Glutamate oxidase mutant

Abstract

The present disclosure addresses the problem of providing an L-glutamate oxidase having heat resistance. Provided is an L-glutamate oxidase mutant having a prescribed amino acid substitution.

Description

Glutamate oxidase variant

[0001] This invention relates to L-glutamate oxidase variants.

[0002] Historically, L-glutamate levels were measured using L-glutamate decarboxylase and L-glutamate dehydrogenase. However, the measurement of carbon dioxide was cumbersome in the decarboxylase measurement system, and the measurement of the absorbance change of the coenzyme in the dehydrogenase measurement system was also cumbersome.

[0003] Subsequently, L-glutamate oxidase that acts solely on L-glutamate was reported through solid culture of actinomycetes (Non-Patent Literature 1). L-glutamate oxidase is encoded as a single polypeptide having α, γ, and β chains, and two polypeptides form a homodimer. This homodimer is cleaved by a protease to become the mature form. The mature form is α ２ β ２ γ ２ It is a heterohexamer structure composed of these elements.

[0004] The recombinant homodimer exhibited weak L-glutamate oxidase activity, inferior substrate affinity compared to the original actinomycete L-glutamate oxidase, and was also thermally unstable. When this recombinant homodimer enzyme was treated with a protease, it acquired the same structure and enzymatic properties as the original actinomycete L-glutamate oxidase (Non-Patent Literature 2). A method of mass-producing this precursor using genetically modified E. coli and treating it with a protease is used industrially.

[0005] Patent Document 1 describes an L-glutamate oxidase mutant. Patent Document 1 does not describe the thermal stability of the L-glutamate oxidase mutant that has been produced.

[0006] Patent document 2 describes a recombinant L-glutamate oxidase mutant, which does not require treatment with a protease.

[0007] International Publication No. 2021 / 193598 Pamphlet International Publication No. 2023 / 140286 Pamphlet

[0008] Kusakabe H et al. , Agric. Biol. Chem. , 47, 1323-1328 (1983) Arima J et al. , J. Biochem. , 134, 805-812 (2003)

[0009] In certain embodiments, the present disclosure aims to provide L-glutamate oxidase with improved heat resistance.

[0010] The inventors have found that the above problems can be solved at least partially by using L-glutamate oxidase having a specific mutation, and have completed the present invention which incorporates this as one embodiment.

[0011] This disclosure provides the following embodiments: [1] An L-glutamate oxidase variant having an amino acid substitution at the position corresponding to position 210 of SEQ ID NO: 1, wherein the substituted amino acid at that position is an amino acid residue other than cysteine. [2] The L-glutamate oxidase variant according to Embodiment 1, having improved heat resistance compared to L-glutamate oxidase without an amino acid substitution at the position corresponding to position 210 of SEQ ID NO: 1. [3] The L-glutamate oxidase variant according to Embodiment 1, wherein the amino acid sequence before the introduction of the amino acid substitution at the position corresponding to position 210 of SEQ ID NO: 1 has 70% or more, 80% or more, or 90% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, or 3. [4] A composition, reagent, electrode, sensor, or kit comprising the L-glutamate oxidase variant according to any one of Embodiments 1 to 3. [5] A polynucleotide encoding the L-glutamate oxidase variant according to any one of Embodiments 1 to 3. [6] A vector comprising the polynucleotide according to Embodiment 5. [7] A host cell comprising the vector described in Embodiment 6. [8] A method for producing an L-glutamate oxidase mutant, comprising the steps of: culturing the host cell described in Embodiment 7 to produce an L-glutamate oxidase mutant described in any of Embodiments 1 to 3; and obtaining the produced L-glutamate oxidase mutant. [9] A method for oxidizing L-glutamate contained in a sample by contacting an L-glutamate oxidase mutant described in any of Embodiments 1 to 3, or a composition, reagent, electrode, sensor, or kit described in Embodiment 4.

[10] The method according to Embodiment 9 for detecting L-glutamate.

[0012] This specification includes the disclosures of Japanese Patent Application No. 2025-053311, which forms the basis of the priority claim of this application.

[0013] In certain embodiments, an effect of the present invention is to obtain L-glutamate oxidase with improved heat resistance.

[0014] L-glutamate oxidase is encoded by the GLOD gene as a single polypeptide comprising an α-chain, a γ-chain and a β-chain, and two polypeptides form a homodimer. In nature, this homodimer is cleaved by a protease to become a mature form. The mature form comprises α ２ β ２ γ ２ , which forms a heterohexameric structure.

[0015] In the present specification, unless otherwise specified, the term GLOD refers to L-glutamate oxidase. L-glutamate oxidase (GLOD) is an oxidoreductase classified under EC 1.4.3.11, which catalyzes the following chemical reaction. [Chemical 1] L-glutamic acid + O ２ + H ２ O → 2-oxoglutaric acid + NH ３ + H ２ O ２

[0016] GLOD uses flavin adenine dinucleotide (FAD) as a coenzyme. Oxidoreductases that use FAD as a coenzyme are known to have an FAD binding motif sequence, for example, a Gly-Xaa-Gly-Xaa-Xaa-Gly motif (wherein Xaa is any amino acid). For example, when SEQ ID NO: 1 is taken as a reference sequence, the amino acid sequence "Gly-Ala-Gly-Ile-Ala-Gly" at positions 51 to 56 of SEQ ID NO: 1 corresponds to the FAD binding motif sequence of Gly-Xaa-Gly-Xaa-Xaa-Gly. In one embodiment, with respect to the variant of the present disclosure, Gly at positions respectively corresponding to position 51, position 53, and position 56 based on SEQ ID NO: 1 does not need to undergo amino acid substitution. In one embodiment, Gly at position 51, position 53, and position 56 of SEQ ID NO: 1 which is GLOD does not undergo amino acid substitution.

[0017] GLOD is known to recognize its substrate, L-glutamic acid, via an arginine residue. For example, using SEQ ID NO: 1 as a reference, Arg(R) at position 291 is an important position for L-glutamic acid recognition. In some embodiments, with respect to the mutants of this disclosure, the position corresponding to position 291 of SEQ ID NO: 1 does not need to be substituted with an amino acid. In some embodiments, with respect to the mutants of this disclosure, if the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is Arg(R), then that amino acid is not substituted.

[0018] (Reference Sequence) For convenience, in this specification, Sequence ID No. 1 is used as the reference sequence to define each position of GLOD. Sequence ID No. 1 is the amino acid sequence of GLOD (M7GLOD) derived from Streptomyces sp. MOE7.

[0019] As a mutant based on SEQ ID NO: 1, M7GLODΔ49C, which has the amino acid sequence of SEQ ID NO: 2, can be cited. M7GLODΔ49C can be seen as having 56 amino acids deleted from positions 456 to 511 of M7GLOD, which has the amino acid sequence of SEQ ID NO: 1, and 7 amino acids different from the sequence of M7GLOD inserted in those positions.

[0020] An example of GLOD is StGLOD derived from Streptomyces sp. X-119-6, which has the amino acid sequence of SEQ ID NO: 3. The secretory signal sequence at the N-terminal end of the natural sequence may be removed (it is removed in SEQ ID NO: 3). In addition, in SEQ ID NO: 3, the first amino acid in the α-chain region immediately after the cleavage of the secretory signal sequence in the wild type has been changed from Ala in the wild type to Met. In this specification, the position of this Met corresponds to position 1 in SEQ ID NO: 3.

[0021] An example of a mutant based on SEQ ID NO: 3 is the StGLOD mutant having the amino acid sequence of SEQ ID NO: 4, as described in Patent Document 1. This mutant can be seen as having the regions from positions 467 to 506 and 670 to 687 of StGLOD having the amino acid sequence of SEQ ID NO: 3 deleted.

[0022] In one embodiment, the variant may be produced based on Sequence ID No. 1. In one embodiment, the variant may be produced based on a GLOD having 70% or more sequence identity with Sequence ID No. 1, for example, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0023] In another embodiment, the variant may be prepared based on Sequence ID No. 2. In one embodiment, the variant may be prepared based on a GLOD having 70% or more sequence identity with Sequence ID No. 2, for example, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0024] In another embodiment, the variant may be prepared based on Sequence ID No. 3. In one embodiment, the variant may be prepared based on a GLOD having 70% or more sequence identity with Sequence ID No. 3, for example, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0025] In another embodiment, the variant can be prepared based on a known GLOD or a GLOD having 70% or more, for example, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity therewith.

[0026] (Heat resistance-enhancing mutation) In one embodiment, the present disclosure provides a GLOD variant into which a heat resistance-enhancing mutation is introduced. This variant has improved heat resistance as compared to GLOD before introduction of the mutation. Herein, for the GLOD variant of the present disclosure, the expression that "heat resistance is improved" means that when GLOD is subjected to heat treatment at a predetermined temperature and for a predetermined time, the residual activity of the GLOD variant after the heat treatment is improved as compared with the residual activity of GLOD before introduction of the mutation after the same heat treatment. For example, the residual activity refers to the activity value of a GLOD sample after heat treatment, when the activity of the GLOD sample stored under refrigeration (e.g., at 4°C) is defined as 1. The residual activity can be a value of 0 or more and 1 or less, but can be a value exceeding 1 when GLOD is activated by the heat treatment.

[0027] In one embodiment, when the residual activity of unmodified GLOD after heat treatment is defined as 100%, the residual activity of the modified GLOD variant after heat treatment can be improved by, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, 200% or more, 250% or more, 300% or more, 350% or more, for example 400% or more. Herein, the expression that "the residual activity is improved by 10%" means that when the residual activity of unmodified GLOD after heat treatment is defined as 100%, the residual activity of the modified GLOD variant after heat treatment is 110%.

[0028] In one embodiment, the GLOD variant of this disclosure has an amino acid substitution at the position corresponding to position 210 of SEQ ID NO: 1, compared to the cysteine ​​residue of SEQ ID NO: 1, and the thermal stability of the mutant after amino acid substitution is improved compared to the mutant before substitution. With respect to the above position, "amino acid substitution (having an amino acid substitution)" means that the wild-type amino acid is replaced with another amino acid, and therefore the wild-type amino acid is excluded from the substituted amino acid. In SEQ ID NO: 1, position 210 is cysteine. Therefore, the substituted amino acid at the position corresponding to position 210 of SEQ ID NO: 1 is an amino acid residue other than cysteine. The corresponding position will be described later.

[0029] In one embodiment, with respect to the GLOD variant of the present disclosure, the substituted amino acid (substituted amino acid) introduced at the position corresponding to position 210 of SEQ ID NO: 1 may be alanine, glutamic acid, aspartic acid, lysine, arginine, histidine, glycine, leucine, isoleucine, valine, methionine, glutamine, asparagine, serine, threonine, tryptophan, phenylalanine, tyrosine, or proline. The inventors of the present invention were able to obtain a GLOD with a complete quaternary structure by substituting a cysteine ​​residue with an amino acid residue other than cysteine ​​at the position corresponding to position 210 of SEQ ID NO: 1. Although we do not wish to be bound by any particular theory, it is thought that in a GLOD, the cysteine ​​residue at the position corresponding to position 210 of SEQ ID NO: 1 may form a disulfide bond with the corresponding cysteine ​​residue of another GLOD. Furthermore, although we do not wish to be bound by any particular theory, it is thought that substituting the cysteine ​​residue at position 210 of Sequence ID No. 1 with an amino acid residue other than cysteine ​​can prevent the formation of a disulfide bond, thereby suppressing the polymerization of GLOD. Therefore, it is reasonably understood that by substituting the amino acid (cysteine ​​residue) at position 210 of Sequence ID No. 1 with any of the 19 other natural amino acid residues, it is possible to obtain GLOD with a uniform quaternary structure (i.e., obtain GLOD that does not polymerize). In this specification, unless otherwise specified, amino acid residues other than cysteine ​​refer to the 19 other natural amino acid residues other than cysteine, namely alanine, glutamic acid, aspartic acid, lysine, arginine, histidine, glycine, leucine, isoleucine, valine, methionine, glutamine, asparagine, serine, threonine, tryptophan, phenylalanine, tyrosine, and proline.

[0030] GLOD is widely distributed in nature, and can be obtained by searching for enzymes of microbial, animal or plant origin. In microorganisms, it can be obtained from, for example, actinomycetes, filamentous fungi, yeasts, or bacteria. In the present specification, the origin of GLOD is not particularly limited, and it means GLOD derived from microorganisms of, for example, the genus Streptomyces, such as Streptomyces sp. X-119-6, Streptomyces sp. MOE7, Streptomyces lydicus, the genus Azotobacter, the genus Embleya, the genus Kitasatospora, the genus Saccharothrix, the genus Alloactinosynnema, the genus Streptoalloteichus, the genus Actinoalloteichus, the genus Catenulispora, the genus Nannocystis, the genus Actinobacteria, the genus Actinophytocola, the genus Sphaerisporangium, the genus Microbispora, the genus Streptosporangium, the genus Phytohabitans, the genus Haliangium, the genus Archangium, the genus Streptacidiphilus, the genus Saccharothrix or the genus Trichoderma, and includes both wild-type and variants thereof, unless otherwise specified.

[0031] (Acquisition of Gene Encoding GLOD) To obtain a gene encoding GLOD (hereinafter, also simply referred to as "GLOD gene"), commonly used gene cloning methods are used. For example, chromosomal DNA or mRNA can be extracted by conventional methods from microbial cells having GLOD-producing ability or various cells. Furthermore, cDNA can be synthesized using mRNA as a template. A chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA obtained in this manner.

[0032] Next, based on the amino acid sequence of GLOD, a suitable probe DNA can be synthesized and used to select the GLOD gene from a library of chromosomal DNA or cDNA. Alternatively, a suitable primer DNA can be prepared based on the amino acid sequence, and the DNA containing the target gene fragment encoding GLOD can be amplified by a suitable polymerase chain reaction (PCR) such as the 5'RACE method or the 3'RACE method. These DNA fragments can then be ligated to obtain DNA containing the full length of the target GLOD gene.

[0033] Examples of GLOD genes include, but are not limited to, the GLOD gene derived from Streptomyces sp. X-119-6, the GLOD gene derived from Streptomyces sp. MOE7, and the GLOD gene derived from Streptomyces lydicus.

[0034] The GLOD gene may be ligated to a vector. Examples of vectors include plasmids, bacteriophages, and cosmids; for example, pBluescriptII SK+ (Stratagene). Plasmids can be obtained by conventional methods. For example, the GLOD gene-containing plasmid can be extracted and purified using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich). The obtained GLOD gene can be manipulated to create GLOD mutant genes, and purified enzymes can be obtained.

[0035] (Mutation of the GLOD gene) Mutation of the GLOD gene can be carried out by any known method depending on the desired mutation form. Specifically, methods such as contacting and acting on the GLOD gene or recombinant DNA containing the gene with a mutagenic drug; ultraviolet irradiation; genetic engineering techniques; or protein engineering techniques can be widely used.

[0036] Examples of mutagenic drugs used in the above-mentioned mutagenesis treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrite, sulfite, hydrazine, formic acid, or 5-bromouracil.

[0037] The conditions for this contact and action can be adjusted according to the type of drug used, and are not particularly limited as long as the desired mutation can be induced in the GLOD gene. Typically, the desired mutation can be induced by contact and action for 10 minutes or more, preferably 10 to 180 minutes, at a drug concentration of 0.5 to 12 M and a reaction temperature of 20 to 80°C. Even when ultraviolet irradiation is performed, it can be carried out according to the conventional method as described above.

[0038] One method that utilizes protein engineering techniques is generally known as Site-Specific Mutagenesis.

[0039] In addition to the gene modification methods described above, the desired modified GLOD gene can also be directly synthesized by organic synthesis or enzymatic synthesis.

[0040] The nucleotide sequence of the GLOD gene can be confirmed, for example, using a multi-capillary DNA analysis system such as the Applied Biosystems 3730xl DNA analyzer (Thermo Fisher Scientific).

[0041] (Transformation and Transduction) The GLOD gene can be incorporated into vectors such as bacteriophages, cosmids, or plasmids used for the transformation of prokaryotic or eukaryotic cells by conventional methods. Using these vectors, the host corresponding to each vector can be transformed or transduced by conventional methods.

[0042] In one embodiment, GLOD can be expressed using prokaryotic cells, such as microorganisms of the genus Escherichia, such as Escherichia coli; microorganisms of the genus Brevibacillus, such as Brevibacillus choshinensis; microorganisms of the genus Corynebacterium, such as Corynebacterium glutamicum; and microorganisms of the genus Streptomyces, such as Streptomyces violaceoruber. Examples of Escherichia coli hosts include, but are not limited to, various strains of Escherichia coli, such as strain K-12, strain JM109, strain DH5α, strain BL21, strain JM109(DE3), strain DH5α(DE3), strain BL21(DE3), strain TG1, strain 1100, strain W3110, and strain C600. Host cells (transformed organisms) into which the GLOD gene has been introduced are obtained by transforming the host or transduction into the host. Methods for introducing recombinant vectors into these host cells include, for example, if the host cell is a microorganism belonging to Escherichia kory, a method of introducing recombinant DNA in the presence of calcium ions can be employed, and electroporation may also be used. Furthermore, commercially available competent cells (e.g., ECOS Competent Escherichia kory BL21(DE3); manufactured by Nippon Gene) may be used. The GLOD gene may be codon-optimized according to the host in which it is expressed.

[0043] In one embodiment, GLOD can be expressed using eukaryotic cells. An example of a eukaryotic host cell is yeast. Examples of microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Schizosaccharomyces, Saccharomyces, Pichia, and Candida. The inserted gene may include a marker gene to enable the selection of transformed cells. Examples of marker genes include genes that complement the host's nutritional requirements, such as URA3 and TRP1. Furthermore, it is desirable that the inserted gene includes a promoter or other regulatory sequence (e.g., enhancer sequence, terminator sequence, polyadenylation sequence, etc.) that can express the target gene in the host cell. Specific examples of promoters include the GAL1 promoter and the ADH1 promoter. As a method for transforming yeast, known methods such as the lithium acetate method (Methods Mol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55, (2003) 481-484) can be suitably used, but are not limited to these. Transformation can be carried out using any method, including the spheroplast method and the glass bead method.

[0044] Other examples of eukaryotic host cells include fungal cells (including filamentous fungi) such as those of the genera Aspergillus and Trichoderma. The method for producing fungal cell transformants is not particularly limited, and one example is to insert the gene encoding GLOD into the host filamentous fungus in a manner that expresses it, according to a conventional method. Specifically, a DNA construct is made in which the gene encoding GLOD is inserted between the expression induction promoter and the terminator, and then the host filamentous fungus is transformed with the DNA construct containing the gene encoding GLOD to obtain a transformant that overexpresses the gene encoding GLOD.

[0045] The method for inserting the gene encoding GLOD into a host filamentous fungus in a manner in which it is expressed is not particularly limited, but examples include methods of directly inserting it onto the chromosome of the host organism using homologous recombination, and methods of introducing it into the host filamentous fungus by ligating it onto a plasmid vector.

[0046] In methods utilizing homologous recombination, a DNA construct can be ligated between sequences homologous to the upstream and downstream regions of a recombination site on a chromosome and inserted into the genome of the host filamentous fungus. By overexpressing this construct within the host filamentous fungus under the control of its own high-expression promoter, a transformant can be obtained through self-cloning. The high-expression promoter is not particularly limited, but examples include the promoter region of the translation elongation factor TEF1 gene (tef1), the promoter region of the α-amylase gene (amy), and the promoter region of the alkaline protease gene (alp).

[0047] In the vector-based method, a DNA construct can be incorporated into a plasmid vector used for filamentous fungal transformation using a standard method, and the corresponding host filamentous fungus can then be transformed using a standard method.

[0048] Such suitable vector-host systems are not particularly limited as long as they are capable of producing GLOD in the host filamentous fungus, and examples include the pUC19 and filamentous fungus system, and the pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and filamentous fungus system.

[0049] While it is preferable to introduce the DNA construct into the chromosome of the host filamentous fungus, another method is to incorporate the DNA construct into an autonomously replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)), thereby allowing it to be used without being introduced into the chromosome.

[0050] The DNA construct may include marker genes to enable the selection of transformed cells. The marker genes are not particularly limited and include, for example, genes that complement the host's nutritional requirements, such as pyrG, niaD, and adeA; and drug resistance genes to drugs such as pyrithiamine and hygromycin B oligomycin. The DNA construct also preferably includes promoters, terminators, and other regulatory sequences (e.g., enhancers, polyadenylation sequences) that enable the overexpression of the gene encoding GLOD in the host cell. The promoter is not particularly limited but may include appropriate inductive or constitutive promoters, such as the tef1 promoter, alp promoter, and amy promoter. The terminator is also not particularly limited but may include, for example, the alp terminator, amy terminator, and tef1 terminator.

[0051] In a DNA construct, the expression regulatory sequence of the gene encoding GLOD is not necessarily required if the DNA fragment containing the GLOD-encoding gene to be inserted contains a sequence with expression regulatory function. Furthermore, when transformation is performed using cotransformation, the DNA construct may not need to contain a marker gene.

[0052] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, the gene encoding GLOD, the alp gene terminator, and the pyrG marker gene are linked to the In-Fusion Cloning Site located at the multi-cloning site of pUC19.

[0053] As a method for transforming filamentous fungi, a method known to those skilled in the art can be appropriately selected. For example, after preparing protoplasts of the host filamentous fungus, the protoplast PEG method using polyethylene glycol and calcium chloride can be used (see, for example, Mol. Gen. Genet. 218, 99-104, 1989, Japanese Patent Publication No. 2007-222055). The culture medium for regenerating the transformed filamentous fungus should be appropriate depending on the host filamentous fungus and the transformation marker gene used. For example, if Aspergillus soybean is used as the host filamentous fungus and the pyrG gene is used as the transformation marker gene, the regeneration of the transformed filamentous fungus can be carried out, for example, in Czapek-Dox minimal medium (DIFCO) containing 0.5% agar and 1.2 M sorbitol.

[0054] Furthermore, for example, in order to obtain the transformed filamentous fungus of the present invention, homologous recombination may be used to replace the promoter of the gene encoding GLOD, which is originally present on the chromosome of the host filamentous fungus, with a high-expression promoter such as tef1. In this case as well, it is preferable to insert a transformation marker gene such as pyrG in addition to the high-expression promoter. For example, for this purpose, referring to Example 1 and Figure 1 described in Japanese Patent Application Publication No. 2011-239681, a transformation cassette consisting of the upstream region of the gene encoding GLOD - transformation marker gene - high-expression promoter - all or part of the gene encoding GLOD can be used. In this case, the upstream region of the gene encoding GLOD and all or part of the gene encoding GLOD are used for homologous recombination. All or part of the gene encoding GLOD can include the region from the start codon to the intermediate region. The length of the region suitable for homologous recombination is preferably 0.5 kb or more.

[0055] The successful production of a transformed filamentous fungus can be confirmed by culturing the transformed filamentous fungus of the present invention under conditions in which GLOD enzyme activity is observed, and then confirming the GLOD activity in the culture obtained after culturing.

[0056] Alternatively, confirmation that transformed filamentous fungi have been produced can be performed by extracting chromosomal DNA from the transformed filamentous fungi, using this as a template for PCR, and confirming that a PCR product capable of amplification is produced when transformation occurs.

[0057] For example, PCR is performed using a combination of a forward primer for the promoter sequence and a reverse primer for the transformation marker gene sequence to confirm that a product of the expected length is produced.

[0058] The host may be a known microorganism, a known strain, or an equivalent or similar of a known microorganism or strain described herein. An equivalent is a host that exhibits equivalent function with respect to the recombinant expression of proteins. Equivalents include hosts created and modified based on hosts that were known at the time of filing of this application, which were developed after the filing of this application, and hosts that are similar in nature to hosts that were known at the time of filing of this application and were discovered after the filing of this application. With regard to the scientific name or classification of a microorganism, if there is a change in the scientific name, genus name, classification, etc. after the filing of this application, the description in this specification shall take precedence, and the time of filing of this application shall be the basis.

[0059] (High-throughput screening) GLOD can also be subjected to high-throughput screening to obtain functional GLOD variants. For example, a library of transformed or transduced strains containing the mutant GLOD gene may be prepared and subjected to high-throughput screening based on microtiter plates, or to ultra-high-throughput screening based on droplet microfluidics. An example is a method of constructing a combinatorial library of mutant genes encoding variants, and then screening a large population of mutant GLODs using phage display (e.g., Chem. Rev. 105 (11): 4056-72, 2005), yeast display (e.g., Comb Chem High Throughput Screen. 2008;11(2): 127-34), or bacterial display (e.g., Curr Opin Struct Biol 17: 474-80, 2007). See also Agresti et al, "Ultrahigh-throughput screening in drop-based microfluidics for directed evolution," Proceedings of the National Academy of Sciences 107 (9): 4004-4009 (Mar, 2010). The description of ultrahigh-throughput screening methods that can be used for screening GLOD variants from that document is incorporated herein by reference. For example, a library can be constructed by error-prone PCR. Alternatively, a library may be constructed by introducing mutations targeting the regions and locations described herein or their corresponding regions and locations using saturated mutagenesis. Using the library, suitable cells such as electrocompetent EBY-100 cells can be transformed to obtain approximately 10^7 variants (10 million). Yeast cells transformed with the library can then be subjected to cell sorting. Polydimethoxysiloxane (PDMS) microfluidic devices prepared using standard soft lithography may also be used. Monodisperse droplets can be formed using a flow focus device.The formed droplets containing individual mutants can be subjected to a suitable sorting device. When selecting cells, the presence or absence of GLOD activity can be utilized. For example, a reaction solution with a composition that develops color when GLOD acts may be used. For example, when detecting hydrogen peroxide produced by the GLOD reaction using the dye formation reaction of TOOS and 4-aminoantipyrine by POD, the absorbance at 555 nm may be measured using a 96-well plate, 192-well plate, 384-well plate, 9600-well plate, etc., and a plate reader. Mutation introduction and selection may be repeated multiple times. Mutation here includes amino acid substitution, insertion, deletion, and / or addition.

[0060] For example, GLOD activity can be confirmed by introducing 1 to 10 mutations into GLOD. Then, starting from the GLOD mutant confirmed to have activity, another 1 to 10 mutations can be introduced and activity can be confirmed. A series of high-throughput screenings (for example, the above method of obtaining and screening approximately 10^7 mutants) can be repeated for 2 or more rounds, 5 or more rounds, 10 or more rounds, 15 or more rounds, for example, 20 or more rounds. By repeating a high-throughput screening, in which 1 or more mutations, 5 or more mutations, for example, 10 or more mutations are introduced in each round, for example, 10 rounds, mutants with 10 or more mutations, 50 or more mutations, for example, 100 or more mutations introduced from the starting GLOD, and which also have activity, can be rapidly obtained. Furthermore, by repeating for 20 rounds, mutants with 20 or more mutations, 100 or more mutations, for example, 200 or more mutations introduced from the starting GLOD, and which also have activity, can be rapidly obtained. Such work can be performed by repeating a routine process.

[0061] Mutations can be introduced at one or more positions in the full amino acid sequence of GLOD, from the first to the last amino acid. However, functionally important regions of the enzyme, such as the active site, substrate recognition site, coenzyme recognition motif, and their vicinity, are excluded. GLOD is widely used in industry, and those skilled in the art are familiar with functionally important regions of the enzyme, including its active site, substrate recognition site, and coenzyme recognition motif. In certain embodiments, for example, one or more mutations can be introduced first in the 1st to 10th positions of the full GLOD sequence. Then, starting from a GLOD mutant that has been confirmed to have activity, one or more mutations can be introduced further in the 11th to 20th positions, and activity can be confirmed. This can be repeated n times (n ≤ 68). For example, one or more mutations can be introduced in the 68th iteration in the 680th to 687th positions. Functionally important regions of the enzyme and regions not intended to be modified may be skipped as appropriate. This allows for the introduction of arbitrary mutations at any position in the full-length sequence, excluding regions crucial to enzyme function. Furthermore, it enables the rapid acquisition of active GLOD mutants having, for example, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, or even 200 or more mutations.

[0062] Mutations may be introduced randomly or by rational design. If mutations are introduced randomly, mutants with further mutations at the mutation site of the disclosed mutation may be excluded from subsequent selection. Alternatively, the disclosed mutation may be introduced into mutants that have been confirmed to have enzyme activity after random mutations have been introduced. In some embodiments, mutations introduced by rational design or randomly may be conservative amino acid substitutions. Conservative amino acid substitutions include amino acid substitutions in which the amino acid before substitution and the amino acid after substitution have similar chemical properties (e.g., Stryer et al., Biochemistry, 5th edition, 2002, pp. 44-49). For example, conservative amino acid substitutions may be selected from the group consisting of (i) substitution of a basic amino acid with a different type of basic amino acid; (ii) substitution of an acidic amino acid with a different type of acidic amino acid; (iii) substitution of an aromatic amino acid with a different type of aromatic amino acid; (iv) substitution of a nonpolar aliphatic amino acid with a different type of nonpolar aliphatic amino acid; and (v) substitution of a polar uncharged amino acid with a different type of polar uncharged amino acid. Basic amino acids may be selected from, for example, arginine, histidine, and lysine. Acidic amino acids may be, for example, aspartic acid or glutamic acid. Aromatic amino acids may be selected from, for example, phenylalanine, tyrosine, and tryptophan. Nonpolar aliphatic amino acids may be selected from, for example, glycine, alanine, valine, leucine, methionine, and isoleucine. Polar uncharged amino acids may be selected from, for example, serine, threonine, cysteine, proline, asparagine, and glutamine.

[0063] In some embodiments, mutations introduced by rational design or randomly introduced include substitutions of functionally similar amino acids. A table of functionally similar amino acids is widely known in the art. In some embodiments, substitutions of functionally similar amino acids may involve the original amino acid and the substituted amino acid belonging to one of the following amino acid classifications: 1) glycine (G), alanine (A); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K), histidine (H); 5) isoleucine (I), leucine (L), valine (V), proline (P); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0064] In a typical embodiment, conservative amino acid substitutions, or substitutions with functionally similar amino acids, are not present in functionally important regions of the enzyme, such as the GLOD active site, substrate recognition site, coenzyme recognition motif, and their vicinity, and therefore do not significantly affect the enzyme's activity.

[0065] GLOD variants may also include those in which additional amino acids are inserted compared to the pre-mutation sequence. In typical embodiments, the amino acid insertions are not located in functionally important regions of the enzyme, such as the active site, substrate recognition site, coenzyme recognition motif, or their vicinity, and therefore do not significantly affect the enzyme's activity. GLOD variants may also include those in which additional amino acids are added compared to the pre-mutation sequence. In some embodiments, the amino acid addition occurs at the N-terminus or C-terminus of GLOD and does not significantly affect the enzyme's activity. Examples of additions include, but are not limited to, short stretches of histidine residues (e.g., 2-6 histidine residues) to aid in the purification of GLOD. Other examples of additions include, but are not limited to, the addition of signal peptides to aid in GLOD expression. Examples of signal peptides include known signal sequences or their functional equivalents.

[0066] GLOD variants may also contain amino acid deletions compared to the pre-mutation sequence. In typical embodiments, the amino acid deletions are not located in regions critical to the enzyme's function and therefore do not significantly affect the enzyme's activity. In some embodiments, the deletions may be short, consisting of one or two amino acids. In some embodiments, if the amino acid sequence of one GLOD is compared to the amino acid sequence of another GLOD and an amino acid is deleted in one sequence, that deletion may be introduced into the other GLOD. Since both GLODs exhibit activity, such deletions are unlikely to significantly affect the enzyme's activity.

[0067] Mutations into GLODs can be introduced in a way that does not disrupt secondary structures or structural motifs such as α-helix structures and β-sheet structures. Regions of secondary structures can be identified, for example, by secondary structure prediction algorithms. Examples of such algorithms include, but are not limited to, NetSurfP-2.0. The same applies to other structural motifs such as nests and niches.

[0068] In one embodiment, the above-mentioned heat-resistant mutation can be introduced into wild-type GLOD or conventional GLOD. Conventional GLOD refers to conventional GLOD that requires protease treatment for the expression of active protein. GLOD into which such a heat-resistant mutation has been introduced is expected to exhibit not only activity after protease treatment, similar to wild-type GLOD and conventional GLOD, but also improved heat resistance compared to GLOD before the mutation was introduced.

[0069] The heat-resistant mutation may further have the thermal stability-enhancing mutation described in Patent Document 2. The heat-resistant mutation may further have the amino acid substitution described in Patent Document 1.

[0070] (Corresponding Position) In this specification, when a specific position in a reference amino acid sequence corresponds to a specific position in another similar amino acid sequence, this is referred to as a corresponding position. The amino acid at the corresponding position is also referred to as the corresponding amino acid. For convenience, this specification uses the amino acid sequence of Streptomyces sp. MOE7-derived GLOD (M7GLOD), shown in Sequence ID No. 1, as a reference. In this case, the "corresponding position" in the amino acid sequence refers to the position in the amino acid sequence of GLOD from another species that corresponds to the specific position in the amino acid sequence of Sequence ID No. 1.

[0071] One method for identifying "corresponding positions" in amino acid sequences is to compare amino acid sequences using known algorithms such as the Lippmann-Parson method, thereby maximizing the identity of conserved amino acid residues present in the amino acid sequences of each GLOD. By aligning the amino acid sequences of GLODs in this way, it is possible to determine the positions of homologous amino acid residues in each GLOD sequence, regardless of insertions or deletions. Corresponding positions (homologous positions) are thought to exist at the same position in the three-dimensional structure and are presumed to have similar effects on the specific function of the target GLOD.

[0072] (Corresponding Mutation Location) In this specification, "the location corresponding to position 210 of the amino acid sequence of SEQ ID NO: 1" refers to the location corresponding to position 210 of SEQ ID NO: 1 when the amino acid sequence of the target GLOD is compared with the amino acid sequence of SEQ ID NO: 1. For example, the location corresponding to position 210 of the amino acid sequence of SEQ ID NO: 1 is position 210 in SEQ ID NO: 2, position 210 in SEQ ID NO: 3, and position 210 in SEQ ID NO: 4.

[0073] (Homogeneity, Identity, or Similarity of Amino Acid Sequences) The homology, identity, or similarity of amino acid sequences can be calculated using programs such as Maximum Matching and Search Homology in GENETYX (manufactured by GENETYX Corporation), or Maximum Matching and Multiple Alignment in DNASIS Pro (manufactured by Hitachi Solutions Corporation), or Multiple Alignment in CLUSTAL W. To calculate amino acid sequence identity, when two or more GLODs are aligned, the positions of identical amino acids in those two or more GLODs can be examined. Based on this information, identical regions in the amino acid sequences can be determined. Here, for two or more amino acid sequences, identity % refers to the percentage obtained when the total number of amino acids in the alignable region is used as the denominator and the number of positions occupied by identical amino acids is used as the numerator, when two or more amino acid sequences are aligned using an algorithm such as Blosum62. Therefore, if there is a region in two or more amino acid sequences that shows no identity whatsoever, for example, if one of the amino acid sequences has an additional sequence at the C-terminus that shows no identity whatsoever, then that region of no identity is impossible to align and is therefore not used in calculating the identity percentage.

[0074] Furthermore, the positions of amino acids that are similar in two or more GLODs can be investigated. For example, multiple amino acid sequences can be aligned using CLUSTALW, in which case Blosum62 is used as the algorithm, and amino acids that are judged to be similar when multiple amino acid sequences are aligned are sometimes called similar amino acids. In the variants of this disclosure, amino acid substitutions may be due to substitutions between such similar amino acids. Through such alignment, it is possible to investigate the regions where the amino acid sequences are identical and the positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, homology regions (conserved regions) in the amino acid sequences can be determined.

[0075] In one embodiment, the GLOD variant of the present disclosure has an amino acid substitution at the position corresponding to position 210 of SEQ ID NO: 1, wherein the substituted amino acid at that position is an amino acid other than cysteine, and when aligned with a GLOD having the amino acid sequence shown in SEQ ID NO: 1, 2, or 3, it has 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more of full-length amino acid sequence identity, and has improved heat resistance compared to the GLOD before amino acid substitution.

[0076] In one embodiment, the GLOD variant of the present disclosure has an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted at a position other than the position corresponding to position 210 of SEQ ID NO: 1, and has improved thermal stability compared to the original GLOD. Here, one or more amino acids means 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 4, for example 1 to 3, for example 1 or 2 amino acids.

[0077] In one embodiment, the GLOD variant of the present disclosure has an amino acid at the position corresponding to position 291 of SEQ ID NO: 1, which is arginine, and an amino acid sequence at positions 51-56 of SEQ ID NO: 1, which is Gly-Xaa-Gly-Xaa-Xaa-Gly (where Xaa represents any amino acid).

[0078] (GLOD Production) In one embodiment, the present invention provides a method for producing GLOD, comprising the steps of culturing a GLOD-producing strain under conditions in which GLOD can be expressed, and isolating GLOD from the culture or culture medium. This method may use host cells transformed with a vector incorporating the gene encoding the GLOD of the present disclosure. Herein, conditions in which GLOD can be expressed mean that the GLOD gene is transcribed and translated, and the polypeptide encoded by the gene is produced.

[0079] Furthermore, the culture medium used for culturing the above-mentioned bacterial strains may include, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or extract from soybeans or wheat bran, to which one or more inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate are added, and carbohydrate raw materials, vitamins, etc. are added as needed.

[0080] Furthermore, the amount of the target enzyme produced can be increased by adding substrates or similar compounds on which the GLOD can act, such as glycated amino acids, glycated peptides, glycated protein hydrolysates, or glycated proteins such as glycated hemoglobin or glycated albumin, to the culture medium.

[0081] The initial pH of the culture medium should be adjusted to pH 7-9. Culturing should be carried out at a culture temperature of 20-42°C, preferably around 25-37°C, for 4-24 hours, and more preferably around 25-37°C, for 8-16 hours, using methods such as deep culture with aeration and stirring, shaking culture, or static culture.

[0082] After the culturing is complete, GLOD can be collected from the culture using conventional enzyme collection methods. For example, the bacterial cells can be subjected to ultrasonic disruption or grinding by conventional methods, or the enzyme can be extracted using a lytic enzyme such as lysozyme, or the enzyme can be released from the bacterial cells by lysis by shaking or standing in the presence of toluene or the like. Then, the solid portion of this solution can be removed by filtration or centrifugation, and if necessary, nucleic acids can be removed with streptomycin sulfate, protamine sulfate, or manganese sulfate, etc. After that, ammonium sulfate, alcohol, acetone, etc., are added and fractionated, and the precipitate is collected to obtain the crude enzyme.

[0083] To obtain a further purified enzyme preparation from the crude enzyme, a purified GLOD enzyme preparation can be obtained by appropriately selecting or combining methods such as gel filtration using Sephadex, Superdex, or Ultrogel; adsorption elution using ion exchange carriers, hydrophobic carriers, or hydroxyapatite; electrophoresis using polyacrylamide gel; sedimentation methods such as sucrose density gradient centrifugation; affinity chromatography; or fractionation using molecular sieve membranes or hollow fiber membranes.

[0084] In one embodiment, the GLOD variant of the present disclosure may, for example, (i) use FAD as a coenzyme, (ii) recognize L-glutamic acid as a substrate, and (iii) oxidize L-glutamic acid to produce 2-oxoglutaric acid, ammonia, and hydrogen peroxide.

[0085] In one embodiment, the GLOD variant of the present disclosure may have a residual activity of 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, for example 90% or more, after heat treatment at 30-40°C, for example 35°C for 30 or 35 minutes, with the activity before heat treatment set to 100%. In one embodiment, the GLOD variant of the present disclosure may have a residual activity of 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, for example 90% or more, after heat treatment at 60°C for 30 or 35 minutes, with the activity before heat treatment set to 100%. In one embodiment, the GLOD variant of the Disclosure may have a residual activity of 50% or more, 60% or more, 65% or more, 70% or more, for example 75% or more, after heat treatment at 65°C for 30 or 35 minutes, with the activity before heat treatment set to 100%. In one embodiment, the GLOD variant of the Disclosure may have a residual activity of 10% or more, 20% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, for example 55% or more, after heat treatment at 70°C for 30 or 35 minutes, with the activity before heat treatment set to 100%.

[0086] The GLOD variants described herein exclude GLODs that do not exhibit activity toward L-glutamic acid.

[0087] (Compositions, Reagents, Electrodes, Sensors, and Kits) In one embodiment, the present invention provides a reagent composition, measuring reagent, electrode, sensor, or kit for measuring L-glutamic acid, comprising GLOD. The composition, reagent, electrode, sensor, or kit may contain a reagent for measuring reduced compounds, a reagent for measuring hydrogen peroxide, a buffer, a surfactant, salts, a preservative, etc. Solubilizers, stabilizers, reactivity enhancers, glycated hemoglobin denaturants, reducing agents, bovine serum albumin, sugars (glycerin, lactose, sucrose, etc.), etc. may also be added. The composition, reagent, electrode, sensor, or kit may be further modified as needed to include other known stabilizers, contaminant elimination systems, etc. Technologies used in various conventional reagents, electrodes, sensors, and kits can be appropriately modified and used in the compositions, reagents, electrodes, sensors, or kits of this disclosure.

[0088] Examples of surfactants include nonionic surfactants and ionic surfactants, such as cationic surfactants, anionic surfactants, and amphoteric surfactants.

[0089] Examples of nonionic surfactants include polyoxyethylene alkyl ethers, fatty acid sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides, and alkyl monoglyceryl ethers.

[0090] Examples of cationic surfactants include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkylbenzyldimethylammonium salts, pyridinium salts (e.g., alkylpyridinium salts), phosphonium salts (e.g., alkylphosphonium salts), imidazolium salts (e.g., alkylimidazolium salts), and isoquinonium salts (e.g., alkylisoquinonium salts).

[0091] The reagent for measuring hydrogen peroxide may contain peroxidase and / or a chromogenic substrate. Examples of chromogenic substrates, in addition to 4-aminoantipyrine, include ADOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), ALOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline), TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine), and DA-64 (N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine).

[0092] (Method for Measuring L-Glutamate) In one embodiment, the present disclosure provides a method for measuring L-glutamate. The measurement of L-glutamate may be qualitative or quantitative. The quantitative method may include the steps of contacting a sample containing L-glutamate with the GLOD of the present disclosure, and measuring the reaction product or consumption. Contact as used in the quantitative method encompasses all forms of physically bringing the enzyme and the sample together so that the GLOD can catalyze the oxidation reaction of L-glutamate, and includes not only, for example, mixing free enzyme and L-glutamate in solution, but also forms of adding or dropping a solution sample containing L-glutamate to an enzyme supported on a solid support.

[0093] The sample used for measurement can be any sample that may contain L-glutamic acid. The sample may be processed as appropriate.

[0094] By keeping the amount of enzyme and reaction time constant and varying the amount of L-glutamic acid added, the minimum detectable L-glutamic acid concentration (detection limit) when using the GLOD can be determined by investigating the L-glutamic acid concentration range in which the absorbance of the detected chromogenic substrate decreases proportionally as the amount of added L-glutamic acid decreases. The amount of enzyme and reaction time can be set so that the detection limit is lower than the L-glutamic acid concentration in the sample or blood.

[0095] For quantitative measurements, a calibration curve can be created beforehand by performing regression analysis, such as the least squares method, from measured values ​​such as absorbance of a control containing L-glutamic acid of known concentration. By plotting the measured values ​​of a sample with an unknown L-glutamic acid concentration against the created calibration curve, the L-glutamic acid concentration in the sample can be quantified.

[0096] The reaction time for GLOD on a sample containing L-glutamic acid can be, for example, 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, less than 60 minutes, less than 30 minutes, less than 10 minutes, for example less than 5 minutes, for example 0.5 minutes or more to less than 60 minutes, 1 minute or more to less than 30 minutes, 1 minute or more to less than 20 minutes, for example 1 minute or more to less than 10 minutes, for example 1 minute or more to less than 5 minutes. The reaction temperature depends on the optimal temperature of the enzyme used, but for example 20 to 45°C, and temperatures used in normal enzymatic reactions can be appropriately selected.

[0097] The amount of GLOD enzyme used depends on the amount of substrate in the sample solution, but it can be added so that the final concentration is, for example, 0.1 to 50 U / ml, or 0.2 to 10 U / ml. The pH during the reaction can be adjusted using a buffer, taking into consideration the pH at which GLOD can act, for example, the optimal pH. The reaction pH is, for example, 3 to 11, 5 to 9, or 6 to 8.

[0098] The measurement of hydrogen peroxide can be performed simultaneously with the hydrogen peroxide generation process and can proceed concurrently with the action of GLOD. Instead of the product, a consuming substance may be measured; for example, dissolved oxygen can be measured, and the amount of dissolved oxygen in the reaction solution can be measured using a dissolved oxygen meter or the like.

[0099] (Method for measuring GLOD activity) The following is an example of a method for measuring GLOD activity using L-glutamic acid as a substrate, but the method is not limited to this. L-glutamic acid may be commercially available. In this specification, unless otherwise specified, enzyme titer is defined as the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured with L-glutamic acid as a substrate at 30°C and pH 7.4, with 1 U being the amount of enzyme.

[0100] A: Reagents for activity measurement (Reagent 1) 250 mM potassium phosphate buffer pH 7.4 (Reagent 2) 30 mM 4-aminoantipyrine (4-AA) solution (Reagent 3) 15 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) solution (Reagent 4) 300 U / ml horseradish peroxidase (POD) solution (Reagent 5) 300 mM L-sodium hydrogen glutamate solution B: Activity measurement method Mix 300 μl of Reagent 1, 12.5 μl of Reagent 2, 25 μl of Reagent 3, 12.5 μl of Reagent 4, deionized water (375-V) μl, and V μl of GLOD solution, and keep warm at 30°C for 5 minutes. Then, after adding 25 μl of reagent 5 and mixing well, the absorbance of light at a wavelength of 555 nm (A) was measured using a U-3900 spectrophotometer (manufactured by Hitachi High-Tech Science) with the cell holder kept warm at 30°C. ５５５ ) Measure and A per minute ５５５ Change (ΔA) Ｓ The absorbance of light at a wavelength of 555 nm (A) was calculated. As a control experiment, 25 μl of deionized water was added instead of 25 μl of reagent 5. ５５５ ) Measure and A per minute ５５５ Change (ΔA) ０ ) was calculated.

[0101] The oxidase activity (U / ml) can be calculated based on the following formula. In the formula, "39.2" is the millimolar extinction coefficient (mM) of the quinone imine dye formed by the condensation of 4-AA and TOOS for light at a wavelength of 555 nm. －１ cm －１ ) shows. [Formula] U / ml = (ΔA Ｓ -ΔA ０ )×600×df / (39.2×0.5×V) =30.6×(ΔA Ｓ -ΔA ０ ) × df / V When using different color-developing reagents, the wavelength and millimolar extinction coefficient at that wavelength corresponding to the color-developing reagent may be used.

[0102] In one embodiment, the Disclosure provides a polynucleotide encoding a GLOD variant. In one embodiment, the Disclosure provides a vector having such a polynucleotide. In one embodiment, the Disclosure provides a host cell transformed with such a vector, i.e., a host cell containing such a vector. In one embodiment, the Disclosure provides a method for producing a GLOD variant, comprising the steps of culturing such host cells to produce a GLOD variant and obtaining the produced GLOD variant. In one embodiment, the GLOD variant of the Disclosure may exhibit reduced polymerization beyond dimers compared to unmodified GLOD. In one embodiment, the Disclosure provides a method for contacting a sample containing L-glutamic acid with a GLOD variant, or a composition, reagent, electrode, sensor, or kit containing the same, to oxidize the L-glutamic acid contained in the sample. In one embodiment, the method may detect L-glutamic acid. In one embodiment, the method may measure L-glutamic acid.

[0103] The GLOD of this disclosure is further illustrated by the following embodiments, however, these are for illustrative purposes only and the disclosure is not limited thereto.

[0104] [Example 1] Improvement of heat resistance of GLOD (M7GLOD) derived from Streptomyces sp. MOE7 by introduction of amino acid substitution 1. Construction of plasmid for GLOD mutant expression The plasmid for expressing the mutant M7GLODΔ49C-T7E (SEQ ID NO: 5) was created by introducing site-directed mutagenesis using "pET22b-M7GLODΔ49C-T7E", which is the plasmid for expressing the GLOD "M7GLODΔ49C-T7 / R186E" described in Patent Document 2, as a template. Hereafter, M7GLODΔ49C-T7E will be described as M7GLOD without mutation.

[0105] The StGLOD (SEQ ID NO: 3) mutant expression plasmid was prepared by introducing site-directed mutagenesis using the StGLOD expression plasmid (pET22b-StGLOD) described in Patent Document 2 as a template.

[0106] For site-directed mutagenesis, the PCR reaction mixture was prepared by mixing 10 μl of KOD one PCR Master Mix (Toyobo), 3 μl of 2 μM Fw primer, 3 μl of 2 μM Rv primer, 0.5 μl of 40 μg / ml template DNA, and 3.5 μl of deionized water. The PCR reaction conditions were 98°C for 10 seconds → 55°C for 5 seconds → 68°C for 35 seconds, repeated 15 times. After PCR, 1 μl of DpnI was added to the reaction mixture and incubated at 37°C for 30 minutes. This solution was used to transform E. coli JM109 strain. The resulting transformants were cultured, and the plasmid sequences extracted were confirmed to be as intended by DNA sequencing analysis. The names of the created mutants and the combinations of Fw and Rv primers used in PCR are shown in Table 1.

[0107]

[0108] 2. Recombinant production of GLOD E. coli BL21 (DE3) strain was transformed with pET22b-M7GLODΔ49C-T7E or a constructed plasmid for expressing a GLOD mutant to create a GLOD-producing strain.

[0109] GLOD-producing strains were inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 μg / ml) in a test tube and cultured overnight at 37°C and 160 rpm. 2.5 ml of the seed culture solution was then inoculated into 250 ml of LB-amp medium (ampicillin concentration 50 μg / ml) containing 0.1 mM IPTG in a Sakaguchi flask and cultured at 25°C and 130 rpm for 16 hours.

[0110] The culture medium was centrifuged at 8,000 rpm for 10 minutes to obtain a pellet, which was then resuspended in 4 ml of 10 mM potassium phosphate buffer (PPB) at pH 6.0. After sonication of the bacterial suspension, the supernatant was collected from centrifugation at 15,000 rpm for 15 minutes and prepared as the crude GLOD enzyme solution.

[0111] (Enzyme Purification) Modified M7GLOD crude enzyme solution was mixed with 250 mM potassium phosphate buffer (PPB) pH 6.0, and the buffer concentration was adjusted to 100 mM. The mixture was then incubated in a 60°C water bath for 30 minutes. The supernatant was collected after centrifugation at 10,000 rpm for 15 minutes, and ammonium sulfate was added to a final concentration of 0.7 M. The enzyme solution was filtered through a 0.22 μm filter, and the filtrate was subjected to hydrophobic interaction chromatography.

[0112] A HiScreen-Butyl-FF column (Cytiva) was used for hydrophobic interaction chromatography. The enzyme solution was loaded onto the column, which had been equilibrated with 20 mM potassium phosphate buffer (PPB) pH 6.0 containing 0.7 M ammonium sulfate. After washing with the buffer used for equilibration, gradient elution was performed while decreasing the ammonium sulfate concentration to 0 M. The purity of each fraction was evaluated by SDS-PAGE, and the high-purity fraction was recovered. The enzyme solution was concentrated to the appropriate concentration by ultrafiltration.

[0113] 3. Measurement of GLOD Activity The reagents used to measure GLOD activity were 4-aminoantipyrine (4-AA) (manufactured by Fujifilm Wako Pure Chemical Industries), TOOS (manufactured by Dojin Chemical Laboratories), and horseradish peroxidase (POD) (manufactured by Toyobo). The reagent compositions for activity measurement are shown in Table 2. For dilution of the GLOD solution, 10 mM PPB (pH 7.4) containing 0.15% bovine serum albumin (BSA, manufactured by Sigma-Aldrich) was used.

[0114]

[0115] After incubating 725 μl of the reagents listed in Table 2 above at 30°C for 5 minutes, 25 μl of 300 mM L-sodium hydrogen glutamate (GluNa, manufactured by Fujifilm Wako Pure Chemical Industries) solution was added and mixed. The absorbance of light at a wavelength of 555 nm was measured using a U-3900 spectrophotometer (manufactured by Hitachi High-Tech Science) with the cell holder incubated at 30°C. ５５５ ) Measure and A per minute ５５５ Change (ΔA) ＳThe following was calculated: Measurements were also performed with 25 μl of deionized water added instead of the substrate solution (GluNa solution), and the A per minute was calculated. ５５５ Change (ΔA) ０ ) was calculated.

[0116] The oxidase activity (U / ml) was calculated based on the following formula. In the formula, "39.2" is the millimolar extinction coefficient (mM) of the quinone imine dye formed by the condensation of 4-AA and TOOS for light at a wavelength of 555 nm. －１ cm －１ ) shows. [Formula] U / ml = (ΔA Ｓ -ΔA ０ )×600×df / (39.2×0.5×V) =30.6×(ΔA Ｓ -ΔA ０ ) × df / V

[0117] 4. Evaluation of Thermal Stability of GLOD Crude GLOD enzyme solution was diluted with 10 mM PPB pH 6.0 to a final concentration of 0.05 U / ml. Subsequently, 240 μl of the 0.05 U / ml GLOD solution was mixed with 160 μl of 250 mM PPB at a predetermined pH, and the mixture was heated in a water bath maintained at a predetermined temperature for 30 minutes. After heating, the GLOD solution was quickly cooled on ice, and the activity was measured using 375 μl of the GLOD solution. The activity of the sample cooled on ice without heating was set to 1, and the residual activity of the heated sample was calculated. The residual activity was calculated three times for each GLOD, and the thermal stability was evaluated using the average value.

[0118] Tables 3 and 4 show the residual activity of the M7GLOD mutant after heating at 70°C for 30 minutes under pH 6.5 conditions. For example, M7GLOD-C210A refers to the C210A mutant of M7GLODΔ49C-T7E.

[0119]

[0120]

[0121] Table 5 shows the residual activity of the GLOD mutant after heating at 60°C for 30 minutes under pH 7.5 conditions.

[0122]

[0123] Table 5-2 shows the residual activity of the StGLOD mutant after heating at 45°C for 30 minutes under pH 6.5 conditions.

[0124]

[0125] The residual activity of various M7GLOD mutants increased by 102% to 176% compared to the residual activity of GLOD before amino acid substitution, indicating improved heat resistance in all GLOD mutants. The residual activity of various StGLOD mutants increased by 146% to 316% compared to the residual activity of GLOD before amino acid substitution, indicating improved heat resistance in all GLOD mutants. It is thought that similar improvements in heat resistance would occur if this amino acid substitution were introduced into GLOD from other sources or into GLOD with high sequence identity. Furthermore, when the prepared GLOD mutants were subjected to gel filtration chromatography, only dimers were obtained, without further polymerization, thus allowing for the acquisition of GLOD with a uniform quaternary structure (uniform molecular weight). This was further confirmed by SDS-PAGE. It is thought that similar results would occur if this amino acid substitution were introduced into GLOD from other sources or into GLOD with high sequence identity.

[0126] 5. Evaluation of GLOD Stability The GLOD purified solution was diluted with 10 mM PPB at pH 6.0 to a final GLOD concentration of 2 U / ml. Subsequently, the prepared GLOD purified solution, 200 mM N-ethylmaleimide (NEM), and 250 mM PPB at a predetermined pH were mixed to prepare solutions with final concentrations of GLOD 0.5 U / mL, NEM 50 mM, and PPB 100 mM, respectively. These solutions were then incubated in an incubator maintained at a predetermined temperature. After heating, the GLOD solution was rapidly cooled on ice, and the activity was measured using 50 μl of the GLOD solution. The residual activity of the sample after heating was calculated by setting the activity of the sample incubated similarly with MilliQ water instead of NEM as 1. For each GLOD, residual activity was calculated three times, and thermal stability was evaluated using the average value.

[0127] Tables 6-1 and 6-2 show the residual activity of the GLOD mutant after heating at 37°C for 120 minutes under pH 7.0 conditions.

[0128]

[0129] The residual activity of various GLOD mutants increased to 103% to 109% compared to the residual activity of GLOD before amino acid substitution, indicating improved stability in all GLOD mutants. It is expected that similar improvements in heat resistance will occur when this amino acid substitution is introduced into GLOD from other sources or into GLOD with high sequence identity.

[0130] [Sequence Listing] Amino acid sequence of Streptomyces sp. MOE7-derived glutamate oxidase (M7GLOD) (Sequence No. 1): MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPAPFTDPAQYAEAGAMRLPSFHPLTLALIDKLGLKRRLFFNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNRTQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFFHSFLGRSDIDPSATYWEIEGGSRQLPEALAKDLRDQIVMGQRMVRLEY YDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPFSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEFSRRWWEFTEEDWKRELDAIAPGLYEYYQRWGEDDAEAALTVPESVRNLPTGLLGAHPSVDEQLIDDEQVEYLRNSTLRGGVRPATQVHG GGSTTDNPNRFMYYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERYGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0131] The amino acid sequence of the deletion-type M7GLOD (M7GLODΔ49C) of Sequence ID No. 2 is MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPAPFTDPAQYAEAGAMRLPSFHPLTLALIDKLGLKRRLFFNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNRTQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFFHSFLGRSDIDPSATYWEIEGGSR QLPEALAKDLRDQIVMGQRMVRLEYYDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPFSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEFSRRWWEFTEEDWKRELDAIAPGLYEYYQDAAEPPATQVHGGGSTTDNPNRFM YYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERYGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0132] Sequence ID 3: Amino acid sequence of glutamate oxidase (StGLOD) derived from Streptomyces sp. X-119-6 MNEMTYEQLARELLLVGPAPTNEDLKLRYLDVLIDNGLNPPGPPKRILIVGAGIAGLVAG DLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPSPFADPAQYAEAGAMRLPSFHPLTLA LIDKLGLKRRLFFNVDIDPQTGNQDAPVPPVFYKSFKDGKTWTNGAPSPEFKEPDKRNHT WIRTNREQVRRAQYATDPSSINEGFHLTGCETRLTVSDMVNQALEPVRDYYSVKQDDGTR VNKPFKEWLAGWADVVRDFDGYSMGRFLREYAEFSDEAVEAIGTIENMTSRLHLAFFHSF LGRSDIDPRATYWEIEGGSRMLPETLAKDLRDQIVMGQRMVRLEYYDPGRDGHHGELTGP GGPAVAIQTVPEGEPYAATQTWTGDLAIVTIPFSSLRFVKVTPPFSYKKRRAVIETHYDQ ATKVLLEFSRRWWEFTEADWKRELDAIAPGLYDYYQQWGEDDAEAALLPQSVRNLPTGL LGAHPSVDESRIGEEQVEYYRNSELRGGVRPATNAYGGGSTTDNPNRFMYYPSHPVPGTQ GGVVLAAYSWSDDAARWDSFDDAERYGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYA CGEAAVYTPHQMTAFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVG DTGVTAAAGRRGAAAATEPMREEALTS

[0133] Sequence ID 4: Amino acid sequence of deletion-type StGLOD MNEMTYEQLARELLLVGPAPTNEDLKLRYLDVLIDNGLNPPGPPKRILIVGAGIAGLVAG DLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPSPFADPAQYAEAGAMRLPSFHPLTLA LIDKLGLKRRLFFNVDIDPQTGNQDAPVPPVFYKSFKDGKTWTNGAPSPEFKEPDKRNHT WIRTNREQVRRAQYATDPSSINEGFHLTGCETRLTVSDMVNQALEPVRDYYSVKQDDGTR VNKPFKEWLAGWADVVRDFDGYSMGRFLREYAEFSDEAVEAIGTIENMTSRLHLAFFHSF LGRSDIDPRATYWEIEGGSRMLPETLAKDLRDQIVMGQRMVRLEYYDPGRDGHHGELTGP GGPAVAIQTVPEGEPYAATQTWTGDLAIVTIPFSSLRFVKVTPPFSYKKRRAVIETHYDQ ATKVLLEFSRRWWEFTEADWKRELDAIAPGLYDYYQQWGEDDAEAAGVRPATNAYGGGS TTDNPNRFMYYPSHPVPGTQGGVVLAAYSWSDDAARWDSFDDAERYGYALENLQSVHGRR IEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTAFHLDVVRPEGPVYFAGEHVSLKHAWI EGAVETAVRAAIAVNEAPVGDTGVTAAAG

[0134] The amino acid sequence of Sequence ID No. 5, M7GLODΔ49C-T7 / R186E, is MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTYHAKKGEPAPFTDPAQIAEAGAMRLPSFHPLTLALIDKLGLKRRLFYNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNETQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFMHSFLGRSDIDPSATYWEIEGGSR QLPEALAKDLRDQIVMGQRMVRLEYYDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPLSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEYSRRWWEFTEEDWKRELDAIAPGLYEYYQDAAEPPATQVHGGGSTTDNPNRFM YYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERFGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0135] Sequence IDs 6-16: Forward Primer Sequence ID 6 CACCTCACCGGCgccGAATCGCGGCTGACC Sequence ID 7 CACCTCACCGGCgacGAATCGCGGCTGACC Sequence ID 8 CACCTCACCGGCgaaGAATCGCGGCTGACC Sequence ID 9 CACCTCACCGGCaagGAATCGCGGCTGACC Sequence ID 10 CACCTCACCGGCcgcGAATCGCGGCTGACC Sequence ID 11 CACCTCACCGGCccgGAATCGCGGCTGACC Sequence ID 12 CACCTCACCGGCaccGAATCGCGGCTGACC Sequence ID 13 CACCTCACCGGCcacGAATCGCGGCTGACC Sequence ID 14 CACCTCACCGGCaacGAATCGCGGCTGACC Sequence ID 15 CACCTCACCGGCcagGAATCGCGGCTGACC Sequence ID 16 CACCTCACCGGCggcGAATCGCGGCTGACC Sequence ID 17: Reverse Primer Sequence ID 17 GCCGGTGAGGTGGAAGCCCTC

[0136] Sequence IDs 18-25: Forward primers (additional for M7GLOD mutation) Sequence ID 18: CACCTCACCGGCttcGAATCGCGGCTGACC Sequence ID 19: CACCTCACCGGCctgGAATCGCGGCTGACC Sequence ID 20: CACCTCACCGGCatcGAATCGCGGCTGACC Sequence ID 21: CACCTCACCGGCatgGAATCGCGGCTGACC Sequence ID 22: CACCTCACCGGCtacGAATCGCGGCTGACC Sequence ID 23: CACCTCACCGGCtggGAATCGCGGCTGACC Sequence ID 24: CACCTCACCGGCagcGAATCGCGGCTGACC Sequence ID 25: CACCTCACCGGCgtcGAATCGCGGCTGACC

[0137] Sequence IDs 26-35: Forward primers (for StGLOD mutations) Sequence ID 26: CATCTGACGGGAgccGAGACCCGCCTTACTGTCTC Sequence ID 27: CATCTGACGGGAgaaGAGACCCGCCTTACTGTCTC Sequence ID 28: CATCTGACGGGAccgGAGACCCGCCTTACTGTCTC Sequence ID 29: CATCTGACGGGAacgGAGACCCGCCTTACTGTCTC Sequence ID 30: CATCTGACGGGAcacGAGACCCGCCTTACTGTCTC Sequence ID 31: CATCTGACGGGAaacGAGACCCGCCTTACTGTCTC Sequence ID 32: CATCTGACGGGAcagGAGACCCGCCTTACTGTCTC Sequence ID 33: CATCTGACGGGAgatGAGACCCGCCTTACTGTCTC Sequence ID 34: CATCTGACGGGAaaaGAGACCCGCCTTACTGTCTC Sequence ID 35: CATCTGACGGGAcgcGAGACCCGCCTTACTGTCTC

[0138] Sequence ID 36: Reverse Primer (for StGLOD mutations) Sequence ID 36 TCCCGTCAGATGGAAGCCTTC

Claims

1. An L-glutamate oxidase mutant having an amino acid substitution at position 210 of SEQ ID NO: 1, wherein the substituted amino acid at that position is an amino acid residue other than cysteine.

2. The L-glutamate oxidase variant according to claim 1, which has improved heat resistance compared to L-glutamate oxidase that does not have an amino acid substitution at the position corresponding to position 210 of SEQ ID NO:

1.

3. The L-glutamate oxidase variant according to claim 1, wherein the amino acid sequence before introducing an amino acid substitution at the position corresponding to position 210 of SEQ ID NO: 1 has 70% or more, 80% or more, or 90% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, or 3.

4. A composition, reagent, electrode, sensor, or kit comprising the L-glutamate oxidase variant described in any one of claims 1 to 3.

5. A polynucleotide encoding an L-glutamate oxidase variant according to any one of claims 1 to 3.

6. A vector comprising the polynucleotide described in claim 5.

7. A host cell comprising the vector according to claim 6.

8. A method for producing an L-glutamate oxidase mutant, comprising the steps of: culturing the host cells described in claim 7 to produce the L-glutamate oxidase mutant described in any one of claims 1 to 3; and obtaining the produced L-glutamate oxidase mutant.

9. A method for oxidizing L-glutamic acid contained in a sample by contacting an L-glutamic acid oxidase variant according to any one of claims 1 to 3, or a composition, reagent, electrode, sensor, or kit according to claim 4, with a sample containing L-glutamic acid.

10. The method according to claim 9, for detecting L-glutamic acid.

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