of pET-16b-GluOX-BL21(DE3) was inoculated onto an LB agar plate containing 100 μg / mL ampicillin and incubated at 37°C overnight. 3 mL of LB liquid medium containing 100 μg / mL ampicillin was placed in a 14 mL round tube, and single colonies from the LB plate were inoculated onto it and incubated at 37°C overnight. 6 mL of LB liquid medium containing 100 μg / mL ampicillin was placed in a 50 mL tube, and then 60 μL of culture medium was added, followed by OD (Oxygen Diffusion). 600 The cells were cultured at 37°C with swirling and shaking until the value reached approximately 0.6. After standing at 16°C for 30 minutes, IPTG was added to a final concentration of 1.0 mM, and the cells were cultured overnight at 16°C with swirling and shaking before being collected.

[0063] The bacterial cells were suspended in a disruption buffer (50 mM HEPES, 100 mM NaCl, pH 7.5) and disrupted using an ultrasonic disruptor (BIORUPTOR, Cosmo Bio Co., Ltd.) at intensity H for 10 minutes with 30-second intervals while circulating cooling water. The disrupted solution was centrifuged at 13000 × g for 15 minutes at 4°C, and the supernatant was collected and purified using His SpinTrap (GE Healthcare Japan Co., Ltd.). The equilibration and washing buffer used was (50 mM HEPES, 100 mM NaCl, pH 7.5), and the elution buffer was (50 mM HEPES, 100 mM NaCl, 500 mM imidazole, pH 7.5). The eluted fraction was collected and diluted with elution buffer to 0.01 mg / mL.

[0064] [Example 3] Mutant screening by activity measurement The activity of the reference GluOX and GluOX variants prepared in Example 1 and Example 2 was evaluated using the following procedure. First, 100 μL of 0.2 M HEPES, pH 7.5, 20 μL of 10 mM glutamic acid solution, 20 μL of 30 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline solution (TOOS solution), 2 μL of 0.1 M 4-aminoantipyrine, 2 μL of 1500 U / mL peroxidase, and 36 μL of ultrapure water were added and mixed. The absorbance at 555 nm of this solution was measured using a microplate reader (Varioskan LUX, ThermoFisher SCIENTIFIC). Subsequently, 20 μL of GluOX solution prepared to 0.01 mg / mL was added, and the change in absorbance at 555 nm over time was measured using a microplate reader. Table 1 shows the relative activity of reference GluOX and GluOX mutants (SEQ ID NO: 3) lacking L467-R506 and R670-S687, with reference GluOX as the control. Table 2 shows the relative activity of reference GluOX and GluOX mutants in which certain amino acid residues were inserted or mutated in reference GluOX and the GluOX mutant (SEQ ID NO: 3) lacking L467-R506 and R670-S687, with reference GluOX as the control. Table 3 shows the relative activity (residue numbers in SEQ ID NO: 3) of GluOX mutants in which certain amino acid residues were mutated in GluOX mutant (inserted 6) in which GGGGS (SEQ ID NO: 4) was inserted between Y376 and A377 and between E356 and L357, with inserted GluOX as the control (residue numbers in SEQ ID NO: 3).

[0065] [Table 1]

[0066] [Table 2]

[0067] [Table 3]

[0068] [Example 4] Determination of the boundary vicinity region When displaying the three-dimensional structure (PDB ID: 2E1M) of the heterooctamer (α12α22β2γ2) of natural GluOX using the molecular graphics software PyMOL, a portion of the structure could not be observed. This is because, when the three-dimensional structure of a protein is experimentally determined by X-ray crystallography, the high mobility of these regions prevents them from adopting a fixed three-dimensional structure. Table 4 shows the regions in the three-dimensional structure of the heterooctamer (α12α22β2γ2) of natural GluOX in which the structure of the amino acid residues in SEQ ID NO: 3 could be confirmed. Based on this result, the near-boundary regions were determined to include the regions in which the three-dimensional structure could not be observed. Specifically, the near-boundary regions correspond to the amino acid sequence of SEQ ID NO: 3 as follows: Near-boundary region of the α1 and α2 regions: region consisting of amino acid residues from positions 349 to 363; near-boundary region of the α2 and γ regions: region consisting of amino acid residues from positions 372 to 377; near-boundary region of the γ and β regions: region consisting of amino acid residues from positions 466 to 469.

[0069] [Table 4]

[0070] Based on the above, the L-glutamate oxidase variant of the present invention exhibits improved enzyme activity compared to the wild type, making it useful for rapid and highly sensitive measurement of L-glutamate, and / or production of 2-oxoglutaric acid, and / or as an L-glutamate testing reagent. [Industrial applicability]

[0071] This invention is useful in a wide range of fields, including biological research, health and nutrition, medicine, and food manufacturing.

Claims

1. An L-glutamate oxidase mutant (excluding L-glutamate oxidase containing the amino acid sequence of SEQ ID NO: 1) having L-glutamate oxidative activity, wherein one or more sites selected from the group consisting of (1) a site in the vicinity of the boundary between the α1 and α2 regions, and (2) a site in the vicinity of the boundary between the α2 and γ regions, has an inserted peptide linker consisting of the amino acid sequence of GGGGS (SEQ ID NO: 4) or two repeats of that amino acid sequence, and is an L-glutamate oxidase mutant having L-glutamate oxidative activity, The region near the boundary between the α1 and α2 regions is the region consisting of amino acid residues from position 349 to 363 in SEQ ID NO: 3, and An L-glutamate oxidase mutant in which the region near the boundary between the α2 and γ domains corresponds to the region consisting of amino acid residues from positions 372 to 377 in SEQ ID NO:

3.

2. The L-glutamate oxidase mutant according to claim 1, wherein the L-glutamate oxidase mutant is a mutant of L-glutamate oxidase derived from a microorganism belonging to the genus Streptomyces.

3. The L-glutamate oxidase mutant according to claim 2, wherein the microorganism belonging to the genus Streptomyces is Streptomyces SP X-119-6.

4. The L-glutamate oxidase mutant according to any one of claims 1 to 3, wherein the peptide linker is inserted in both (1) a site in the vicinity of the boundary between the α1 and α2 regions, and (2) a site in the vicinity of the boundary between the α2 and γ regions.

5. The region near the boundary between the α1 and α2 regions is the region between amino acid residues at positions 356 and 357 in SEQ ID NO: 3, and The L-glutamate oxidase mutant according to any one of claims 1 to 4, wherein the region in the vicinity of the boundary between the α2 region and the γ region is the region between the amino acid residues at positions 376 and 377 in SEQ ID NO:

3.

6. The L-glutamate oxidase mutant according to any one of claims 1 to 5, wherein the L-glutamate oxidase mutant has a mutation in one or more amino acid residues selected from the group consisting of A106, C210, Q235, D236, D237, P244, T311, W313, Q333, I334, M336, Q338, R339, T416, A438, K441, Y455, Q456, Q457, L505, P558, C561, and P569 in SEQ ID NO:

3.

7. The L-glutamate oxidase mutant according to any one of claims 1 to 6, wherein the L-glutamate oxidase mutant has a mutation in one or more amino acid residues selected from the group consisting of A106S, C210S, Q235E, D236E, D237E, P244H, T311S, W313F, Q333E, I334V, I334L, M336L, Q338E, R339K, T416S, A438P, K441E, Y455F, Q456R, Q457E, Q457K, L505I, P558A, C561S, and P569A in SEQ ID NO:

3.

8. A polynucleotide encoding an L-glutamate oxidase variant according to any one of claims 1 to 7.

9. An expression vector comprising the polynucleotide described in claim 8.

10. A transformed microorganism comprising an expression unit comprising a polynucleotide encoding an L-glutamate oxidase mutant according to any one of claims 1 to 7, and a promoter operably linked thereto.

11. A method for producing an L-glutamate oxidase mutant, comprising generating the L-glutamate oxidase mutant according to any one of claims 1 to 7 using the transformed microorganism according to claim 10.

12. An L-glutamate oxidase variant obtained by the manufacturing method described in Claim 11.

13. A method for analyzing L-glutamic acid, comprising measuring the amount of L-glutamic acid contained in a test sample using an L-glutamic acid oxidase variant according to any one of claims 1 to 7 or 12.

14. The method according to claim 13, wherein the measurement of L-glutamic acid is performed using N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (TOOS) and 4-aminoantipyrine, and peroxidase, in addition to the L-glutamic acid oxidase variant described in any one of claims 1 to 7, 12.

15. A method for producing 2-oxoglutaric acid, comprising generating 2-oxoglutaric acid in vitro from L-glutamic acid in the presence of an L-glutamic acid oxidase mutant according to any one of claims 1 to 7, 12.

16. An L-glutamate detection reagent or kit comprising an L-glutamate oxidase variant according to any one of claims 1 to 7 or 12.

17. The L-glutamic acid detection reagent or kit according to claim 16, further comprising one or more selected from the group consisting of a reaction buffer or buffer salt, a hydrogen peroxide detection reagent, an ammonia detection reagent, and a 2-oxoglutaric acid detection reagent.

18. (a) a device, and (b) a detection system for L-glutamate analysis comprising an L-glutamate oxidase variant according to any one of claims 1 to 7, 12.

19. (c) The detection system for L-glutamic acid analysis according to claim 18, further comprising one or more selected from the group consisting of a reaction buffer or buffer salt, a hydrogen peroxide detection reagent, an ammonia detection reagent, and a 2-oxoglutaric acid detection reagent.

20. An enzyme sensor for L-glutamate analysis comprising (a) a detection electrode, and (b) an L-glutamate oxidase variant according to any one of claims 1 to 7, 12, fixed to or positioned on the detection electrode.

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