Recombinant expression glutamate oxidase

Abstract

This invention produces glutamate oxidase that does not need to be treated with a protease.　Provided is a glutamate oxidase mutant obtained by deleting a designated amino acid sequence.

Description

Recombinantly expressed glutamate oxidase

[0001] The present invention relates to recombinantly expressed glutamate oxidase.

[0002] L-glutamic acid was traditionally measured using L-glutamate decarboxylase and L-glutamate dehydrogenase. However, both methods were complicated because the measurement of carbon dioxide in the decarboxylase measurement system was time-consuming, and the measurement of the change in absorbance of the coenzyme in the dehydrogenase measurement system required measurement of the coenzyme.

[0003] Subsequently, an L-glutamate oxidase that acts only on L-glutamic acid was reported by solid culture of actinomycetes (Non-Patent Document 1). L-glutamate oxidase is encoded as a single polypeptide having an α chain, a γ chain, and a β chain, and the two polypeptides form a homodimer. This homodimer is cleaved by a protease to produce the mature form. The mature form is α ２ β ２ gamma ２ It is a heterohexameric structure composed of

[0004] The recombinantly expressed homodimer exhibited weak L-glutamate oxidase activity, inferior substrate affinity, and thermal instability compared to the original actinomycete L-glutamate oxidase. When this recombinantly expressed homodimeric enzyme was treated with a protease, it acquired the same structure as the original actinomycete L-glutamate oxidase and exhibited equivalent enzymatic properties (Non-Patent Document 2). A method in which this precursor is mass-produced using recombinant Escherichia coli and then treated with a protease is used industrially. Patent Document 1 describes L-glutamate oxidase mutants. Patent Document 1 does not disclose the thermostability of the L-glutamate oxidase mutants that have been produced.

[0005] International Publication No. 2021 / 193598 Pamphlet

[0006] Kusakabe H et al. , Agric. Biol. Chem. , 47, 1323-1328 (1983) Arima J et al. , J. Biochem. , 134, 805-812 (2003)

[0007] In certain embodiments, the present disclosure aims to provide a method for conveniently producing L-glutamate oxidase. Also, in certain embodiments, the present disclosure aims to provide an L-glutamate oxidase. Also, in certain embodiments, the present disclosure aims to provide an L-glutamate oxidase having high sequence identity to SEQ ID NO:58.

[0008] When recombinantly expressing L-glutamate oxidase, particularly when expressing L-glutamate oxidase on a large scale for industrial use, conventional industrial production methods have required treatment with protease to convert the L-glutamate oxidase to its mature form. However, the protease treatment step is complicated and results in high costs.

[0009] As a result of extensive research aimed at solving the above problems, the present inventors have found that, as an example, the above problems can be at least partially solved by using a genetically engineered glutamate oxidase derived from actinomycetes, and have completed the present invention, which includes this finding as one embodiment. Furthermore, the present inventors have found that the above problems can be at least partially solved by using a glutamate oxidase having a specific mutation, and have completed the present invention, which includes this finding as one embodiment.

[0010] The present disclosure provides the following embodiments: [1] A glutamate oxidase mutant having a deletion region, wherein all or part of the γ-β region between the γ-chain region and the β-chain region of wild-type glutamate oxidase is deleted, wherein 1 to 10 amino acids are deleted or not deleted from the carboxy-terminal side of the γ-chain region of the wild-type glutamate oxidase, and wherein 1 to 4 amino acids are deleted or not deleted from the amino-terminal side of the β-chain region of the wild-type glutamate oxidase, the deleted region is a single contiguous deleted region, and the glutamate oxidase mutant has glutamate oxidase activity. [2] The glutamate oxidase mutant according to embodiment 1, wherein 31 to 54 contiguous amino acids are deleted. [3] The glutamate oxidase mutant according to embodiment 1, wherein the C-terminus of the deleted region is at the position corresponding to position 510, 508, or 507 of SEQ ID NO: 1. [4] The mutant according to embodiment 1, which lacks the amino acid sequence of the region corresponding to positions 459 to 507 of SEQ ID NO: 1, and has improved thermostability compared to glutamate oxidase in which all or part of the region corresponding to positions 459 to 507 of SEQ ID NO: 1 is not deleted. [5] The mutant according to embodiment 4, which lacks the amino acid sequence of the region corresponding to positions 459 to 507 of SEQ ID NO: 1, and has improved thermostability compared to glutamate oxidase in which the region corresponding to positions 459 to 466 of SEQ ID NO: 1 is not deleted. [6] The mutant according to embodiment 4, wherein the improved thermal stability is evaluated based on the residual activity after heat treatment at 45°C for 30 or 35 minutes, or at 65°C for 30 minutes, and the residual activity of the mutant is 110% or more when the residual activity of glutamate oxidase without the amino acid sequence deletion after heat treatment at 45°C for 30 or 35 minutes, or at 65°C for 30 minutes, is taken as 100%; or the mutant according to embodiment 5, wherein the residual activity of the mutant is 110% or more when the residual activity of glutamate oxidase without the amino acid sequence deletion after heat treatment at 45°C for 30 or 35 minutes, or at 65°C for 30 minutes, is taken as 100%.[7] A mutant according to any one of embodiments 1 to 6, which has an amino acid sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 12, or SEQ ID NO: 13, lacks the amino acid sequence, and has glutamate oxidase activity. [8] The variant of any one of embodiments 1 to 7, further comprising an amino acid substitution compared to the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 58 at one or more positions selected from the group consisting of: a position corresponding to position 87 of SEQ ID NO: 1, a position corresponding to position 103 of SEQ ID NO: 1, a position corresponding to position 133 of SEQ ID NO: 1, a position corresponding to position 186 of SEQ ID NO: 1, a position corresponding to position 297 of SEQ ID NO: 1, a position corresponding to position 376 of SEQ ID NO: 1, a position corresponding to position 393 of SEQ ID NO: 1, a position corresponding to position 428 of SEQ ID NO: 1, a position corresponding to position 516 of SEQ ID NO: 1, a position corresponding to position 566 of SEQ ID NO: 1, a position corresponding to position 568 of SEQ ID NO: 1, a position corresponding to position 585 of SEQ ID NO: 1, and a position corresponding to position 615 of SEQ ID NO: 1, wherein the variant has improved thermostability after amino acid substitution compared to glutamate oxidase before substitution.[9] The amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 1 is tyrosine; The amino acid substitution at the position corresponding to position 103 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine; The amino acid substitution at the position corresponding to position 133 of SEQ ID NO: 1 is selected from the group consisting of leucine and tyrosine; The amino acid substitution at the position corresponding to position 186 of SEQ ID NO: 1 is selected from the group consisting of glutamic acid, aspartic acid, tyrosine, glutamine, asparagine, alanine, leucine, cysteine, methionine, phenylalanine, serine, histidine, and threonine; The amino acid substitution at the position corresponding to position 297 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, isoleucine, and methionine; The amino acid substitution at the position corresponding to position 376 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine. 1 is selected from the group consisting of tyrosine and methionine; the amino acid substitution at position corresponding to position 428 of SEQ ID NO:1 is selected from the group consisting of tyrosine and methionine; the amino acid substitution at position corresponding to position 516 of SEQ ID NO:1 is phenylalanine; the amino acid substitution at position corresponding to position 566 of SEQ ID NO:1 is selected from the group consisting of phenylalanine, leucine, valine and methionine; the amino acid substitution at position corresponding to position 568 of SEQ ID NO:1 is selected from the group consisting of isoleucine and methionine; the amino acid substitution at position corresponding to position 585 of SEQ ID NO:1 is selected from the group consisting of leucine and methionine; or the amino acid substitution at position corresponding to position 615 of SEQ ID NO:1 is selected from the group consisting of leucine, valine and phenylalanine.

[10] The variant of embodiment 9, which has 90% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:69, and which lacks an amino acid sequence, and which has an amino acid substitution compared to the amino acid sequence of SEQ ID NO:58 at one or more positions selected from the group consisting of: the position corresponding to position 87 of SEQ ID NO:1, the position corresponding to position 103 of SEQ ID NO:1, the position corresponding to position 133 of SEQ ID NO:1, the position corresponding to position 186 of SEQ ID NO:1, the position corresponding to position 297 of SEQ ID NO:1, the position corresponding to position 376 of SEQ ID NO:1, the position corresponding to position 393 of SEQ ID NO:1, the position corresponding to position 428 of SEQ ID NO:1, the position corresponding to position 516 of SEQ ID NO:1, the position corresponding to position 566 of SEQ ID NO:1, the position corresponding to position 568 of SEQ ID NO:1, the position corresponding to position 585 of SEQ ID NO:1, and the position corresponding to position 615 of SEQ ID NO:1, wherein the amino acid substitution is any of the amino acid substitutions of embodiment 9, and which has glutamate oxidase activity.

[11] The glutamate oxidase variant according to any one of embodiments 1 to 10, further lacking a region of the amino acid sequence corresponding to positions 670 to 687 of SEQ ID NO: 1.

[12] A composition, reagent, electrode, sensor, or kit comprising the glutamate oxidase variant according to any one of embodiments 1 to 10.

[13] A polynucleotide encoding the glutamate oxidase variant according to any one of embodiments 1 to 11.

[14] A vector comprising the polynucleotide according to embodiment 13.

[15] A host cell comprising the vector according to embodiment 14.

[16] A method for producing a glutamate oxidase variant, comprising the steps of: culturing the host cell according to embodiment 15 to produce the glutamate oxidase variant according to any one of embodiments 1 to 11; and obtaining the produced glutamate oxidase variant.

[17] A method for oxidizing glutamic acid contained in a sample by contacting the glutamate oxidase variant according to any one of embodiments 1 to 11, or the composition, reagent, electrode, sensor, or kit according to embodiment 12, with the sample.

[18] The method according to embodiment 17, in which glutamic acid is detected.

[19] A glutamate oxidase mutant comprising an amino acid substitution, wherein the glutamate oxidase mutant after the amino acid substitution has improved thermostability compared to the glutamate oxidase before the substitution, (i) when aligned with the amino acid sequence of SEQ ID NO: 1, an amino acid substitution has been performed at a position corresponding to a position selected from the group consisting of positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1; (ii) in the amino acid sequence of (i), one or more amino acids have been substituted, deleted, or added at a position other than positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1; (iii) (i) or (ii) above, wherein the full-length amino acid sequence of the glutamate oxidase variant 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 or SEQ ID NO: 58; or (iv) (i) or (ii) above, wherein the full-length amino acid sequence of the glutamate oxidase variant 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 or SEQ ID NO: 58, and wherein the amino acid at position 291 of SEQ ID NO: 1 is arginine and the amino acid sequence at positions 51 to 56 of SEQ ID NO: 1 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid).

[20] The amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 1 is tyrosine; The amino acid substitution at the position corresponding to position 103 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine; The amino acid substitution at the position corresponding to position 133 of SEQ ID NO: 1 is selected from the group consisting of leucine and tyrosine; The amino acid substitution at the position corresponding to position 186 of SEQ ID NO: 1 is selected from the group consisting of glutamic acid, aspartic acid, tyrosine, asparagine, glutamine, alanine, leucine, cysteine, methionine, phenylalanine, serine, histidine, and threonine; The amino acid substitution at the position corresponding to position 297 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, isoleucine, and methionine; The amino acid substitution at the position corresponding to position 376 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine. 20. The glutamate oxidase variant according to embodiment 19, wherein the amino acid substitution at position 393 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, isoleucine, and methionine, the amino acid substitution at position 428 of SEQ ID NO: 1 is selected from the group consisting of tyrosine and methionine, the amino acid substitution at position 516 of SEQ ID NO: 1 is phenylalanine, the amino acid substitution at position 566 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, valine, and methionine, the amino acid substitution at position 568 of SEQ ID NO: 1 is selected from the group consisting of isoleucine and methionine, the amino acid substitution at position 585 of SEQ ID NO: 1 is selected from the group consisting of leucine and methionine, or the amino acid substitution at position 615 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, and phenylalanine. This specification incorporates the disclosure of Japanese Patent Application No. 2022-005869, from which the present application claims priority.

[0011] In certain embodiments, the present invention provides a recombinantly expressed glutamate oxidase that does not require treatment with a protease, hi certain embodiments, a glutamate oxidase with improved thermostability.

[0012] L-glutamate oxidase is encoded by the GLOD gene as a single polypeptide having an α chain, a γ chain, and a β chain, and the two polypeptides form a homodimer. In nature, this homodimer is cleaved by a protease to form the mature form. The mature form is α ２ β ２ gamma ２ It is a heterohexameric structure composed of

[0013] In this specification, unless otherwise specified, GLOD refers to L-glutamate oxidase. L-glutamate oxidase is an oxidoreductase classified in EC 1.4.3.11, and catalyzes the following chemical reaction: [Chemical Formula 1] L-glutamic acid + O ２ +H ２ O → 2-oxoglutaric acid + NH ３ +H ２ O ２

[0014] 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, such as a Gly-Xaa-Gly-Xaa-Xaa-Gly motif (where Xaa represents any amino acid). For example, when SEQ ID NO: 1 is used as a reference sequence, the amino acid sequence "Gly-Ala-Gly-Ile-Ala-Gly" from positions 51 to 56 of SEQ ID NO: 1 corresponds to the FAD-binding motif sequence Gly-Xaa-Gly-Xaa-Xaa-Gly. In certain embodiments, with respect to the variants of the present disclosure, the Gly at positions 51, 53, and 56, respectively, based on SEQ ID NO: 1, may not be substituted. In certain embodiments, the Gly at positions 51, 53, and 56 of SEQ ID NO: 1 in L-glutamate oxidase are not substituted.

[0015] GLOD is known to recognize its substrate glutamic acid through an arginine residue. For example, with reference to SEQ ID NO: 1, Arg (R) at position 291 is an important position for glutamic acid recognition. In some embodiments, with respect to the variants of the present disclosure, no amino acid substitution is required at the position corresponding to position 291 of SEQ ID NO: 1. In some embodiments, with respect to the variants of the present disclosure, if the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is Arg (R), then this amino acid is not substituted.

[0016] (Reference Sequence) For convenience, in this specification, each position of GLOD is defined using SEQ ID NO: 1 as the reference sequence. SEQ ID NO: 1 is the amino acid sequence of glutamate oxidase (StGLOD) derived from Streptomyces sp. X-119-6, except that the N-terminal secretory signal sequence (MTTDTARRHTGAER) in the native sequence has been removed. Furthermore, in SEQ ID NO: 1, the first amino acid in the α-chain region immediately after 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: 1.

[0017] (Regarding Each Subunit of GLOD) For convenience, SEQ ID NO: 1 is used as the reference sequence, and the α-chain of GLOD is defined as a polypeptide consisting of 375 amino acid residues from positions 1 to 375 (ANEM...EGEP). For convenience, SEQ ID NO: 1 is used as the reference sequence, and the γ-chain of GLOD is defined as a polypeptide consisting of 91 amino acids from positions 376 to 466 (YAAT...AEAA). For convenience, SEQ ID NO: 1 is used as the reference sequence, and the β-chain of GLOD is defined as a polypeptide consisting of 163 amino acids from positions 507 to 669. For convenience, SEQ ID NO: 1 is used as the reference sequence, and the region from positions 467 to 506 is naturally removed by proteases (LALP...SELR). For convenience, for SEQ ID NO: 1 is used as the reference sequence, and the C-terminal sequence from positions 670 to 687 (RRGAAAATEPMREALTS) is also a region removed by proteases.

[0018] (GLOD Mutant with Deletion of Amino Acid Sequence) The inventors suspected that a region of the GLOD amino acid sequence that can be removed by proteases might be preventing the functional expression of GLOD. They therefore constructed a GLOD mutant sequence in which the amino acid sequence between the γ region and the β region was deleted, and expressed the GLOD mutant. When deleting the region that can be removed by proteases, in one embodiment, the last eight amino acids (GEDDAEAA) of the γ region were also deleted from the mutant. Furthermore, the first amino acid residue, G, of the β chain was also deleted from the mutant. Therefore, the boundary between the γ chain region and the β chain region of the constructed mutant was a sequence of γ chain region...QQW-GVRP...β chain region, linking the C-terminal QQW of the γ chain and the N-terminal GVRP of the β chain. As a result, a mutant exhibiting GLOD activity was obtained. This GLOD mutant with deletion of amino acid sequence does not require protease treatment to express its activity. In certain embodiments, when the amino acid sequence deleted GLOD variant is recombinantly expressed, it is not treated with a protease.

[0019] The inventors further continued their development and created linkage region-substituted mutants in which the border sequence (γ-chain region...QQW-GVRP...β-chain region) was replaced with various amino acid sequences. Specifically, as mutants in which the border sequence (γ-chain region...QQW-GVRP...β-chain region) was modified, mutant M7GLODΔ49C having the amino acid sequence of SEQ ID NO:59, mutant StGLODΔ49Ai having the amino acid sequence of SEQ ID NO:12, and mutant StGLODΔ49C having the amino acid sequence of SEQ ID NO:13 were created, recombinantly expressed, and the activity of the mutants was confirmed. Surprisingly, all of the mutants exhibited GLOD activity. These amino acid sequence-deleted GLOD mutants also do not require protease treatment to express activity.

[0020] The mutant M7GLODΔ49C having the amino acid sequence of SEQ ID NO:59 can also be considered to be obtained by deleting 56 amino acids from positions 456 to 511 of M7GLOD having the amino acid sequence of SEQ ID NO:58, and inserting 7 amino acids (QDAAEPP) different from the sequence of M7GLOD in those positions. In other words, M7GLODΔ49C has deleted positions 456 to 504 of SEQ ID NO:58, and substituted positions 505 to 511 with QDAAEPP. Alternatively, M7GLODΔ49C has deleted positions 457 to 505 of SEQ ID NO:58, substituted positions 456 with Q, and substituted positions 506 to 511 with DAAEPP. Alternatively, M7GLODΔ49C has a deletion of positions 458 to 506 of SEQ ID NO: 58, substitutions of positions 456 to 457 with QD, and substitutions of positions 507 to 511 with AAEPP. Alternatively, M7GLODΔ49C has a deletion of positions 459 to 507 of SEQ ID NO: 58, substitutions of positions 456 to 458 with QDA, and substitutions of positions 508 to 511 with AEPP. Alternatively, M7GLODΔ49C has a deletion of positions 460 to 508 of SEQ ID NO: 58, substitutions of positions 456 to 459 with QDAA, and substitutions of positions 509 to 511 with EPP. Alternatively, M7GLODΔ49C has a deletion of positions 461 to 509 of SEQ ID NO:58, a substitution of positions 456 to 460 with QDAAE, and a substitution of positions 510 to 511 with PP. Alternatively, M7GLODΔ49C has a deletion of positions 462 to 510 of SEQ ID NO:58, a substitution of positions 456 to 461 with QDAAEP, and a substitution of position 511 with P. Alternatively, M7GLODΔ49C has a deletion of positions 462 to 510 of SEQ ID NO:58, and a substitution of positions 456 to 462 with QDAAEPP.

[0021] The mutant StGLODΔ49Ai having the amino acid sequence of SEQ ID NO: 12 can also be considered to have 56 amino acids from positions 456 to 511 of StGLOD having the amino acid sequence of SEQ ID NO: 1 deleted, and 7 amino acids (RKLDKTE) different from the StGLOD sequence inserted at those positions. In other words, StGLODΔ49Ai has positions 456 to 504 of SEQ ID NO: 1 deleted, and positions 505 to 511 replaced with RKLDKTE. Alternatively, StGLODΔ49Ai has positions 457 to 505 of SEQ ID NO: 1 deleted, with position 456 replaced with R and positions 506 to 511 replaced with KLDKTE. Alternatively, StGLODΔ49Ai has a deletion of positions 458 to 506 of SEQ ID NO: 1, a substitution of positions 456 to 457 with RK, and a substitution of positions 507 to 511 with LDKTE. Alternatively, StGLODΔ49Ai has a deletion of positions 459 to 507 of SEQ ID NO: 1, a substitution of positions 456 to 458 with RKL, and a substitution of positions 508 to 511 with DKTE. Alternatively, StGLODΔ49Ai has a deletion of positions 460 to 508 of SEQ ID NO: 1, a substitution of positions 456 to 459 with RKLD, and a substitution of positions 509 to 511 with KTE. Alternatively, StGLODΔ49Ai has a deletion of positions 461 to 509 of SEQ ID NO: 1, a substitution of positions 456 to 460 with RKLDK, and a substitution of positions 510 to 511 with TE. Alternatively, StGLODΔ49Ai has a deletion of positions 462 to 510 of SEQ ID NO: 1, a substitution of positions 456 to 461 with RKLDKT, and a substitution of position 511 with E. Alternatively, StGLODΔ49Ai has a deletion of positions 462 to 510 of SEQ ID NO: 1, and a substitution of positions 456 to 462 with RKLDKTE.

[0022] The mutant StGLODΔ49C having the amino acid sequence of SEQ ID NO: 13 can also be considered to be a mutant in which 56 amino acids from positions 456 to 511 of StGLOD having the amino acid sequence of SEQ ID NO: 1 have been deleted, and 7 amino acids (QDAAEPP) different from the StGLOD sequence have been inserted in those positions. In other words, StGLODΔ49C has deleted positions 456 to 504 of SEQ ID NO: 1, and substituted positions 505 to 511 with QDAAEPP. Alternatively, StGLODΔ49C has deleted positions 457 to 505 of SEQ ID NO: 1, substituted positions 456 with Q, and substituted positions 506 to 511 with DAAEPP. Alternatively, StGLODΔ49C has a deletion of positions 458 to 506 of SEQ ID NO: 1, a substitution of positions 456 to 457 with QD, and a substitution of positions 507 to 511 with AAEPP. Alternatively, StGLODΔ49C has a deletion of positions 459 to 507 of SEQ ID NO: 1, a substitution of positions 456 to 458 with QDA, and a substitution of positions 508 to 511 with AEPP. Alternatively, StGLODΔ49C has a deletion of positions 460 to 508 of SEQ ID NO: 1, a substitution of positions 456 to 459 with QDAA, and a substitution of positions 509 to 511 with EPP. Alternatively, StGLODΔ49C has a deletion of positions 461 to 509 of SEQ ID NO: 1, a substitution of positions 456 to 460 with QDAAE, and a substitution of positions 510 to 511 with PP. Alternatively, StGLODΔ49C has a deletion of positions 462 to 510 of SEQ ID NO: 1, a substitution of positions 456 to 461 with QDAAEP, and a substitution of position 511 with P. Alternatively, StGLODΔ49C has a deletion of positions 462 to 510 of SEQ ID NO: 1, and a substitution of positions 456 to 462 with QDAAEPP.

[0023] The inventors continued their development and created several mutants by varying the length of the deleted region. In one embodiment, the present disclosure provides a glutamate oxidase mutant having a deleted region, wherein all or part of the γ-β interregion between the γ-chain region and the β-chain region of wild-type glutamate oxidase is deleted, 1 to 10 amino acids are deleted or not deleted on the carboxy-terminal side of the γ-chain region of the wild-type glutamate oxidase, and 1 to 4 amino acids are deleted or not deleted on the amino-terminal side of the β-chain region of the wild-type glutamate oxidase, the deleted region is a single contiguous deleted region, and the mutant has glutamate oxidase activity.

[0024] Furthermore, the present inventors continued their development and created many mutants with different lengths of deleted regions. In some embodiments, the deletion mutants include those having a deletion at positions 457 to 510 of SEQ ID NO:1 (dΔ54), a deletion at positions 458 to 510 of SEQ ID NO:1 (dΔ53), a deletion at positions 459 to 510 of SEQ ID NO:1 (dΔ52), a deletion at positions 460 to 510 of SEQ ID NO:1 (dΔ51), a deletion at positions 461 to 510 of SEQ ID NO:1 (dΔ50), a deletion at positions 462 to 510 of SEQ ID NO:1 (dΔ49), a deletion at positions 463 to 510 of SEQ ID NO:1 (dΔ48), a deletion at positions 464 to 510 of SEQ ID NO:1 (dΔ47), a deletion at positions 465 to 510 of SEQ ID NO:1 (dΔ46), Deletion at positions 466 to 510 of SEQ ID NO:1 (dΔ45), Deletion at positions 467 to 510 of SEQ ID NO:1 (dΔ44), Deletion at positions 468 to 510 of SEQ ID NO:1 (dΔ43), Deletion at positions 469 to 510 of SEQ ID NO:1 (dΔ42), Deletion at positions 470 to 510 of SEQ ID NO:1 (dΔ41), Deletion at positions 471 to 510 of SEQ ID NO:1 (dΔ40), Deletion at positions 472 to 510 of SEQ ID NO:1 (dΔ39), Deletion at positions 473 to 510 of SEQ ID NO:1 (dΔ38), Deletion at positions 474 to 510 of SEQ ID NO:1 (dΔ37), A deletion occurs at positions 475 to 510 of SEQ ID NO:1 (dΔ36), a deletion occurs at positions 476 to 510 of SEQ ID NO:1 (dΔ35), a deletion occurs at positions 477 to 510 of SEQ ID NO:1 (dΔ34), a deletion occurs at positions 478 to 510 of SEQ ID NO:1 (dΔ33), a deletion occurs at positions 479 to 510 of SEQ ID NO:1 (dΔ32), or a deletion occurs at positions 480 to 510 of SEQ ID NO:1 (dΔ31).

[0025] In another embodiment, the deletion mutants are selected from the group consisting of: a deletion at positions 455 to 508 of SEQ ID NO:1 (DΔ54), a deletion at positions 456 to 508 of SEQ ID NO:1 (DΔ53), a deletion at positions 457 to 508 of SEQ ID NO:1 (DΔ52), a deletion at positions 458 to 508 of SEQ ID NO:1 (DΔ51), a deletion at positions 459 to 508 of SEQ ID NO:1 (DΔ50), a deletion at positions 460 to 508 of SEQ ID NO:1 (DΔ49), a deletion at positions 461 to 508 of SEQ ID NO:1 (DΔ48), a deletion at positions 462 to 508 of SEQ ID NO:1 (DΔ47), a deletion at positions 463 to 508 of SEQ ID NO:1 (DΔ46), Deletion of positions corresponding to positions 464 to 508 of SEQ ID NO: 1 (DΔ45), Deletion of positions corresponding to positions 465 to 508 of SEQ ID NO: 1 (DΔ44), Deletion of positions corresponding to positions 466 to 508 of SEQ ID NO: 1 (DΔ43), Deletion of positions corresponding to positions 467 to 508 of SEQ ID NO: 1 (DΔ42), Deletion of positions corresponding to positions 468 to 508 of SEQ ID NO: 1 (DΔ41), Deletion of positions corresponding to positions 469 to 508 of SEQ ID NO: 1 (DΔ40), Deletion of positions corresponding to positions 470 to 508 of SEQ ID NO: 1 (DΔ39), Deletion of positions corresponding to positions 471 to 508 of SEQ ID NO: 1 (DΔ38), Deletion of positions corresponding to positions 472 to 508 of SEQ ID NO: 1 (DΔ37), A deletion occurs at positions corresponding to positions 473 to 508 of SEQ ID NO:1 (DΔ36), a deletion occurs at positions corresponding to positions 474 to 508 of SEQ ID NO:1 (DΔ35), a deletion occurs at positions corresponding to positions 475 to 508 of SEQ ID NO:1 (DΔ34), a deletion occurs at positions corresponding to positions 476 to 508 of SEQ ID NO:1 (DΔ33), a deletion occurs at positions corresponding to positions 477 to 508 of SEQ ID NO:1 (DΔ32), or a deletion occurs at positions corresponding to positions 478 to 508 of SEQ ID NO:1 (DΔ31).

[0026] In one embodiment, the present disclosure provides a mutant in which RWGEDDAEAALTVPESVRNLPTGLLGAHPSVDEQLIDDEQVEYLRNSTLRGGVR is deleted based on SEQ ID NO: 58. For convenience in the present specification, this deletion mutant may be referred to as M7GLOD-dΔ54. In one embodiment, the present disclosure provides mutants in which the following deletion regions, which vary in chain length, are deleted based on SEQ ID NO: 58:

[0027] In another embodiment, the present disclosure provides mutants in which the following deletion regions with different chain lengths are deleted based on SEQ ID NO:58:

[0028] In one embodiment, the present disclosure provides mutants in which the following deletion regions with different chain lengths are deleted based on SEQ ID NO: 1:

[0029] In another embodiment, the present disclosure provides mutants in which the following deletion regions with different chain lengths are deleted based on SEQ ID NO: 1:

[0030] The above mutants are intended to be encompassed in the glutamate oxidase mutants lacking 31 to 54 consecutive amino acids as referred to herein.

[0031] In one embodiment, the C-terminus of the deleted region may be a position corresponding to position 512, 511, 510, 509, 508, 507, 506, or 505 of SEQ ID NO: 1. In one embodiment, the C-terminus of the deleted region may be a position corresponding to position 510, 508, or 507 of SEQ ID NO: 1. For convenience, the C-terminus of the deleted region may be referred to herein as the starting point. In one embodiment, the deletion mutant may have 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 consecutive amino acids deleted from the starting point. Because the starting point is deleted, the glutamate oxidase deletion mutant does not have a starting point. Furthermore, the glutamate oxidase deletion mutant does not have consecutive amino acids 31 to 54 from the starting site (ie, does not have amino acids 31 to 54 including the starting amino acid).

[0032] Without wishing to be bound by any particular theory, it is believed that in nature, the γ-β region of glutamate oxidase is excised by a protease. Therefore, it is believed that even if the excised region is deleted in advance from recombinantly expressed glutamate oxidase, an active glutamate oxidase will still be obtained. In this case, since the excised region is not present in the mature protein, it is unlikely that the excised region plays a role in the active protein. Therefore, if activity is confirmed for both long-deleted and short-deleted deletion mutants of the amino acid sequence of the excised region (e.g., activity is confirmed for the Δ54 and Δ31 mutants), it is reasonably likely that mutants with intermediate deletion lengths (e.g., Δ53-Δ32 mutants) will also exhibit similar activity.

[0033] The mutants were constructed and confirmed to have GLOD activity. Based on the disclosures herein, those skilled in the art can construct similar deletion mutants and confirm the presence or absence of their activity through routine testing. Deletion mutants that have no activity are excluded from glutamate oxidase mutants that have glutamate oxidase activity.

[0034] In certain embodiments, the present disclosure provides a GLOD mutant having a deletion in the amino acid sequence, the deletion corresponding to positions 459 to 507 of SEQ ID NO: 1, and having glutamate oxidase activity. In certain embodiments, the mutant may further lack a deletion corresponding to positions 670 to 687.

[0035] In certain embodiments, the present disclosure provides a GLOD mutant lacking an amino acid sequence deletion, which mutant lacks a region corresponding to positions 459 to 507 of SEQ ID NO: 1, has glutamate oxidase activity, and exhibits improved thermostability compared to a glutamate oxidase that does not lack all or part of the region corresponding to positions 459 to 507 of SEQ ID NO: 1. In certain embodiments, the mutant may further lack a region corresponding to positions 670 to 687.

[0036] In certain embodiments, the present disclosure provides a deleted GLOD mutant, which lacks a region corresponding to positions 459 to 507 of SEQ ID NO: 1, and which has glutamate oxidase activity and improved thermostability compared to a glutamate oxidase that does not lack the region corresponding to positions 459 to 466 of SEQ ID NO: 1. In certain embodiments, the mutant may further lack a region corresponding to positions 670 to 687.

[0037] In some embodiments, deletion mutants may be made based on SEQ ID NO: 58. In some embodiments, deletion mutants may be made based on glutamate oxidases that have 90% or more sequence identity to SEQ ID NO: 58, such as 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.

[0038] In another embodiment, deletion mutants may be made based on SEQ ID NO: 1. In certain embodiments, deletion mutants may be made based on glutamate oxidases that have 90% or more sequence identity to SEQ ID NO: 1, such as 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.

[0039] In another embodiment, deletion mutants may be generated based on known glutamate oxidases or glutamate oxidases having 90% or more, for example 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 thereto.

[0040] (Effect of improving thermal stability) Furthermore, when these amino acid sequence-deleted GLOD mutants were heat-treated, it was surprisingly found that the residual activity of each mutant was improved compared to the corresponding GLOD sequence before the amino acid sequence was deleted. The residual activity indicates the activity value of the GLOD sample after heat treatment, when the activity of a GLOD sample stored refrigerated (e.g., 4°C) is set to 1. The residual activity can be a value between 0 and 1, but can exceed 1 if GLOD is activated by heat treatment.

[0041] Herein, mutants in which the amino acid sequence between the γ chain and the β chain has been deleted, such as StGLODΔ49C, M7GLODΔ49C, and StGLODΔ49Ai, as well as the mutants listed in Tables 1-4, may be referred to as γ-chain-to-β-chain amino acid sequence-deleted GLOD mutants, or simply as amino acid sequence-deleted GLOD mutants. An amino acid sequence-deleted GLOD mutant may also be referred to as "GLODΔ." The number of deleted amino acid residues, XX, may also be written after GLODΔ (GLODΔXX). For example, a GLOD mutant in which 49 amino acid residues have been deleted may also be referred to as GLODΔ49. A deletion mutant starting at the position corresponding to position 510 of SEQ ID NO: 1 may also be referred to as "GLOD-dΔ." For example, a mutant based on SEQ ID NO:58 in which 54 amino acids have been deleted, starting from the position corresponding to position 510 in SEQ ID NO:1, may be referred to as M7GLOD-dΔ54. Furthermore, a deletion mutant starting from the position corresponding to position 508 in SEQ ID NO:1 may be referred to as "GLOD-DΔ." For example, a mutant based on SEQ ID NO:58 in which 54 amino acids have been deleted, starting from the position corresponding to position 508 in SEQ ID NO:1, may be referred to as M7GLOD-DΔ54. As used herein, the term "amino acid sequence-deleted GLOD mutant" encompasses not only StGLOD and M7GLOD, but also modified versions thereof, GLODs having 90% or more amino acid sequence identity thereto, and GLODs of other origins in which a region corresponding to the deleted region in SEQ ID NO:1, 12, or 13 has been deleted.

[0042] In certain embodiments, the residual activity of the modified GLOD mutant of the present disclosure after heat treatment can be improved by, for example, 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, 150% or more, 200% or more, 250% or more, 300% or more, 350% or more, e.g., 400% or more, relative to the residual activity of the unmodified GLOD after heat treatment, which is taken as 100%. Here, a 10% improvement in residual activity means that the residual activity of the modified GLOD mutant after heat treatment is 110% relative to the residual activity of the unmodified GLOD after heat treatment, which is taken as 100%.

[0043] In some embodiments, the thermal stability of a GLOD mutant of the present disclosure may be evaluated by measuring the residual activity after heat treatment at 45°C for 30 or 35 minutes, or at 65°C for 30 minutes. In some embodiments, when the residual activity of GLODΔ lacking a predetermined region of SEQ ID NO: 1 after heat treatment at 45°C for 30 or 35 minutes is taken as 100%, the residual activity of the mutant may be 105% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 200% or more, 300% or more, for example, 400% or more. The deleted predetermined region may start at the C-terminal position corresponding to position 510, 508, or 507 of SEQ ID NO: 1, and may contain 31 to 54 consecutive amino acids (including the starting point). In one embodiment, GLODΔ lacking a region corresponding to positions 459 to 507 of SEQ ID NO: 1 may have a residual activity of 105% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 200% or more, 300% or more, for example, 400% or more, when the residual activity of GLOD lacking all or part of positions 459 to 507 of SEQ ID NO: 1 after heat treatment at 45°C for 30 or 35 minutes is taken as 100%. In another embodiment, GLODΔ lacking a region corresponding to positions 459 to 507 of SEQ ID NO: 1 may have a residual activity of 105% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 200% or more, 300% or more, for example, 400% or more, when the residual activity of GLOD lacking the amino acid sequence of positions 459 to 466 of SEQ ID NO: 1 after heat treatment at 45°C for 30 or 35 minutes is taken as 100%. In a specific embodiment, the mutant may further lack a region corresponding to positions 670 to 687.In another embodiment, GLODΔ lacking all or part of the region corresponding to positions 457 to 510 of SEQ ID NO: 1 (e.g., a region consisting of 31 to 54 consecutive amino acids starting from the C-terminal position corresponding to positions 510, 508, or 507 of SEQ ID NO: 1) can have a residual activity of 105% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 200% or more, 300% or more, for example, 400% or more, when the residual activity of GLOD without the deletion after heat treatment at 65°C for 30 minutes is taken as 100%. In a specific embodiment, the mutant can further lack a region corresponding to positions 670 to 687.

[0044] The present inventors have found that GLODΔ mutants, in which a specific region corresponding to positions 459 to 507 of SEQ ID NO: 1 or a specific region consisting of 31 to 54 consecutive amino acids starting from positions 510, 508, or 507 of SEQ ID NO: 1 on the C-terminal side, have been deleted, have improved thermostability compared to unmodified GLOD. Based on these findings, those skilled in the art will understand that delta mutants in which the corresponding amino acid sequences of other GLODs with 90% or more sequence identity have been deleted will also have improved thermostability and can be used in various reactions, even for GLODs of other origins. The same is true for the region corresponding to positions 670 to 687 of SEQ ID NO: 1.

[0045] In certain embodiments, the present disclosure provides GLODΔ, which has 90% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 59, 12, or 13, lacks a predetermined region, and has glutamate oxidase activity. The predetermined region may be a region consisting of 31 to 54 consecutive amino acids starting from the C-terminal position corresponding to position 510, 508, or 507 of SEQ ID NO: 1. This GLODΔ may further lack a region corresponding to positions 670 to 687 of SEQ ID NO: 1.

[0046] GLOD is widely distributed in nature and can be obtained by searching for enzymes of microbial, animal, or plant origin, such as actinomycetes, filamentous fungi, yeast, or bacteria. As used herein, the origin of GLOD is not particularly limited, and refers to GLOD derived from microorganisms of the genus Streptomyces, such as Streptomyces sp. X-119-6 and Streptomyces sp. MOE7, Azotobacter, Embleya, Kitasatospora, Saccharothrix, Alloactinosynnema, Streptoalloteichus, Actinoalloteichus, Catenulispora, Nannocystis, Actinobacteria, Actinophytocola, Sphaerisporangium, Microbispora, Streptosporangium, Phytohabitans, Haliangium, Archangium, Streptacidiphilus, Saccharothrix, or Trichoderma, and includes both wild-type and modified forms thereof, unless otherwise specified.

[0047] (Obtaining the gene encoding GLOD) To obtain the gene encoding GLOD (hereinafter also simply referred to as the "GLOD gene"), a commonly used gene cloning method is used. For example, chromosomal DNA or mRNA can be extracted by standard methods from microbial cells or various cells capable of producing GLOD. Furthermore, cDNA can be synthesized using the mRNA as a template. The chromosomal DNA or cDNA obtained in this manner can be used to prepare a chromosomal DNA or cDNA library.

[0048] Next, a suitable probe DNA is synthesized based on the amino acid sequence of the GLOD, and the GLOD gene is selected from a chromosomal DNA or cDNA library using this. Alternatively, a suitable primer DNA is prepared based on the amino acid sequence, and a DNA containing a desired gene fragment encoding GLOD is amplified by a suitable polymerase chain reaction (PCR) such as the 5'RACE method or the 3'RACE method. These DNA fragments are then ligated to obtain DNA containing the full-length GLOD gene of interest.

[0049] Examples of the GLOD gene include, but are not limited to, the GLOD gene derived from Streptomyces sp. X-119-6 and the GLOD gene derived from Streptomyces sp. MOE7.

[0050] The GLOD gene may be linked to a vector. Examples of vectors include any vectors such as plasmids, bacteriophages, and cosmids, such as pBluescriptII SK+ (Stratagene). Plasmids can be obtained by standard methods. For example, a plasmid containing the GLOD gene can be extracted and purified using a GenElute Plasmid Miniprep Kit (Sigma-Aldrich). The obtained GLOD gene can be manipulated to prepare a GLOD mutant gene or to obtain a purified enzyme.

[0051] (Mutation of the GLOD gene) The GLOD gene can be mutated by any known method depending on the intended mutation form. That is, a wide variety of methods can be used, including contacting the GLOD gene or a recombinant DNA incorporating the gene with a mutagenic agent, UV irradiation, genetic engineering techniques, or protein engineering techniques.

[0052] Examples of mutagenic agents used in the above mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil.

[0053] The conditions for this contact and action can be adjusted depending on the type of drug used, and are not particularly limited as long as the desired mutation can actually be induced in the GLOD gene. Generally, the desired mutation can be induced by contact and action at a drug concentration of preferably 0.5 to 12 M, at a reaction temperature of 20 to 80°C, for 10 minutes or more, preferably 10 to 180 minutes. When ultraviolet irradiation is performed, it can also be carried out according to the standard method described above.

[0054] Protein engineering techniques can be used, generally known as site-specific mutagenesis, such as the Kramer method (Nucleic Acids Res., 12, 9441-9456 (1984)), the Eckstein method (Nucleic Acids Res., 13, 8749-8764 (1985); Nucleic Acids Res., 13, 8765 (1985); Nucleic Acids Res., 14, 9679 (1986)), and the Kunkel method (Proc. Natl. Acid. Sci. USA, 82, 488-492 (1985)).

[0055] In addition to the above-mentioned gene modification methods, the desired modified GLOD gene can also be directly synthesized by organic synthesis or enzymatic synthesis.

[0056] The nucleotide sequence of the GLOD gene can be confirmed, for example, by using a multi-capillary DNA analysis system, Applied Biosystems 3730x1 DNA Analyzer (manufactured by Thermo Fisher Scientific).

[0057] (Transformation / Transduction) The GLOD gene can be incorporated into a vector such as a bacteriophage, cosmid, or a plasmid used for transforming prokaryotic or eukaryotic cells by standard methods, and then used to transform or transduce a host corresponding to each vector by standard methods.

[0058] In one embodiment, GLOD can be expressed using prokaryotic cells, such as Escherichia microorganisms, e.g., Escherichia coli, Brevibacillus microorganisms, e.g., Brevibacillus choshinensis, Corynebacterium microorganisms, e.g., Corynebacterium glutamicum, or Streptomyces microorganisms, e.g., Streptomyces violaceoruber. Examples of E. coli hosts include, but are not limited to, various E. coli strains, such as K-12, JM109, DH5α, BL21, JM109(DE3), DH5α(DE3), BL21(DE3), TG1, 1100, W3110, and C600. Host cells (transformants) containing the GLOD gene are obtained by transforming or transducing the host. As a method for transferring a recombinant vector into such host cells, for example, when the host cell is a microorganism belonging to Escherichia coli, a method of transferring recombinant DNA in the presence of calcium ions can be used, or electroporation can be used. Furthermore, commercially available competent cells (e.g., ECOS Competent Escherichia coli BL21(DE3); manufactured by Nippon Gene) can also be used. The GLOD gene can be codon-optimized depending on the expression host.

[0059] In certain embodiments, GLOD may be expressed using eukaryotic cells. An example of a eukaryotic host cell is yeast. Microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Schizosaccharomyces, Saccharomyces, Pichia, and Candida. The inserted gene may contain a marker gene to enable selection of transformed cells. Examples of marker genes include genes that complement auxotrophy of the host, such as URA3 and TRP1. It is also desirable for the inserted gene to contain a promoter or other regulatory sequence (e.g., an enhancer sequence, a terminator sequence, a polyadenylation sequence, etc.) capable of expressing the gene of interest in the host cell. Specific examples of promoters include the GAL1 promoter and the ADH1 promoter. Methods for transforming yeast include well-known methods, such as a method using lithium acetate (Methods Mol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003) 481-484), but are not limited thereto. Transformation can be performed using any of a variety of methods, including the spheroplast method and the glass bead method.

[0060] Other examples of eukaryotic host cells include fungal cells (including filamentous fungi) such as those of the genus Aspergillus and Trichoderma. The method for producing a transformant of a fungal cell is not particularly limited, and examples include a method in which a gene encoding GLOD is inserted into a host filamentous fungus in a manner that allows the gene to be expressed, according to a conventional method. Specifically, a DNA construct is prepared in which the gene encoding GLOD is inserted between an expression-inducing promoter and a terminator, and then a host filamentous fungus is transformed with the DNA construct containing the gene encoding GLOD, thereby obtaining a transformant that overexpresses the gene encoding GLOD.

[0061] The method for inserting the gene encoding GLOD into the host filamentous fungus in such a manner that the gene is expressed is not particularly limited, and examples include a method of directly inserting the gene into the chromosome of the host organism by using homologous recombination; and a method of introducing the gene into the host filamentous fungus by linking it onto a plasmid vector.

[0062] In the method using homologous recombination, a DNA construct is ligated between sequences homologous to the upstream and downstream regions of a recombination site on a chromosome and inserted into the genome of a host filamentous fungus. A transformant can be obtained by self-cloning by overexpressing the construct in the host filamentous fungus under the control of its own high-expression promoter. The high-expression promoter is not particularly limited, but examples include the promoter region of the TEF1 gene (tef1), which is a translation elongation factor, the promoter region of the α-amylase gene (amy), and the promoter region of the alkaline protease gene (alp).

[0063] In the method using a vector, the DNA construct can be inserted into a plasmid vector used for transforming filamentous fungi by a conventional method, and the corresponding host filamentous fungus can be transformed by a conventional method.

[0064] Such a suitable vector-host system is not particularly limited as long as it is a system that allows GLOD to be produced in a host filamentous fungus, and examples thereof include a system of pUC19 and a filamentous fungus, and a system of pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and a filamentous fungus.

[0065] The DNA construct is preferably introduced into the chromosome of the host filamentous fungus for use; however, as an alternative method, the DNA construct can be incorporated into an autonomously replicating vector (Ozeki et al., Biosci. Biotechnol. Biochem. 59, 1133 (1995)) for use without being introduced into the chromosome.

[0066] The DNA construct may contain a marker gene that allows for the selection of transformed cells. The marker gene is not particularly limited, and examples include genes that complement auxotrophy of the host, such as pyrG, niaD, and adeA; and drug resistance genes for drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably contains a promoter, terminator, or other control sequence (e.g., enhancer, polyadenylation sequence, etc.) that enables overexpression of the gene encoding GLOD in the host cell. The promoter is not particularly limited, and examples include appropriate expression-inducible promoters and constitutive promoters, such as the tef1 promoter, alp promoter, and amy promoter. The terminator is also not particularly limited, and examples include the alp terminator, amy terminator, and tef1 terminator.

[0067] In the DNA construct, an expression control sequence for the gene encoding GLOD is not necessarily required if the DNA fragment containing the gene encoding GLOD to be inserted contains a sequence having an expression control function. Furthermore, when transformation is performed by co-transformation, the DNA construct may not necessarily have a marker gene.

[0068] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, a gene encoding GLOD, an alp gene terminator, and a pyrG marker gene are ligated to an In-Fusion Cloning Site in the multiple cloning site of pUC19.

[0069] The method for transforming a filamentous fungus can be appropriately selected from methods known to those skilled in the art. For example, the protoplast PEG method, in which protoplasts of a host filamentous fungus are prepared and then transformed with polyethylene glycol and calcium chloride (see, for example, Mol. Gen. Genet. 218, 99-104, 1989; JP 2007-222055 A), can be used. A medium for regenerating the transformed filamentous fungus is selected appropriately depending on the host filamentous fungus and the transformation marker gene used. For example, when Aspergillus sojae is used as the host filamentous fungus and the pyrG gene is used as the transformation marker gene, the transformed filamentous fungus can be regenerated in, for example, Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2 M sorbitol.

[0070] Furthermore, for example, to obtain the transformed filamentous fungus of the present invention, the promoter of the gene encoding GLOD originally present on the chromosome of the host filamentous fungus may be replaced with a high-expression promoter such as tef1 using homologous recombination. In this case, it is also preferable to insert a transformation marker gene such as pyrG in addition to the high-expression promoter. For example, for this purpose, a transformation cassette consisting of the upstream region of the gene encoding GLOD, a transformation marker gene, a high-expression promoter, and all or part of the gene encoding GLOD can be used, as described in Example 1 and FIG. 1 of Japanese Patent Application Laid-Open No. 2011-239681. 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. The all or part of the gene encoding GLOD can include a region extending from the initiation codon. The length of the region suitable for homologous recombination is preferably 0.5 kb or more.

[0071] The production of the transformed filamentous fungus of the present invention can be confirmed by culturing the transformed filamentous fungus of the present invention under conditions in which the enzymatic activity of GLOD is observed, and then confirming the activity of GLOD in the culture obtained after culturing.

[0072] Furthermore, confirmation of the production of the transformed filamentous fungus of the present invention may be carried out by extracting chromosomal DNA from the transformed filamentous fungus, performing PCR using this as a template, and confirming that an amplifiable PCR product is produced when transformation has occurred.

[0073] For example, PCR is carried out using a combination of a forward primer for the nucleotide sequence of the promoter used and a reverse primer for the nucleotide sequence of the transformation marker gene, and it is confirmed that a product of the expected length is produced.

[0074] The host may be a known microorganism, known strain, or an equivalent of the known microorganism or strain described herein. An equivalent refers to a host that exhibits equivalent functions with respect to recombinant expression of a protein. Equivalents include hosts that have been created and modified based on hosts known at the time of filing of this application, which have been developed after the filing of this application, and hosts with properties similar to those known at the time of filing of this application that have been discovered after the filing of this application. Regarding the scientific name or classification of a microorganism, if there is a change in the scientific name, genus name, or classification after the filing of this application, the description in this specification shall take precedence, and the date of filing of this application shall be used as the basis.

[0075] (High-Throughput Screening) GLOD can also be subjected to high-throughput screening to obtain functional GLOD mutants. For example, a library of transformed or transduced strains carrying a mutated GLOD gene may be prepared and subjected to high-throughput screening based on microtiter plates, or ultra-high-throughput screening based on droplet microfluidics. Examples include 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), bacterial display (e.g., Curr Opin Struct Biol 17: 474-80, 2007), or the like. 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 to screen for GLOD variants is incorporated herein by reference. For example, libraries can be constructed using error-prone PCR. Saturation mutagenesis can also be used to construct libraries by targeting the regions and positions described herein or their corresponding regions and positions. The libraries can be transformed into appropriate cells, such as electrocompetent EBY-100 cells, to obtain approximately 10 mutants (10 million). Yeast cells transformed with the libraries can then be subjected to cell sorting. Polydimethoxylsiloxane (PDMS) microfluidic devices fabricated using standard soft lithography techniques can also be used. Monodisperse droplets can be formed using a flow-focusing device.The droplets formed containing individual mutants can be placed in an appropriate sorting device. The presence or absence of GLOD activity can be utilized to select cells. For example, a reaction solution formulated to develop color upon the action of GLOD may be used. For example, when DCIP is used, absorbance at 600 nm may be measured using a 96-well plate, 192-well plate, 384-well plate, 9600-well plate, or the like, and a plate reader. Mutation introduction and selection may be repeated multiple times. Mutations herein include amino acid substitutions, insertions, deletions, and / or additions.

[0076] For example, 1 to 10 mutations can be introduced into GLOD, and GLOD activity can be confirmed. Then, starting from a GLOD mutant confirmed to have activity, 1 to 10 additional mutations can be introduced and activity can be confirmed. A series of high-throughput screening (e.g., the above-described method of obtaining and screening approximately 10 to the power of 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, for example, 10 rounds of high-throughput screening, in which 1 or more, 5 or more, for example, 10 or more mutations are introduced in each round, 10 or more, 50 or more, for example, 100 or more mutations can be introduced from the starting GLOD, and mutants that still have activity can be rapidly obtained. Furthermore, by repeating 20 rounds, 20 or more, 100 or more, for example, 200 or more mutations can be introduced from the starting GLOD, and mutants that still have activity can be rapidly obtained. Such operations can be performed by repeating a routine process.

[0077] Mutations may be introduced at any one or more positions from the first amino acid to the last amino acid in the full-length amino acid sequence of GLOD, excluding regions important for enzyme function, such as the active center, substrate recognition site, coenzyme recognition motif, and their vicinity. GLOD is widely used industrially, and those skilled in the art are familiar with the regions important for enzyme function, including the active center, substrate recognition site, and coenzyme recognition motif. In certain embodiments, for example, one or more mutations may first be introduced at positions 1 to 10 of the full-length GLOD sequence. Next, starting from a GLOD variant confirmed to have activity, one or more mutations may be further introduced at positions 11 to 20, and activity may be confirmed. This process may be repeated n times (n≦68). For example, on the 68th iteration, one or more mutations may be introduced at positions 680 to 687. Regions important for enzyme function or regions not intended to be modified may be skipped along the way, as appropriate. This allows any mutation to be introduced at any position in the full-length sequence, except for regions important for the function of the enzyme, and also makes it possible to rapidly obtain 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, for example, 200 or more mutations.

[0078] Mutations may be introduced randomly or by rational design. In certain 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 and after substitution have similar chemical properties (e.g., Stryer et al., Biochemistry, 5th ed., 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 basic amino acid; (ii) substitution of an acidic amino acid with a different acidic amino acid; (iii) substitution of an aromatic amino acid with a different aromatic amino acid; (iv) substitution of a nonpolar aliphatic amino acid with a different nonpolar aliphatic amino acid; and (v) substitution of a polar uncharged amino acid with a different 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.

[0079] In some embodiments, mutations introduced by rational design or randomly include substitution with a functionally similar amino acid. Tables of functionally similar amino acids are widely known in the art. In some embodiments, the original and substituted amino acids may belong to any of the following amino acid classes: 1) glycine (G), alanine (A); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (N), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V), proline (P); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0080] In typical embodiments, conservative amino acid substitutions or substitutions with functionally similar amino acids are not present in regions critical to the function of the enzyme, such as the active center of GLOD, the substrate recognition site, the coenzyme recognition motif, and their vicinity, and therefore do not significantly affect the activity of the enzyme.

[0081] GLOD variants may also include those in which additional amino acids have been inserted compared to the sequence before mutation. In typical embodiments, the amino acid insertion is not located in an area critical for enzyme function, such as the active center, substrate recognition site, coenzyme recognition motif, or the vicinity thereof, and therefore does not significantly affect enzyme activity. GLOD variants may also include those in which additional amino acids have been added compared to the sequence before mutation. In certain embodiments, the amino acid addition is made to the N-terminus or C-terminus of GLOD and does not significantly affect enzyme activity. Examples of additions include, but are not limited to, a short stretch of histidine residues (e.g., 2 to 6 histidine residues) to aid in purification of GLOD. Examples of additions include, but are not limited to, the addition of a signal peptide to aid in expression of GLOD. Signal peptides include known signal sequences or functional equivalents thereof.

[0082] GLOD variants may also contain amino acid deletions compared to the pre-mutation sequence. In typical embodiments, the amino acid deletions are not in regions critical to the function of the enzyme and therefore do not significantly affect the activity of the enzyme. In some embodiments, the deletions may be as short as one or two amino acids. In some embodiments, the amino acid sequence of one GLOD can be compared to that of another GLOD, and if an amino acid is deleted in one sequence, the deletion can be introduced into the other GLOD. Because both GLODs exhibit activity, such deletions are unlikely to significantly affect the activity of the enzyme.

[0083] Mutations can be introduced into GLOD so as not to disrupt secondary structures or structural motifs, such as α-helices and β-sheets. Regions of secondary structure can be identified, for example, by using secondary structure prediction algorithms. Examples of such prediction algorithms include, but are not limited to, NetSurfP-2.0. The same applies to other structural motifs, such as nests and niches.

[0084] (Mutation Improving Thermostability) In one embodiment, the present disclosure provides a GLOD mutant having a mutation that improves thermostability. The mutant has improved thermostability compared to GLOD before the introduction of the mutation. Here, with respect to the GLOD mutant of the present disclosure, improved thermostability means that when GLOD is subjected to heat treatment at a predetermined temperature for a predetermined time, the residual activity of the GLOD mutant after heat treatment is improved compared to the residual activity of GLOD before the introduction of the mutation.

[0085] In certain embodiments, the GLOD variant of the present disclosure has an amino acid substitution compared to SEQ ID NO: 1 or 58 at one or more positions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, selected from the group consisting of: a position corresponding to position 87 of SEQ ID NO: 1; a position corresponding to position 103 of SEQ ID NO: 1; a position corresponding to position 133 of SEQ ID NO: 1; a position corresponding to position 186 of SEQ ID NO: 1; a position corresponding to position 297 of SEQ ID NO: 1; a position corresponding to position 376 of SEQ ID NO: 1; a position corresponding to position 393 of SEQ ID NO: 1; a position corresponding to position 428 of SEQ ID NO: 1; a position corresponding to position 516 of SEQ ID NO: 1; a position corresponding to position 566 of SEQ ID NO: 1; a position corresponding to position 568 of SEQ ID NO: 1; a position corresponding to position 585 of SEQ ID NO: 1; and a position corresponding to position 615 of SEQ ID NO: 1, and the thermal stability of the variant after amino acid substitution is improved compared to the variant before substitution. With respect to the above positions, an amino acid is substituted (having an amino acid substitution) means that the wild-type amino acid is substituted with another amino acid, and therefore the substituted amino acid excludes the wild-type amino acid.

[0086] In certain embodiments, with respect to the GLOD variants of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 87 of SEQ ID NO: 1 may be tyrosine.

[0087] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 103 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine.

[0088] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 133 of SEQ ID NO: 1 may be selected from the group consisting of leucine and tyrosine.

[0089] In certain embodiments, with respect to the GLOD variants of the present disclosure, the amino acid substitution introduced at the position corresponding to position 186 of SEQ ID NO: 1 may be selected from the group consisting of glutamic acid, aspartic acid, tyrosine, glutamine, asparagine, alanine, leucine, cysteine, methionine, phenylalanine, serine, histidine, and threonine.

[0090] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 297 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, isoleucine, and methionine.

[0091] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 376 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine.

[0092] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 393 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, isoleucine, and methionine.

[0093] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 428 of SEQ ID NO: 1 may be selected from the group consisting of tyrosine and methionine.

[0094] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 516 of SEQ ID NO: 1 can be phenylalanine.

[0095] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 566 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, valine, and methionine.

[0096] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 568 of SEQ ID NO: 1 may be selected from the group consisting of isoleucine and methionine.

[0097] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 585 of SEQ ID NO: 1 may be selected from the group consisting of leucine and methionine.

[0098] In certain embodiments, for GLOD variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 615 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, and phenylalanine. Corresponding positions are described below.

[0099] In certain embodiments, the above-described thermostability-improving mutations can be introduced into wild-type GLOD or conventional GLOD. Conventional GLOD, as used herein, refers to conventional GLOD that requires protease treatment to express active protein. GLOD into which such thermostability-improving mutations have been introduced not only exhibits activity after protease treatment, similar to wild-type GLOD and conventional GLOD, but is also thought to have improved thermostability compared to GLOD before the introduction of the mutations. For convenience, such mutants may be referred to herein as GLOD-T. Furthermore, multiple such thermostability-improving mutations may be introduced. GLOD into which one of the above-described thermostability-improving mutations has been introduced may be referred to herein as GLOD-T1. Similarly, mutants into which 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations have been introduced may be referred to as GLOD-T2, GLOD-T3, GLOD-T4, GLOD-T5, GLOD-T6, GLOD-T7, GLOD-T8, GLOD-T9, GLOD-T10, GLOD-T11, GLOD-T12, or GLOD-T13, respectively.

[0100] The present inventors have found that GLOD mutants in which amino acid substitutions have been introduced at positions corresponding to positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1 have improved thermostability compared to GLOD before modification. Based on these findings, those skilled in the art will understand that GLOD of other origins in which similar amino acid substitutions have been introduced at positions corresponding to position 87, etc. of SEQ ID NO: 1 will also have improved thermostability and can be used in various reactions.

[0101] In certain embodiments, the above-described thermostability-improving mutations may be introduced into an amino acid sequence-deleted GLOD mutant (GLODΔ) of the present disclosure. Unless otherwise specified, the amino acid sequence-deleted GLOD mutant of the present disclosure does not require protease treatment to express active protein. As described above, the amino acid sequence-deleted GLOD mutant of the present disclosure surprisingly has improved thermostability compared to the corresponding GLOD before the amino acid sequence deletion. The above-described thermostability-improving mutations may be further introduced into such an amino acid sequence-deleted GLOD mutant (GLODΔ) of the present disclosure. For convenience, such a mutant may be referred to herein as GLODΔ-T. GLODΔ into which one thermostability-improving mutation has been introduced may be referred to herein as GLODΔ-T1. Similarly, Δ mutants into which 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations have been introduced may be referred to as GLODΔ-T2, GLODΔ-T3, GLODΔ-T4, GLODΔ-T5, GLODΔ-T6, GLODΔ-T7, GLODΔ-T8, GLODΔ-T9, GLODΔ-T10, GLODΔ-T11, GLODΔ-T12, or GLODΔ-T13, respectively.

[0102] (Preliminary Amino Acid Substitutions) In a preliminary embodiment, GLOD variants of the present disclosure, such as thermostability-improved variants such as GLOD-T and GLOD-T1 to GLOD-T13, and GLOD variants with deleted amino acid sequences, such as GLODΔ-T and GLODΔ-T1 to GLODΔ-T13, e.g., SEQ ID NOs: 12, 13, 58, 59, or 60, or GLOD variants having 70% or more, 80% or more, or 90% or more amino acid sequence identity to any of these, may optionally have an amino acid substitution as described in WO 2021 / 193598. For example, with respect to the GLOD variants of the present disclosure, Ala at position 106 of SEQ ID NO: 1 herein may be substituted with Ser. This amino acid substitution is referred to herein as A106S. Similarly, hereinafter, using SEQ ID NO: 1 herein as a reference, a GLOD variant of the present disclosure may have one or more, for example, 1 to 25, amino acid substitutions 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, P598A, C601S, and P609A. The same applies to positions corresponding to these positions.

[0103] For convenience, to distinguish from the thermostability-improving amino acid deletion Δ and thermostability-improving mutation T of the present disclosure, the amino acid substitutions described in WO 2021 / 193598 may be represented as J. That is, J represents a set of amino acid substitutions described in WO 2021 / 193598, and the order of the components does not matter. [Number 1] J={A106S, C210S, Q235E, D236E, D237E, P244H, T311S, W313F, Q333E, I334V, I334L, M336L, Q 338E, R339K, T416S, A438P, K441E, Y455F, Q456R, Q457E, Q457K, L505I, P598A, C601S, P609A}

[0104] Furthermore, a mutant having one mutation in J is designated as J1, a mutant having n mutations in J is designated as Jn, and so on up to J25. In one embodiment, the present disclosure provides the following combination mutants. A person skilled in the art could create these finite combinations of mutants one by one and confirm their activity and thermostability. The Δ to be deleted in the following may be a sequence of 31 to 54 in length. Furthermore, in the following, the starting point on the C-terminal side of the Δ to be deleted may be a position corresponding to position 510, 508, or 507 of SEQ ID NO: 1. [Equation 2] GLOD-T ∩ J [Equation 3] GLOD-T ∩ {J1 to J25} [Equation 4] {GLOD-T1 to GLOD-T13} ∩ J [Equation 5] {GLOD-T1 to GLOD-T13} ∩ {J1 to J25} [Equation 6] GLODΔ-T ∩ J [Equation 7] GLODΔ-T ∩ {J1 to J25} [Equation 8] {GLODΔ-T1 to GLODΔ-T13} ∩ J [Equation 9] {GLODΔ-T1 to GLODΔ-T13} ∩ {J1 to J25}

[0105] (GLOD Mutant) By introducing the amino acid sequence deletion of the present disclosure into the GLOD gene, a GLODΔ mutant can be created, and GLOD, for example, highly thermostable GLOD, can be produced without protease treatment. By introducing the amino acid substitution T of the present disclosure into the GLOD gene, a GLOD-T mutant can be created, and highly thermostable GLOD can be produced. By introducing the amino acid sequence deletion and amino acid substitution of the present disclosure into the GLOD gene, a GLODΔ-T mutant can be created, and GLOD, for example, highly thermostable GLOD can be produced without protease treatment.

[0106] In some limiting embodiments, the substituted amino acid is excluded from back-mutation to an amino acid in the native GLOD sequence (natural amino acid). In other embodiments, the substituted amino acid at a position corresponding to, for example, position 87 of SEQ ID NO: 1, can be identical to the amino acid at that position in the native GLOD sequence (natural amino acid).

[0107] (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. Furthermore, an amino acid at a corresponding position is referred to as a corresponding amino acid. For convenience, this specification will be described using the amino acid sequence of GLOD derived from Streptomyces sp. X-119-6 shown in SEQ ID NO: 1 as a reference. In this case, the "corresponding position" in an amino acid sequence refers to a position in the amino acid sequence of GLOD derived from another organism species that corresponds to a specific position in the amino acid sequence of GLOD derived from Streptomyces sp. X-119-6 shown in SEQ ID NO: 1.

[0108] A method for identifying "corresponding positions" in amino acid sequences is, for example, to compare amino acid sequences using a known algorithm such as the Lippmann-Parson method, and assign maximum identity to conserved amino acid residues present in the amino acid sequences of each GLOD. By aligning the GLOD amino acid sequences in this manner, it is possible to determine the positions of homologous amino acid residues in each GLOD sequence, regardless of insertions or deletions in the amino acid sequences. Corresponding positions (homologous positions) are considered to be at the same positions in the three-dimensional structure, and can be predicted to have similar effects on the specific function of the target GLOD.

[0109] (Corresponding Position of Mutation) As used herein, "a position corresponding to position 87 in the amino acid sequence of SEQ ID NO: 1" refers to the position corresponding to position 87 in SEQ ID NO: 1 when the amino acid sequence of a subject GLOD is compared with the amino acid sequence of SEQ ID NO: 1. The same applies to other positions in SEQ ID NO: 1, such as positions 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615. The same applies to positions 106, 210, 235, 236, 237, 244, 311, 313, 333, 334, 334, 336, 338, 339, 416, 438, 441, 455, 456, 457, 505, 598, 601, and 609 in SEQ ID NO: 1. For example, the position corresponding to position 87 in the amino acid sequence of SEQ ID NO: 1 is position 87 in SEQ ID NO: 58.

[0110] (Corresponding Region) A "corresponding region" in an amino acid sequence is defined in the same manner as the above-mentioned "corresponding position." For example, the region corresponding to positions 459 to 507 in SEQ ID NO: 1 is positions 459 to 507 in SEQ ID NO: 58. Furthermore, the region corresponding to positions 459 to 466 in SEQ ID NO: 1 is positions 459 to 466 in SEQ ID NO: 58. Furthermore, the region corresponding to positions 457 to 510 in SEQ ID NO: 1 is positions 457 to 510 in SEQ ID NO: 58. Furthermore, the region corresponding to positions 670 to 687 in SEQ ID NO: 1 is positions 673 to 690 in SEQ ID NO: 58.

[0111] (Homology, Identity, or Similarity of Amino Acid Sequences) Amino acid sequence homology, identity, or similarity can be calculated using programs such as GENETYX (GENETYX) maximum matching and search homology, DNASIS Pro (Hitachi Solutions) maximum matching and multiple alignment, or CLUSTAL W multiple alignment. To calculate amino acid sequence identity, two or more GLODs can be aligned and the positions of identical amino acids in the 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, the percent identity refers to the percentage calculated by aligning two or more amino acid sequences using an algorithm such as Blosum62, where the denominator is the total number of amino acids in the alignable regions, and the numerator is the number of positions occupied by identical amino acids. Therefore, normally, when two or more amino acid sequences have a region where no identity is observed, for example, when one of the amino acid sequences has an additional sequence at the C-terminus where no identity is observed, the region where no identity is observed cannot be aligned and is therefore not used in calculating the percent identity.

[0112] It is also possible to examine the positions of similar amino acids in two or more GLODs. For example, multiple amino acid sequences can be aligned using CLUSTALW. In this case, the Blosum62 algorithm is used, and amino acids that are determined to be similar when multiple amino acid sequences are aligned may be referred to as similar amino acids. In the variants disclosed herein, amino acid substitutions may be due to substitutions between such similar amino acids. Such alignments allow for the examination of regions of identical amino acid sequences and positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, regions of homology (conserved regions) in the amino acid sequences can be determined.

[0113] In one embodiment, the GLOD variant of the present disclosure has a full-length amino acid sequence identity of 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 when aligned with GLOD having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 58, and has improved thermal stability compared to GLOD before modification.

[0114] In one embodiment, the GLOD variant of the present disclosure has an amino acid sequence in which one or several amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted, at positions other than those corresponding to positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1, and has improved thermal stability compared to the GLOD before modification. Here, "one or several amino acids" refers to 1 to 15, 1 to 10, 1 to 7, 1 to 5, or 1 to 4, for example, 1 to 3, for example, 1 or 2 amino acids.

[0115] In one embodiment, GLODΔ of the present disclosure: (i) when aligned with the amino acid sequence of SEQ ID NO: 1, lacks the amino acid sequence of a predetermined region of SEQ ID NO: 1, and optionally lacks the amino acid sequence of a region corresponding to positions 670 to 687 of SEQ ID NO: 1, and has glutamate oxidase activity, wherein the predetermined region is a region consisting of 31 to 54 consecutive amino acids starting from the C-terminal side at a position corresponding to positions 510, 508, or 507 of SEQ ID NO: 1; (ii) in (i), consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, or added at a position other than the predetermined region of SEQ ID NO: 1, or consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, or added at a position other than the predetermined region and the region corresponding to positions 670 to 687 of SEQ ID NO: 1; (iii) In the above (i) or (ii), the full-length amino acid sequence of the GLODΔ has a sequence identity of 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, with the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 59.(iv) In (i) or (ii) above, the full-length amino acid sequence of the GLODΔ has 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 sequence identity with the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 59, and the amino acid at the position 291 of SEQ ID NO: 1 in the GLODΔ is arginine, and the amino acid sequence at positions 51 to 56 of SEQ ID NO: 1 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid), or (v) In (i) or (ii) above, the full-length amino acid sequence of the GLODΔ has 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 sequence identity with the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 59, the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is arginine, and the amino acid sequence at positions 51 to 56 of SEQ ID NO: 1 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid), and the thermal stability is improved compared to GLOD before modification.

[0116] In certain embodiments, GLOD-T of the present disclosure comprises: (i) an amino acid substitution at a position corresponding to a position selected from the group consisting of positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1, when aligned with the amino acid sequence of SEQ ID NO: 1; (ii) an amino acid sequence in (i) in which one or several amino acids have been substituted, deleted, or added at a position other than positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1; (iii) In the above (i) or (ii), the full-length amino acid sequence of the GLOD-T has a sequence identity of 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, with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 58; (iv) In the above (i) or (ii), the full-length amino acid sequence of said GLOD-T has 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 sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 58, and the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 in said GLOD-T is arginine, and the amino acid sequence at positions 51 to 56 of SEQ ID NO: 1 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid), or(v) In (i) or (ii) above, the full-length amino acid sequence of GLOD-T has 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 sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 58, the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is arginine, and the amino acid sequence at positions 51 to 56 of SEQ ID NO: 1 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid), and the thermal stability is improved compared to GLOD before modification.

[0117] In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 87 of SEQ ID NO: 1 may be tyrosine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 103 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 133 of SEQ ID NO: 1 may be selected from the group consisting of leucine and tyrosine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 186 of SEQ ID NO: 1 may be selected from the group consisting of glutamic acid, aspartic acid, tyrosine, glutamine, asparagine, alanine, leucine, cysteine, methionine, phenylalanine, serine, histidine, and threonine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 297 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, isoleucine, and methionine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 376 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 393 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, isoleucine, and methionine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 428 of SEQ ID NO: 1 may be selected from the group consisting of tyrosine and methionine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 516 of SEQ ID NO: 1 may be phenylalanine. In certain embodiments, for GLOD-T variants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 566 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, valine, and methionine.In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 568 of SEQ ID NO: 1 may be selected from the group consisting of isoleucine and methionine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 585 of SEQ ID NO: 1 may be selected from the group consisting of leucine and methionine. In certain embodiments, for a GLOD-T variant of the present disclosure, the replacement amino acid introduced at a position corresponding to position 615 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, and phenylalanine.

[0118] In one embodiment, GLODΔ-T of the present disclosure: (i) when aligned with the amino acid sequence of SEQ ID NO: 1, lacks a predetermined region of the amino acid sequence of SEQ ID NO: 1, optionally lacking a region of the amino acid sequence corresponding to positions 670 to 687 of SEQ ID NO: 1, and has glutamate oxidase activity, wherein the predetermined region is a region consisting of 31 to 54 consecutive amino acids starting from the C-terminal position corresponding to positions 510, 508, or 507 of SEQ ID NO: 1; and (ii) when aligned with the amino acid sequence of SEQ ID NO: 1, amino acids at positions corresponding to positions selected from the group consisting of 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1 have been substituted, for example, modified to an amino acid selected from the group consisting of tyrosine, phenylalanine, tryptophan, leucine, isoleucine, valine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, alanine, and threonine; (iii) (i) above, the amino acid sequence is one or more amino acids substituted, deleted or added at positions corresponding to positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585 and 615 of SEQ ID NO: 1 and at positions other than the predetermined region of SEQ ID NO: 1, or the amino acid sequence is one or more amino acids substituted, deleted or added at positions corresponding to positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585 and 615 of SEQ ID NO: 1 and at positions other than the predetermined region and the region corresponding to positions 670 to 687 of SEQ ID NO: 1; In the above (i) or (ii), the full-length amino acid sequence of the GLODΔ-T has a sequence identity of 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, with the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 69.(iv) In (i) or (ii) above, the full-length amino acid sequence of the GLODΔ-T has 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 sequence identity with the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 69, and the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 in the GLODΔ-T is arginine, and the amino acid sequence at positions 51 to 56 of SEQ ID NO: 1 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid), or (v) In the above (i) or (ii), the full-length amino acid sequence of the GLODΔ-T has 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 sequence identity with the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 69, the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is arginine, and the amino acid sequence at positions 51 to 56 of SEQ ID NO: 1 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid), and the thermal stability is improved compared to the GLOD before modification.

[0119] In an embodiment, the present disclosure provides a polypeptide having 70% or more, 75% or more, 80% or more, 85% or more, for example, 90% or more, for example, 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:69, and lacking a predetermined region of SEQ ID NO:1, wherein the predetermined region is a region consisting of 31 to 54 consecutive amino acids starting from the C-terminal side at a position corresponding to position 510, 508, or 507 of SEQ ID NO:1, and including at least one of the following amino acids: a position corresponding to position 87 of SEQ ID NO:1, a position corresponding to position 103 of SEQ ID NO:1, a position corresponding to position 133 of SEQ ID NO:1, a position corresponding to position 186 of SEQ ID NO:1, a position corresponding to position 297 of SEQ ID NO:1, a position corresponding to position 376 of SEQ ID NO:1, a position corresponding to position 393 of SEQ ID NO:1, a position corresponding to position 428 of SEQ ID NO:1, a position corresponding to position 516 of SEQ ID NO:1, a position corresponding to position 566 of SEQ ID NO:1, a position corresponding to position 568 of SEQ ID NO:1, The present invention provides a GLOD variant having an amino acid substitution compared to the amino acid sequence of SEQ ID NO:58 at one or more positions selected from the group consisting of: a position corresponding to position 585 of SEQ ID NO:1; and a position corresponding to position 615 of SEQ ID NO:1, wherein the GLOD variant has glutamate oxidase activity, provided that in certain embodiments, the substituted amino acids exclude wild-type amino acids. In certain embodiments, the variant may further lack a region corresponding to positions 670-687.

[0120] In certain embodiments, for a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 87 of SEQ ID NO: 1 may be tyrosine. In certain embodiments, for a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 103 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine. In certain embodiments, for a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 133 of SEQ ID NO: 1 may be selected from the group consisting of leucine and tyrosine. In certain embodiments, for a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 186 of SEQ ID NO: 1 may be selected from the group consisting of glutamic acid, aspartic acid, tyrosine, glutamine, asparagine, alanine, leucine, cysteine, methionine, phenylalanine, serine, histidine, and threonine. In certain embodiments, with respect to a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 297 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, isoleucine, and methionine. In certain embodiments, with respect to a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 376 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine. In certain embodiments, with respect to a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 393 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, isoleucine, and methionine. In certain embodiments, with respect to a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 428 of SEQ ID NO: 1 may be selected from the group consisting of tyrosine and methionine. In certain embodiments, with respect to a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 516 of SEQ ID NO: 1 may be phenylalanine. In certain embodiments, with respect to the GLODΔ-T mutants of the present disclosure, the replacement amino acid introduced at the position corresponding to position 566 of SEQ ID NO: 1 may be selected from the group consisting of phenylalanine, leucine, valine, and methionine.In certain embodiments, for a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 568 of SEQ ID NO: 1 may be selected from the group consisting of isoleucine and methionine. In certain embodiments, for a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 585 of SEQ ID NO: 1 may be selected from the group consisting of leucine and methionine. In certain embodiments, for a GLOD delta-T mutant of the present disclosure, the substituted amino acid introduced at a position corresponding to position 615 of SEQ ID NO: 1 may be selected from the group consisting of leucine, valine, and phenylalanine.

[0121] (Production of GLOD) In ​​one embodiment, the present invention provides a method for producing GLOD, comprising culturing a strain capable of producing GLOD under conditions conducive to expression of the GLOD, and isolating GLOD from the culture or culture broth. This method can use a host cell transformed with a vector incorporating a gene encoding the GLOD of the present disclosure. Here, conditions conducive to expression of GLOD refer to conditions in which the GLOD gene is transcribed and translated, and a polypeptide encoded by the gene is produced.

[0122] The medium for culturing the above-mentioned strains may contain, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran infusion, 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 have been added, and further contains, as necessary, carbohydrate raw materials, vitamins, and the like.

[0123] In addition, the production amount of the target enzyme can be improved by adding to the culture medium substrates on which the GLOD can act or similar compounds thereof, such as glycated amino acids, glycated peptides, glycated protein hydrolysates, or glycated proteins such as glycated hemoglobin and glycated albumin.

[0124] The initial pH of the medium is suitably adjusted to pH 7 to 9. Cultivation is preferably carried out at a culture temperature of 20 to 42°C, preferably around 25 to 37°C, for 4 to 24 hours, more preferably around 25 to 37°C, for 8 to 16 hours by aeration and agitation submerged culture, shaking culture, static culture, or the like.

[0125] After the culture is completed, GLOD can be collected from the culture by conventional enzyme collection methods. For example, the cells can be subjected to ultrasonic disruption, grinding, etc., or the enzyme can be extracted using a lytic enzyme such as lysozyme, or the cells can be lysed by shaking or standing in the presence of toluene, etc., to excrete the enzyme from the cells. The solution can then be filtered, centrifuged, etc. to remove solids, and, if necessary, nucleic acids can be removed using streptomycin sulfate, protamine sulfate, manganese sulfate, etc. After that, the solution is fractionated by adding ammonium sulfate, alcohol, acetone, etc., and the precipitate can be collected to obtain the crude enzyme.

[0126] To obtain a more purified enzyme preparation from the crude enzyme, for example, gel filtration using Sephadex, Superdex, Ultrogel, etc., adsorption elution using an ion exchange carrier, a hydrophobic carrier, or hydroxyapatite, electrophoresis using polyacrylamide gel, etc., sedimentation methods such as sucrose density gradient centrifugation, affinity chromatography, fractionation methods using molecular sieve membranes or hollow fiber membranes, etc. can be appropriately selected or combined to obtain a purified GLOD enzyme preparation.

[0127] In certain embodiments, the GLOD mutant of the present disclosure may be, for example, one that (i) uses FAD as a coenzyme, (ii) recognizes glutamic acid as a substrate, and (iii) oxidizes glutamic acid to produce 2-oxoglutarate, ammonia, and hydrogen peroxide.

[0128] In some embodiments, the residual activity of a GLOD variant of the present disclosure after heat treatment at 30 to 40°C, for example at 35°C, for 30 or 35 minutes may be 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, relative to the activity before heat treatment, taken as 100%. In some embodiments, the residual activity of a GLOD variant of the present disclosure after heat treatment at, for example, 60°C for 30 or 35 minutes may be 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, relative to the activity before heat treatment, taken as 100%. In some embodiments, the residual activity of a GLOD variant of the present disclosure after heat treatment, for example, at 65°C for 30 or 35 minutes, can be 50% or more, 60% or more, 65% or more, 70% or more, for example, 75% or more, relative to the activity before heat treatment, taken as 100%. In some embodiments, the residual activity of a GLOD variant of the present disclosure after heat treatment, for example, at 70°C for 30 or 35 minutes, can be 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, for example, 35% or more, relative to the activity before heat treatment, taken as 100%.

[0129] The GLOD variants of the present disclosure exclude GLODs that do not exhibit activity towards glutamate.

[0130] (Composition, Reagent, Electrode, Sensor, and Kit) In certain embodiments, the present invention provides a reagent composition, measurement reagent, electrode, sensor, or kit for measuring glutamate, 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, or the like. In addition, a solubilizer, a stabilizer, a reactivity enhancer, a glycated hemoglobin denaturant, a reducing agent, bovine serum albumin, a sugar (glycerin, lactose, sucrose, etc.), or the like may be added. Other known stabilizers, systems for eliminating impurities, etc. may be added to the composition, reagent, electrode, sensor, or kit as needed. Techniques used in various conventional reagents, electrodes, sensors, and kits can be appropriately modified and used in the composition, reagent, electrode, sensor, or kit of the present disclosure.

[0131] Examples of surfactants include nonionic surfactants and ionic surfactants, such as cationic surfactants, anionic surfactants, and amphoteric surfactants.

[0132] Examples of nonionic surfactants include polyoxyethylene alkyl ethers, fatty acid sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides, and alkyl monoglyceryl ethers.

[0133] Examples of cationic surfactants include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkylbenzyldimethylammonium salts, pyridinium salts such as alkylpyridinium salts, phosphonium salts such as alkylphosphonium salts, imidazolium salts such as alkylimidazolium salts, and isoquinonium salts such as alkylisoquinonium salts.

[0134] The reagent for measuring hydrogen peroxide may contain peroxidase and / or a chromogenic substrate. Examples of the chromogenic substrate include, in addition to 4-aminoantipyrine, 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).

[0135] (Method for Measuring Glutamic Acid) In certain embodiments, the present disclosure provides a method for measuring glutamic acid. Glutamic acid measurement can be a qualitative or quantitative method. A quantitative method can include contacting a sample containing glutamic acid with GLOD of the present disclosure and measuring the reaction product or consumed product. The term "contact" in this quantitative method encompasses any manner in which the enzyme and the sample are physically brought together so that GLOD can catalyze the oxidation reaction of glutamic acid. For example, this includes not only mixing free enzyme and glutamic acid in a solution, but also adding or dropping a solution sample containing glutamic acid to an enzyme supported on a solid support.

[0136] The sample used for the measurement can be any sample that may contain glutamic acid. The sample can be appropriately processed.

[0137] The minimum detectable glutamic acid concentration (detection limit concentration) can be determined by examining the glutamic acid concentration range in which the absorbance of the detected chromogenic substrate decreases proportionally as the amount of glutamic acid added decreases, while the amount of enzyme used and reaction time are kept constant. The amount of enzyme and reaction time can be set so that the detection limit is lower than the glutamic acid concentration in the measurement sample or blood.

[0138] In quantitative measurements, a calibration curve can be prepared in advance by performing regression analysis such as the least squares method on the measured values ​​of absorbance of a control containing glutamic acid at a known concentration. The glutamic acid concentration in a sample can be quantified by plotting the measured values ​​of the sample with an unknown glutamic acid concentration against the prepared calibration curve.

[0139] The time for allowing GLOD to act on a sample containing glutamic acid may be, for example, 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, but less than 60 minutes, less than 30 minutes, or less than 10 minutes, for example, less than 5 minutes, for example, 0.5 minutes or more but less than 60 minutes, 1 minute or more but less than 30 minutes, 1 minute or more but less than 20 minutes, for example, 1 minute or more but less than 10 minutes, or for example, 1 minute or more but less than 5 minutes. The reaction temperature varies depending on the optimum temperature of the enzyme used, but is, for example, 20 to 45°C, and can be appropriately selected from temperatures used in ordinary enzymatic reactions.

[0140] The amount of GLOD enzyme used will depend on the amount of substrate contained in the sample solution, but may be added so that the final concentration is, for example, 0.1 to 50 U / ml, e.g., 0.2 to 10 U / ml. The pH during reaction may be adjusted using a buffer, taking into consideration the pH at which GLOD can act, e.g., the optimal pH. The reaction pH is, for example, 3 to 11, 5 to 9, e.g., 6 to 8.

[0141] The measurement of hydrogen peroxide can be carried out simultaneously with the step of generating hydrogen peroxide, and can proceed simultaneously with the action of GLOD. Consumed substances may be measured instead of produced substances. An example of the consumed substance to be measured is dissolved oxygen. The amount of dissolved oxygen in the reaction solution can be measured using a dissolved oxygen meter or the like.

[0142] (Method for measuring GLOD activity) Below, an example of a method for measuring GLOD activity using glutamic acid as a substrate is described, but the measurement method is not limited to this. Glutamic acid may be commercially available. Unless otherwise specified, in this specification, the enzyme activity is defined as the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured using glutamic acid as a substrate at 30° C. and pH 7.4.

[0143] 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 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 incubate at 30°C for 5 minutes. Then, 25 μl of Reagent 5 was added and mixed well, and the absorbance (A) of light at a wavelength of 555 nm was measured using a spectrophotometer U-3900 (Hitachi High-Tech Science) with the cell holder kept at 30°C. ５５５ ) was measured and the A per minute ５５５ Amount of change (ΔA Ｓ As a control experiment, 25 μl of deionized water was added instead of 25 μl of Reagent 5, and the absorbance (A) of light with a wavelength of 555 nm was calculated. ５５５ ) was measured and the A per minute ５５５ Amount of change (ΔA ０ ) was calculated.

[0144] The oxidase activity (U / ml) can be calculated based on the following formula: "39.2" in the formula is the millimolar extinction coefficient (mM) of the quinoneimine dye formed by condensation of 4-AA and TOOS with respect to light having a wavelength of 555 nm. －１ cm －１ ) [Formula] U / ml = (ΔA Ｓ -ΔA ０ )×600×df / (39.2×0.5×V) =30.6×(ΔA Ｓ -ΔA ０ )×df / V When a different color-developing reagent is used, the wavelength and millimolar extinction coefficient at that wavelength according to the color-developing reagent can be used.

[0145] In some embodiments, the present disclosure provides a polynucleotide encoding a glutamate oxidase variant. In some embodiments, the present disclosure provides a vector comprising such a polynucleotide. In some embodiments, the present disclosure provides a host cell transformed with such a vector, i.e., a host cell comprising such a vector. In some embodiments, the present disclosure provides a method for producing a glutamate oxidase variant, comprising culturing such a host cell to produce the glutamate oxidase variant, and obtaining the produced glutamate oxidase variant. In some embodiments, the present disclosure provides a method for oxidizing glutamate in a sample containing glutamate by contacting the glutamate oxidase variant, or a composition, reagent, electrode, sensor, or kit comprising the same, with the sample. In some embodiments, the method is capable of detecting glutamate. In some embodiments, the method is capable of measuring glutamate.

[0146] (Further Methods for Producing GLOD) The present inventors have produced GLODΔ by introducing a specific amino acid sequence deletion into GLOD. They have also produced GLODΔ-T by introducing a specific amino acid substitution into GLODΔ. The deletions and mutations disclosed herein can be combined as appropriate. Further mutants can also be produced based on the findings of the present disclosure. Thus, in one embodiment, the present invention provides a method for producing a GLOD mutant with improved thermostability by modifying GLOD or GLODΔ, the method comprising the following steps: (i) obtaining a GLOD gene or a GLODΔ gene; (ii) incorporating the GLOD gene or the GLODΔ gene into a vector, transforming a host cell, expressing GLOD or GLODΔ, and isolating the expression product; (iii) confirming the activity of the expression product; (iv) measuring the residual activity of the expression product after heat treatment; and (v) (vi) modifying the GLOD gene or GLODΔ gene so that, when the amino acid sequence of GLOD or GLODΔ is aligned with the amino acid sequence set forth in SEQ ID NO: 1, an amino acid at a position corresponding to a position selected from the group consisting of positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1 is substituted, for example, with an amino acid selected from the group consisting of tyrosine, phenylalanine, tryptophan, leucine, isoleucine, valine, and methionine; (vi) incorporating the modified gene into a vector, transforming host cells, expressing the modified gene, and isolating the expression product; (vii) measuring the residual activity of the expression product of the modified gene after heat treatment and comparing it with the value in step (iv); (viii) If the residual activity of the modified variant after heat treatment is increased by 5%, 6%, 7%, 8%, 9%, or 10% or more compared to the residual activity of GLOD or GLODΔ before modification, the modified variant is designated as a GLOD variant with improved thermostability; (ix) if necessary, repeating steps (v) to (vii) for the variant of step (viii); and (x) incorporating the GLOD variant with improved thermostability of step (viii) or (ix) into a kit or composition.

[0147] In certain embodiments, a peptide linker may be inserted into the region of the GLOD variant of the present disclosure, such as GLODΔ or GLODΔ-T, where the amino acid sequence has been deleted. The peptide linker may be, for example, a linker composed of 1 to 20 amino acid residues. The peptide linker may be composed of an amino acid sequence different from the partial amino acid sequence of GLOD. The peptide linker may be composed of, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. The peptide linker may be composed of, for example, 2-19, 3-18, 4-17, 5 to 16, 6-15, e.g., 7-14 amino acid residues. The amino acid residues constituting the peptide linker may be natural amino acids or glycine. Examples of the peptide linker include Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Asp, Glu, Arg, His, and Lys. Examples of the peptide linker include Gly, Ala, Ser, and Thr. In some embodiments, the peptide linker may be GGGGS or a repeat thereof. The number of repeats may be 2, 3, or 4. In some embodiments, the GLOD variants of the present disclosure, such as GLODΔ or GLODΔ-T, do not have a peptide linker.

[0148] In one embodiment, a GLOD variant of the present disclosure, e.g., GLODΔ or GLODΔ-T, has 90% or more amino acid sequence identity with SEQ ID NO: 1. The deletion Δ, if present, shall not be included in the calculation of sequence identity. In another embodiment, a GLOD variant of the present disclosure, e.g., GLODΔ or GLODΔ-T, excludes sequences that have 90% or more amino acid sequence identity with SEQ ID NO: 1.

[0149] In some embodiments, a GLOD variant of the present disclosure, e.g., GLODΔ or GLODΔ-T, has 90% or greater amino acid sequence identity with SEQ ID NO: 58. The deletion Δ, if present, shall not be included in the calculation of sequence identity. In another embodiment, a GLOD variant of the present disclosure, e.g., GLODΔ or GLODΔ-T, excludes sequences that have 90% or greater amino acid sequence identity with SEQ ID NO: 58. In some embodiments, a GLOD variant of the present disclosure, e.g., GLODΔ or GLODΔ-T, excludes known GLODs and known GLOD variants.

[0150] In a mutant in which positions 459 to 507 of SEQ ID NO: 1 have been deleted, the amino acid numbering is shifted. The position corresponding to position 516 of SEQ ID NO: 1 is the 467th position counting from the N-terminus in the deletion mutant. The position corresponding to position 566 of SEQ ID NO: 1 is the 517th position counting from the N-terminus in the deletion mutant. The position corresponding to position 568 of SEQ ID NO: 1 is the 519th position counting from the N-terminus in the deletion mutant. The position corresponding to position 585 of SEQ ID NO: 1 is the 536th position counting from the N-terminus in the deletion mutant. The position corresponding to position 615 of SEQ ID NO: 1 is the 566th position counting from the N-terminus in the deletion mutant.

[0151] The GLOD of the present disclosure is further illustrated by the following examples, which are for illustrative purposes only and are not intended to limit the present disclosure in any way.

[0152] Example 1 Improvement of Heat Resistance of Glutamate Oxidase (StGLOD) Derived from Streptomyces sp. X-119-6 by Deleting Amino Acid Sequence 1. Construction of Plasmid for Expression of Glutamate Oxidase (GLOD) A plasmid (pKK223-3-StGLOD) for expression of glutamate oxidase (StGLOD) derived from Streptomyces sp. X-119-6 having the amino acid sequence of SEQ ID NO: 1 was prepared using NEBuilder HiFi DNA Assembly (New England Biolabs).

[0153] The StGLOD gene having the base sequence of SEQ ID NO: 2 was divided into three fragments, SEQ ID NO: 3 (StGLOD-f1), SEQ ID NO: 4 (StGLOD-f2), and SEQ ID NO: 5 (StGLOD-f3), and synthesis was outsourced to Integrated DNA Technologies. The 15 bases on the 3' end of StGLOD-f1 and the 15 bases on the 5' end of StGLOD-f2 are overlapping sequences for gene assembly. The 15 bases on the 3' end of StGLOD-f2 and the 15 bases on the 5' end of StGLOD-f3 are overlapping sequences for gene assembly.

[0154] The plasmid fragment was prepared by PCR using the pKK223 plasmid as a template and primers of SEQ ID NO: 6 (ggtcatttcattcatgaattctgtttcctgtgtgaaattg) and SEQ ID NO: 7 (gcgttaacttcttaagcttggctgttttggcggatgag). The SEQ ID NO: 6 or SEQ ID NO: 7 primers each contain a 15-base sequence overlapping with StGLOD-f1 or StGLOD-f3 at the 5' end of the sequence that anneals to pKK223-3. After PCR, 1.0 μl of DpnI (New England BioLabs) was added to the solution and incubated at 37°C for 1 hour. The amplified fragment was then purified using a GFX PCR DNA and Gel Band Purification Kit (Cytiva).

[0155] The composition shown in the table below was reacted at 50°C for 60 minutes to obtain a plasmid for expressing StGLOD (pKK223-3-StGLOD). The resulting plasmid was transformed into E. coli JM109. The resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed by DNA sequence analysis to be the desired sequence.

[0156]

[0157] 2. Preparation of StGLOD deletion mutants Site-directed mutagenesis was performed using pKK223-3-StGLOD as a template to obtain a plasmid carrying a gene encoding the StGLOD deletion mutant. 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 (pKK223-3-StGLOD), and 3.5 μl of ion-exchanged water. A deletion mutant prepared using primers of SEQ ID NO: 8 (gataagaccgaagcgaccaatgcgtacgga) and SEQ ID NO: 9 (cagcttacgataatagtcgtacagacccgg) was designated StGLODΔ49Ai, and a deletion mutant prepared using primers of SEQ ID NO: 10 (gcagagccacctgcgaccaatgcgtacgga) and SEQ ID NO: 11 (cgcatcttgataatagtcgtacagacccgg) was designated StGLODΔ49C. The amino acid sequences of StGLODΔ49Ai and StGLODΔ49C are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The PCR reaction conditions were a cycle of 98°C for 10 seconds, 55°C for 5 seconds, and 68°C for 35 seconds, repeated seven times.

[0158] 1 μl of DpnI was added to the post-PCR solution and treated at 37°C for 1 hour to degrade the template pKK223-3-StGLOD. 2 μl of the resulting DpnI-treated solution was mixed with 5 μl of Ligation High Ver. 2 (Toyobo), 1 μl of 5 U / μl T4 polynucleotide kinase, and 7 μl of ion-exchanged water, and the mixture was reacted at 16°C for 1 hour. The reaction solution was then used to transform E. coli JM109 strain. The resulting transformants were cultured, and the base sequence of the extracted plasmid was confirmed to be the desired sequence by DNA sequence analysis.

[0159] 1 μl of DpnI was added to the post-PCR solution and treated at 37°C for 1 hour to degrade the template pKK223-3-StGLODΔ49C. The resulting DpnI-treated solution was used to transform E. coli JM109. The resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed to be the desired sequence by DNA sequence analysis.

[0160] 3. Recombinant Production of GLOD The GLOD-producing strain was inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 μg / ml) placed in a test tube and cultured overnight at 37°C and 160 rpm. 2.5 ml of the seed culture was inoculated into 250 ml of LB-amp medium (ampicillin concentration 50 μg / ml) containing 0.1 mM IPTG placed in a Sakaguchi flask and cultured at 25°C and 130 rpm for 16 hours.

[0161] The culture medium was centrifuged at 8,000 rpm for 10 minutes, and the resulting pellet was resuspended in 4 ml of 10 mM potassium phosphate buffer (PPB) pH 7.5. The bacterial cell suspension was then sonicated and centrifuged at 15,000 rpm for 15 minutes. The supernatant was collected and used as the GLOD crude enzyme solution.

[0162] 4. Measurement of GLOD activity The reagents used to measure GLOD activity were 4-aminoantipyrine (4-AA) (Fujifilm Wako Pure Chemical Industries), TOOS (Dojindo Laboratories), and horseradish peroxidase (POD) (Toyobo). The composition of the activity measurement reagent is shown in Table 2. The GLOD solution was diluted with 10 mM PPB (pH 7.4) containing 0.15% bovine serum albumin (BSA, Sigma-Aldrich).

[0163]

[0164] 725 μl of the reagent in the above table was incubated at 30°C for 5 minutes, and then 25 μl of 300 mM sodium hydrogen glutamate (GluNa, manufactured by Fujifilm Wako Pure Chemical Industries) solution was added and mixed. The absorbance (A) of light at a wavelength of 555 nm was measured using a spectrophotometer U-3900 (manufactured by Hitachi High-Tech Science) with the cell holder kept at 30°C. ５５５ ) was measured and the A per minute ５５５ Amount of change (ΔA ＳMeasurements were also carried out by adding 25 μl of ion-exchanged water instead of the substrate solution (GluNa solution), and the A per minute was calculated. ５５５ Amount of change (ΔA ０ ) was calculated.

[0165] The oxidase activity (U / ml) was calculated based on the following formula: "39.2" in the formula is the millimolar extinction coefficient (mM) of the quinoneimine dye formed by condensation of 4-AA and TOOS with respect to light with a wavelength of 555 nm. －１ cm －１ ) [Formula] U / ml = (ΔA Ｓ -ΔA ０ )×600×df / (39.2×0.5×V) =30.6×(ΔA Ｓ -ΔA ０ ) × df / V

[0166] 5. Evaluation of GLOD Thermal Stability The GLOD crude enzyme solution was diluted with 10 mM PPB, pH 6.0, to a final GLOD concentration of 0.05 U / ml. Subsequently, 240 μl of 0.05 U / ml GLOD solution was mixed with 160 μl of 250 mM PPB, pH 6.0, and heated for 30 or 35 minutes in a water bath maintained at a predetermined temperature. After heating, the GLOD solution was quickly cooled on ice, and activity was measured using 375 μl of the GLOD solution. The activity of the sample cooled on ice without heating was set as 1, and the residual activity of the heated sample was calculated. Residual activity was calculated three times for each GLOD, and the average value was used to evaluate thermal stability.

[0167] Table 3 shows the residual activity of the StGLOD deletion mutants after heating at 45° C. for 30 minutes.

[0168]

[0169] The residual activity of the StGLOD deletion mutants shown in the table above was increased by 0.11 to 0.37 compared to StGLOD, and all of the StGLOD deletion mutants had improved thermostability. In other words, the residual activity was improved by approximately 20% and approximately 65% ​​compared to the wild-type.

[0170] [Example 2] Improvement of thermostability of StGLODΔ49C by amino acid substitution 6. Preparation of modified StGLODΔ49C The expression and residual activity of StGLODΔ49C were confirmed. Therefore, further modified mutants based on StGLODΔ49C were prepared.

[0171] Site-directed mutagenesis was performed using the StGLODΔ49C expression plasmid (pKK223-3-StGLODΔ49C) as a template to obtain a plasmid carrying a gene encoding modified StGLOD. 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 (pKK223-3-StGLOD), and 3.5 μl of ion-exchanged water. The names of the mutants and the combinations of Fw primer and Rv primer are shown in Table 4. The PCR reaction conditions were a cycle of 98°C for 10 seconds, 55°C for 5 seconds, and 68°C for 35 seconds, repeated 15 times. A plasmid for expressing the multiple mutant StGLODΔ49C was constructed by repeatedly introducing single mutations. For example, a plasmid for expressing the double mutant StGLODΔ49C / F87Y / Y103I was constructed by PCR using pKK223-3-StGLODΔ49C / F87Y as a template and primers of SEQ ID NO: 20 and SEQ ID NO: 16.

[0172]

[0173] PCR of the prepared mutants, recombinant production of GLOD, activity measurement, and evaluation of thermal stability were all performed in the same manner as for StGLODΔ49C described above. The residual activity of the modified StGLODΔ49C after heating at 50°C for 35 minutes is shown in the table below.

[0174]

[0175] The residual activity of the modified StGLODΔ49C shown in the table above was increased by 0.02 to 0.72 compared to StGLODΔ49C, and all of the modified StGLODΔ49C enzymes had improved thermostability. In other words, the residual activity of the modified enzymes produced was improved by about 9% or more to about 325% or more compared to the unmodified StGLODΔ49C enzyme.

[0176] An octuplet mutant was prepared by combining eight amino acid substitutions (F87Y, Y103I, F133Y, F297M, F393L, F428Y, Y517F, and Y536L) that contributed to the improvement of the thermal stability of StGLODΔ49C. The residual activity of the StGLOD deletion mutant after heating under heat treatment conditions that were more severe than those listed in Table 9 (60°C, 65°C, or 70°C for 35 minutes) is shown in the table below.

[0177]

[0178] The octamutant of StGLODΔ49C shown in the above table was stable even when heated at 60° C. for 35 minutes, and retained more than one-third of its activity even when heated at 70° C. for 35 minutes. It was revealed that the thermal stability of StGLODΔ49C was dramatically improved by combining the amino acid substitutions shown in Table 9.

[0179] Example 3: Obtaining a thermostability-improved StGLOD homologue 7. Construction of a plasmid for expressing a StGLOD homologue having a thermostability-improving mutation An amino acid sequence (M7GLODΔ49C, SEQ ID NO: 59) was designed in which a putative glutamate oxidase (M7GLOD) derived from Streptomyces sp. MOE7 having the amino acid sequence of SEQ ID NO: 58 had been deleted in the same manner as StGLODΔ49C, and an amino acid sequence (M7GLODΔ49C-T8, SEQ ID NO: 60) was designed in which eight amino acid substitutions (F87Y, Y103I, F133Y, F297M, F393L, F428Y, Y517F, Y536L) had been introduced.

[0180] The M7GLODΔ49C-T8 expression plasmid (pET22b-M7GLODΔ49C-T8) was prepared using In-Fusion HD Cloning Kit (Clontech). The M7GLODΔ49C-T8 gene having the nucleotide sequence of SEQ ID NO: 61 was divided into three fragments of SEQ ID NO: 62 (M7GLODΔ49C-T8-f1), SEQ ID NO: 63 (M7GLODΔ49C-T8-f2), and SEQ ID NO: 64 (M7GLODΔ49C-T8-f3), and synthesis was entrusted to Integrated DNA Technologies. The 15 bases on the 5'-end of M7GLODΔ49C-T8-f1 are a duplicated sequence for assembly into pET-22b(+). The 15 bases on the 3' end of M7GLODΔ49C-T8-f1 and the 15 bases on the 5' end of M7GLODΔ49C-T8-f2 are overlapping sequences for gene assembly. The 15 bases on the 3' end of M7GLODΔ49C-T8-f2 and the 15 bases on the 5' end of M7GLODΔ49C-T8-f3 are overlapping sequences for gene assembly. The 15 bases on the 3' end of M7GLODΔ49C-T8-f3 are overlapping sequences for assembly into pET-22b(+).

[0181] The plasmid fragment was prepared by PCR using the pET-22b(+) plasmid as a template and primers of SEQ ID NO: 65 (catatgtatatctccttcttaaag) and SEQ ID NO: 66 (taacaaagcccgaaaggaag). 1.0 μl of DpnI (New England BioLabs) was added to the PCR solution, which was then incubated at 37°C for 1 hour. The amplified fragment was then purified using a GFX PCR DNA and Gel Band Purification Kit (Cytiva).

[0182] The composition shown in the table below was reacted at 50°C for 15 minutes to obtain a plasmid for expressing M7GLODΔ49C-T8 (pET22b-M7GLODΔ49C-T8). The resulting plasmid was transformed into E. coli JM109. The resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed by DNA sequence analysis to be the intended sequence. Subsequently, pET22b-M7GLODΔ49C-T8 was transformed into E. coli BL21(DE3) to prepare a strain producing M7GLODΔ49C-T8.

[0183]

[0184] Using pET22b-M7GLODΔ49C-T8 as a template and primers of SEQ ID NO: 67 (atcgaggtcttttatactggagctggacaa) and SEQ ID NO: 68 (aaagacctcgatgcggcggccatggaccga), a plasmid (pET22b-M7GLODΔ49C-T7) carrying a gene encoding M7GLODΔ49C-T8 without the Y536L amino acid substitution was obtained according to the method described in "6. Preparation of modified StGLODΔ49C." M7GLODΔ49C-T8 without the Y536L amino acid substitution is referred to as M7GLODΔ49C-T7 (SEQ ID NO: 69).

[0185] Using pET22b-M7GLODΔ49C-T7 as a template and the primers shown in Table 12, plasmids for expressing various modified M7GLODΔ49C-T7 were constructed in the same manner as in "6. Preparation of modified StGLODΔ49C." The names of the mutants prepared and the combinations of Fw primer and Rv primer are shown in Table 12.

[0186]

[0187] Subsequently, E. coli BL21(DE3) strain was transformed with pET22b-M7GLODΔ49C-T7 or an expression plasmid for various modified M7GLODΔ49C-T7 to prepare strains producing M7GLODΔ49C-T7 and various modified M7GLODΔ49C-T7.

[0188] 8. Recombinant production and thermostability evaluation of M7GLODΔ49C-T7 M7GLODΔ49C-T7 and modified M7GLODΔ49C-T7 production 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 180 rpm. 2.5 ml of the seed culture was inoculated into 250 ml of LB-amp medium (ampicillin concentration 100 μg / ml) in a Sakaguchi flask and cultured at 37 ° C and 130 rpm. When the OD600 of the culture reached 0.6 to 1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM or 0.1 mM, and the culture was cultured at 15 ° C and 130 rpm for 16 hours.

[0189] The culture medium was centrifuged at 8,000 rpm for 10 minutes, and the resulting pellet was resuspended in 4 to 25 ml of 10 mM potassium phosphate buffer (PPB) pH 6.0. The bacterial cell suspension was sonicated and then centrifuged at 15,000 rpm for 15 minutes. The supernatant was collected and used as M7GLODΔ49C-T7 and modified M7GLODΔ49C-T7 crude enzyme solutions.

[0190] M7GLODΔ49C-T7 and modified M7GLODΔ49C-T7 were heated at 70°C for 30 minutes, and their residual activity was calculated according to the method described above (Table 13). M7GLODΔ49C-T7 was also heated at 60°C or 65°C for 30 minutes, and their residual activity was calculated.

[0191] M7GLODΔ49C-T7 exhibited high thermostability, with a residual activity of 0.93 after heating at 60°C for 30 minutes and a residual activity of 0.69 after heating at 65°C for 30 minutes. It was demonstrated that the thermostability of GLOD, including that of StGLOD, can be improved by combining the amino acid substitutions shown in Table 9.

[0192]

[0193] Furthermore, the residual activity of each of the modified M7GLODΔ49C-T7 enzymes was increased by 0.05 to 0.58 compared to M7GLODΔ49C-T7, and the thermostability of each of the modified M7GLODΔ49C-T7 enzymes was improved. In other words, the residual activity of the modified enzymes was improved by about 29% to about 341% compared to the unmodified M7GLODΔ49C-T7 enzyme.

[0194] Example 4: Verification of the effect of deletion on improving thermostability in M7GLOD-T8 9. Construction of a plasmid for expressing M7GLOD The expression plasmid (pET22b-M7GLOD) for M7GLOD (SEQ ID NO: 58) was prepared in the same manner as in "7. Construction of a plasmid for expressing StGLOD homologues with mutations that improve thermostability." Synthesis of a DNA fragment (SEQ ID NO: 86) containing the M7GLOD gene (SEQ ID NO: 85) was outsourced to Integrated DNA Technologies. The 15 bases on the 5'-end and 15 bases on the 3'-end of SEQ ID NO: 86 are overlapping sequences used for assembly into pET-22b(+). The pET-22b(+) plasmid fragment amplified using SEQ ID NO: 65 and SEQ ID NO: 66 was ligated to the base sequence of SEQ ID NO: 86 by in-fusion reaction to obtain pET22b-M7GLOD. The resulting plasmid was used to transform E. coli JM109 strain, and the resulting transformant was cultured. The base sequence of the extracted plasmid was confirmed by DNA sequence analysis to be the desired sequence.

[0195] 10. Construction of Plasmid for Expression of M7GLOD-T8 Using a similar method, a plasmid (pET22b-M7GLOD-T8) for expression of M7GLOD-T8 (SEQ ID NO: 87) was prepared, in which eight amino acid substitutions (F87Y, Y103I, F133Y, F297M, F393L, F428Y, Y566F, Y585L) were introduced into M7GLOD. Note that the amino acid substitutions Y566F and Y585L were introduced as Y517F and Y536L in M7GLODΔ49C-T8 (SEQ ID NO: 60).

[0196] Using pET22b-M7GLOD as a template, a fragment was amplified by PCR using primers SEQ ID NO: 88 (ccgtcgttggtgggaattc) and SEQ ID NO: 89 (gcgctcagcatcgtcaaaag). Using pET22b-M7GLODΔ49C-T8 as a template, a fragment was amplified by PCR using primers SEQ ID NO: 90 (cttttgacgatgctgagcgc) and SEQ ID NO: 91 (gaattcccaccaacgacgg). Both fragments were treated with DpnI, purified, and ligated by in-fusion reaction to obtain pET22b-M7GLOD-T8. The resulting plasmid was transformed into E. coli JM109. The resulting transformant was cultured, and the nucleotide sequence of the extracted plasmid was confirmed to be the desired sequence by DNA sequence analysis.

[0197] 11. Preparation of Various Deletion-Type M7GLOD-T8s and Evaluation of Thermal Stability Various deletion-type M7GLOD-T8s were prepared by performing PCR in the same manner as in "6. Preparation of Modified StGLODΔ49C" using pET22b-M7GLOD-T8 as a template to delete all or part of the region encoding positions 460 to 508 of SEQ ID NO:87. PCR was performed using 35 cycles of "98°C for 10 seconds, 68°C for 40 seconds." The primers used are listed in Table 14.

[0198]

[0199] The PCR product was treated with DpnI, and 2 μL of the resulting mixture was mixed with 5 μL of Ligation High Ver. 2 (Toyobo Co., Ltd.), 1 μL of T4 polynucleotide kinase (Toyobo Co., Ltd.), and 7 μL of ion-exchanged water, and a ligation reaction was carried out at 16°C for 60 minutes. The resulting reaction product was transformed into E. coli JM109 strain. The resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed to be the desired sequence by DNA sequence analysis.

[0200] Subsequently, E. coli BL21(DE3) was transformed with the expression plasmids for M7GLOD-T8 and various deletion-type M7GLOD-T8 to prepare strains producing M7GLOD-T8 and various deletion-type M7GLOD-T8, and these strains were recombinantly produced according to "8. Recombinant production of M7GLODΔ49C-T7." M7GLOD-T8 and various deletion-type M7GLOD-T8 were heated at 65°C for 30 minutes, and their residual activity was calculated according to the method described above (Table 15).

[0201]

[0202] The residual activity of each of the deletion types of M7GLOD-T8 was increased by 0.05 to 0.54 compared to M7GLOD-T8, and the thermostability of each of the deletion types of M7GLOD-T8 was improved. In other words, the residual activity of the modified enzymes produced was improved by about 38% to about 415% compared to the unmodified M7GLODΔ49C-T7.

[0203] [Example 5] Improvement of thermostability of StGLOD by amino acid substitution 12. Construction of a plasmid for expressing StGLOD A plasmid (pET22b-StGLOD) for expressing StGLOD (SEQ ID NO: 1) was prepared in the same manner as in "7. Construction of a plasmid for expressing StGLOD homologue having a mutation for improving thermostability." Using pKK223-3-StGLOD as a template and primers of SEQ ID NO: 99 (gaaggagatatacatatgaatgaaatgacctacgagcaattg) and SEQ ID NO: 100 (tcctttcgggctttgttaagaagttaacgcctcctc), a fragment containing the StGLOD gene was amplified by PCR. PCR amplification was performed using the pET-22b(+) plasmid as a template and primers of SEQ ID NO: 101 (caaagcccgaaaggaagctgagttggctgc) and SEQ ID NO: 102 (tcctttcgggctttgttaagaagttaacgcctcctc). PCR was performed in the same manner as in "2. Preparation of StGLOD deletion mutants."

[0204] Both fragments were treated with DpnI, purified, and ligated by in-fusion reaction to obtain pET22b-StGLOD. The resulting plasmid was transformed into E. coli JM109. The resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed to be the desired sequence by DNA sequence analysis.

[0205] 13. Construction of StGLOD Mutants Site-directed mutagenesis was performed using the StGLOD expression plasmid (pET22b-StGLOD) as a template to construct expression plasmids for various modified StGLODs in the same manner as in "6. Construction of modified StGLOD Δ49C." The names of the mutants constructed and the combinations of Fw primer and Rv primer are shown in Table 16.

[0206]

[0207] Subsequently, E. coli BL21(DE3) was transformed with pET22b-StGLOD or the expression plasmids for various modified StGLODs to prepare strains capable of producing StGLOD and various modified StGLODs, and these strains were recombinantly produced according to "8. Recombinant Production of M7GLODΔ49C-T7." StGLOD and various modified StGLODs were heated at 50°C for 30 minutes, and their residual activity was calculated according to the method described above (Table 17).

[0208]

[0209] The residual activity of each modified StGLOD was increased by 0.01 to 0.76 compared to StGLOD, and all of the modified StGLODs had improved thermostability. In other words, the residual activity of the modified enzymes produced was improved by about 6% to about 422% compared to StGLOD before modification. It was demonstrated that the amino acid substitutions found to improve thermostability in StGLODΔ49C or M7GLODΔ49C-T7 are not limited to their parent enzymes, and also contribute to the improvement of thermostability of StGLOD. Therefore, it is expected that similar improvements in thermostability will be achieved when these amino acid substitutions are introduced into GLODs of other origins or GLODs with high sequence identity thereto.

[0210] The glutamate oxidase variants of the present disclosure can be produced on a large scale without requiring treatment with a protease, and can be used in the oxidation reaction of glutamate.

[0211] Various documents, including patent applications and manufacturer's manuals, are cited herein. The disclosures of these documents are not considered relevant to the patentability of this disclosure, but are incorporated herein by reference in their entirety. More particularly, all referenced documents are incorporated herein by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

[0212] [Sequence Listing] SEQ ID NO: 1: 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

[0213]

[0214] Sequence number 3 StGLOD-f1 atgaatgaaatgacctacgagcaattggcccgcaattggttggtccggccccgacgaacgaggactttaaattacgctatttggatgtgctgatgataacgggctgaacccggggtccgcctaagcgtatcttgatcgtcggggctggtttgcaggccttgttgccggagatttgttgacccgcgctgggcatgatgttactattcttgaagctaatgccaaccgtgtgggaggcgtatcaagacgttccatgcaaaaaaaaaggagagccatctccattcgctgatccagcgcagttcggaagctggggcgatgcgtcttccttcctttcacccacttacc ttggcacttatcgacaagctgggcttaaaacgccgcctgttttttaatgtagattattgacccacaaactggaaatcaagatgctccagtcccaccggtcttctataagtcttttaaggacggcaagacgtggacaaatggagccccgagccctgagtttaaagagcccgataaacgt aatcacacctggatccgtacgaatcgcgaacaagtacgccgcgcccaatacgccacagacccgagctccatcaatgaaggcttccatctgacgggatgtgagacccgccttactgtctccgacatggttaaccaagcgcttgaaccggtacgcgactattattcagtcaagcaggac

[0215] Sequence number 4: StGLOD-f2 tcagtcaagcaggacgatggaactcgtgtgaataagcctttcaaagaatggcttgcagggtgggccgacgtcgtgcgtgatttcgacggttattctatgggccgcttcttacgtgaatatgccgagttttcggatgaggcagtggaagcaattggcacaattgaaaacatgacctcgcgcctgcaccttgccttttttcattcgtttttgggccgttccgatatcgatccgcgcgccacttactgggagatcgagggggggtcacgcatgcttcctgaaacactggcaaaggacctgcgcgatcagatcgttatgggtcagcgcatggtacgcttagagtactatgacccgggccgcgatggccatcatggtgagcttactgggcctgggggtccggccgtggccattcagacggttccagagggagagccttacgccgcaactcaaacgtggacaggagacctggcgattgttactatcccctttagctctttacgctttgttaaagtaacgccacccttctcttataaaaagcgccgcgcggtaattgagacccattatgatcaggccaccaaagtcttacttgaatttagccgtcgttggtgggaattcacagaggcggactggaagcgtgagcttgatgctatcgccccgggtctgtacgactattatcaacagtggggtgaagac

[0216] Accession number 5: StGLOD-f3 cagtggggtgaagacgacgccgaagcagcgcttgcactgccgcagtcagtccgcaacttgcctaccggcttgcttggggcccacccaagcgtcgacgaatcgcgtatcggagaggagcaagttgagtattatcgtaacagcgaactgcgtggcggagtgcgccctgcgaccaatgcgtacggagggggtagtacgaccgataatcctaatcgtttcatgtactatccctcccaccccgttccaggcacgcaggggggggtcgtacttgcagcatacagttggagcgatgatgcagctcgttgggattcttttgacgatgctgagcgctatggctatgcgcttgaaaacttacagtctgtccacggccgtcgtattgaggtgttctatacaggagcgggacagacccaatcatggctgcgcgatccgtacgcatgcggcgaggcagctgtctatacaccccatcaaatgaccgcgtttcacttagatgtggtgcgtcccgaggggcccgtctattttgcgggtgagcacgtttcattaaaacatgcctggatcgagggggcggtggaaacagccgttcgtgcggcgatcgctgtaaatgaagcccccgtaggtgacactggggtcacagccgctgctgggcgccgtggggcggccgccgctactgagcctatgcgcgaggaggcgttaacttcttaa

[0217] Accession numbers 6 to 11: Primer sequences

[0218] SEQ ID NO: 12 Amino acid sequence of StGLODΔ49Ai MNEMTYEQLARELLLVGPAPTNEDLKLRYLDVLIDNGLNPPGPPKRILIVGAGIAGLVAGDLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPSPFADPAQYAEAGAMRLPSFHPLTLALIDKLGLKRRLFFNVDIDPQTGNQDAPVPPVFYKSFKDGKTWTNGAPSPEFKEPDKRNHTWIRTNREQVRRAQYATDPSSINEGFHLTGCETRLTVSDMVNQALEPVRDYYSVKQDDGTRVNKPFKEWLAGWADVVRDFDGYSMGRFLREYAEFSDEAVEAIGTIENMTSRLHLAFFHSFLGRSDIDPRATYWEIEGGS RMLPETLAKDLRDQIVMGQRMVRLEYYDPGRDGHHGELTGPGGPAVAIQTVPEGEPYAATQTWTGDLAIVTIPFSSLRFVKVTPPFSYKKRRAVIETHYDQATKVLLEFSRRWWEFTEADWKRELDAIAPGLYDYYRKLDKTEATNAYGGGSTTDNPNR FMYYPSHPVPGTQGGVVLAAYSWSDDAARWDSFDDAERYGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTAFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVGDTGVTAAAGRRGAAAATEPMREEALTS

[0219] SEQ ID NO: 13 Amino acid sequence of StGLODΔ49C MNEMTYEQLARELLLVGPAPTNEDLKLRYLDVLIDNGLNPPGPPKRILIVGAGIAGLVAGDLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPSPFADPAQYAEAGAMRLPSFHPLTLALIDKLGLKRRLFFNVDIDPQTGNQDAPVPPVFYKSFKDGKTWTNGAPSPEFKEPDKRNHTWIRTNREQVRRAQYATDPSSINEGFHLTGCETRLTVSDMVNQALEPVRDYYSVKQDDGTRVNKPFKEWLAGWADVVRDFDGYSMGRFLREYAEFSDEAVEAIGTIENMTSRLHLAFFHSFLGRSDIDPRATYWEIEGGS RMLPETLAKDLRDQIVMGQRMVRLEYYDPGRDGHHGELTGPGGPAVAIQTVPEGEPYAATQTWTGDLAIVTIPFSSLRFVKVTPPFSYKKRRAVIETHYDQATKVLLEFSRRWWEFTEADWKRELDAIAPGLYDYYQDAAEPPATNAYGGGSTTDNPNR FMYYPSHPVPGTQGGVVLAAYSWSDDAARWDSFDDAERYGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTAFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVGDTGVTAAAGRRGAAAATEPMREEALTS

[0220] SEQ ID NOs: 14 to 57 Primer sequences

[0221] SEQ ID NO: 58: Amino acid sequence of putative glutamate oxidase (M7GLOD) from Streptomyces sp. MOE7: MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPAPFTDPAQYAEAGAMRLPSFHPLTLALIDKLGLKRRLFFNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNRTQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFFHSFLGRSDIDPSATYWEIEGGSRQLPEALAKDLRDQIVMGQRMVRLEY YDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPFSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEFSRRWWEFTEEDWKRELDAIAPGLYEYYQRWGEDDAEAALTVPESVRNLPTGLLGAHPSVDEQLIDDEQVEYLRNSTLRGGVRPATQVHG GGSTTDNPNRFMYYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERYGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0222] SEQ ID NO: 59: Amino acid sequence of deleted M7GLOD (M7GLODΔ49C) MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPAPFTDPAQYAEAGAMRLPSFHPLTLALIDKLGLKRRLFFNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNRTQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFFHSFLGRSDIDPSATYWEIEGGSR QLPEALAKDLRDQIVMGQRMVRLEYYDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPFSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEFSRRWWEFTEEDWKRELDAIAPGLYEYYQDAAEPPATQVHGGGSTTDNPNRFM YYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERYGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0223] SEQ ID NO: 60: Amino acid sequence of M7GLODΔ49C (M7GLODΔ49C-T8) into which eight thermostabilizing mutations have been introduced MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTYHAKKGEPAPFTDPAQIAEAGAMRLPSFHPLTLALIDKLGLKRRLFYNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNRTQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFMHSFLGRSDIDPSATYWEIEGGSR QLPEALAKDLRDQIVMGQRMVRLEYYDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPLSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEYSRRWWEFTEEDWKRELDAIAPGLYEYYQDAAEPPATQVHGGGSTTDNPNRFM YYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERFGYALENLQSVHGRRIEVFLTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0224]

[0225] Sequence number 62 M7GLODΔ49C-T8-f1 ggagatatacatatggacgacaagacctatcagcagttagcacgcgaactgctgcttgtagggcccgagccagcaatgaagatcttaaattgcgctatttagacgtacttatgataacgggctggagcccccagtcgatcgtaaacgtatcttaattgtgggggcagggatcgccccggcctggtcgcgggacatttgttgacacgcgcgggacacgatgtcaccatcctggaggcgaatgcaaatcgcgttggaggtccattaagacatatcatgcaaagaaaggcgaacggccccttcaccgacccggctcaaattgcagag gcgggagctatgcgtttgccttcattccatcccttaacgttagcgctgattgataaattagggttgaagcgtcgcttgttctataatgttgacattgaccctaagacgggcaatcaaggtgcagcattgccgcctgtcgtttacaagagctttaaggacggtaa aacttggacgtatggtaaaccttctccggaattccgtgaaccagacaaacgtaaccacacttggattcgtaccaatcgcactcaagtacgtcgcgcccagtatgtgaaggacccgagtgcgattaacgaagggttccatttaaccgggtgcgagtcgcgtttga

[0226] SEQ ID NO: 63 M7GLODΔ49C-T8-f2 gcgagtcgcgtttgacagtttctgatatggtgaaccaagctttagaaccggttcgtgactactattctgtattgcagagcgacgggcgccgcgtaaataagccatttaaggagtggttagacggctgggccggtgtaatccgcgatttcgacggattctcaatgggacgttttctgcgcgagtacgctgggttttcggacgaggcggtagaagcaatcggtaccatcgagaatatgacaagtcgtttgcatttggcatttatgcacagcttcttaggtcgcagtgacattgaccccagcgcgacatattgggagattgaaggcggtagccgtcagctgcctgaggctctggcaaaagacctgcgtgatcagattgtaatgggccaacgcatggtgcgtttagagtactacgacccaggacgtgatggacaccatggaggtctggcaggaccctctggtcctgcggttgcaatcgaaactgtacccgagaacgagccaagtgcagagccgcagacgtggactgcggacctggcgattgtcactgtaccactttcgtctcttcgctttgttgcggtgactcccccattcagttataaaaaacgtcgtgcagttatcgaaactcactatgatcaggcaacaaaagtgctgttagagtattctcgtcgctggtg

[0227] SEQ ID NO: 64 M7GLODΔ49C-T8-f3 ttctcgtcgctggtgggagtttacagaagaggactggaagcgtgagttggacgcgattgcccccggcctgtatgaatactaccaggatgctgcagaaccgccggcaactcaagttcatggaggaggttcaacaactgacaatccaaaccgttttatgtattatccgagtcacgcggtgcctggttctaagggtggtgtggttttggccgcgtatagttggagtgacgatgcagcgcgttgggattcctttgatgacgccgaacgttttggctacgccctggaaaaccttcaatcggtccatggccgccgcatcgaggtctttttaactggagctggacaaactcagtcatggttgcgcgacccctacgcctgcggtgaggccgcagtttacacaccacatcagatgacatcttttcacttagacgtagtgcgcccggaggggccagtatactttgcgggagagcatgtctctcttaagcatgcgtggatcgagggggccgttgaaaccgcggtccgtgctgcgatcgcggttaacgaagcccccgtaccatacgacacagctgctgctcgtgcggaggcccctcgcgaacgtgccggtacagcatcagccactcgcacgcgcgagaaggccgtgacttcctaacaaagcccgaaa

[0228] SEQ ID NOs: 65 to 68 Primer Sequences

[0229] SEQ ID NO: 69 Amino acid sequence of M7GLODΔ49C (M7GLODΔ49C-T7) into which seven thermostabilizing mutations have been introduced MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTYHAKKGEPAPFTDPAQIAEAGAMRLPSFHPLTLALIDKLGLKRRLFYNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNRTQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFMHSFLGRSDIDPSATYWEIEGGSR QLPEALAKDLRDQIVMGQRMVRLEYYDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPLSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEYSRRWWEFTEEDWKRELDAIAPGLYEYYQDAAEPPATQVHGGGSTTDNPNRFM YYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERFGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0230] SEQ ID NOs: 70 to 84 Primer sequences

[0231]

[0232]

[0233] SEQ ID NO: 87: Amino acid sequence of M7GLOD-T8 MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTYHAKKGEPAPFTDPAQIAEAGAMRLPSFHPLTLALIDKLGLKRRLFNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNRTQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFMHSFLGRSDIDPSATYWEIEGGSRQLPEALAKDLRDQIVMGQRMVRLEY YDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPLSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEYSRRWWEFTEEDWKRELDAIAPGLYEYYQRWGEDDAEAALTVPESVRNLPTGLLGAHPSVDEQLIDDEQVEYLRNSTLRGGVRPATQVHG GGSTTDNPNRFMYYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERFGYALENLQSVHGRRIEVFLTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0234] SEQ ID NOs: 88 to 117 Primer sequences

[0235] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A glutamate oxidase mutant having a deleted region, the wild-type glutamate oxidase lacks all or part of the γ-β region between the γ-chain region and the β-chain region, The carboxy-terminal amino acids of the gamma chain region of wild-type glutamate oxidase may or may not be deleted by 1 to 10 amino acids, The amino acid sequence of the wild-type glutamate oxidase is deleted with or without 1 to 4 amino acids at the amino terminal end of the β-chain region, A glutamate oxidase mutant having one continuous deleted region and having glutamate oxidase activity.

2. The glutamate oxidase mutant according to claim 1, wherein 31 to 54 consecutive amino acids are deleted.

3. 2. The glutamate oxidase mutant according to claim 1, wherein the C-terminus of the deleted region is at a position corresponding to position 510, 508, or 507 of SEQ ID NO:

1.

4. 2. The mutant according to claim 1, wherein the amino acid sequence of the region corresponding to positions 459 to 507 of SEQ ID NO: 1 is deleted, and the mutant has improved thermostability compared to a glutamate oxidase in which all or part of the region corresponding to positions 459 to 507 of SEQ ID NO: 1 is not deleted.

5. 5. The mutant according to claim 4, wherein the amino acid sequence of the region corresponding to positions 459 to 507 of SEQ ID NO: 1 is deleted, and the mutant has improved thermostability compared to a glutamate oxidase that does not have the amino acid sequence of the region corresponding to positions 459 to 466 of SEQ ID NO: 1 deleted.

6. The improvement in thermal stability is evaluated based on the residual activity after heat treatment at 45°C for 30 or 35 minutes, or at 65°C for 30 minutes; 5. The mutant according to claim 4, wherein the residual activity of the mutant is 110% or more when the residual activity of glutamate oxidase without the amino acid sequence deletion after heat treatment at 45°C for 30 or 35 minutes, or after heat treatment at 65°C for 30 minutes is taken as 100%.

7. The improvement in thermal stability is evaluated by the residual activity after heat treatment at 45°C for 30 or 35 minutes, or at 65°C for 30 minutes, 6. The mutant according to claim 5, wherein the residual activity of the mutant is 110% or more when the residual activity of glutamate oxidase without the deleted amino acid sequence after heat treatment at 45°C for 30 or 35 minutes, or after heat treatment at 65°C for 30 minutes is taken as 100%.

8. The mutant of claim 1, which has an amino acid sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 12 or SEQ ID NO: 13, lacks an amino acid sequence, and has glutamate oxidase activity.

9. moreover, a position corresponding to position 87 of SEQ ID NO: 1; a position corresponding to position 103 of SEQ ID NO: 1; a position corresponding to position 133 of SEQ ID NO: 1; a position corresponding to position 186 of SEQ ID NO: 1; a position corresponding to position 297 of SEQ ID NO: 1; a position corresponding to position 376 of SEQ ID NO: 1; a position corresponding to position 393 of SEQ ID NO: 1; a position corresponding to position 428 of SEQ ID NO: 1; a position corresponding to position 516 of SEQ ID NO: 1; a position corresponding to position 566 of SEQ ID NO: 1; a position corresponding to position 568 of SEQ ID NO: 1; a position corresponding to position 585 of SEQ ID NO: 1; and a position corresponding to position 615 of SEQ ID NO: 1; and having an amino acid substitution compared to the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 58 at one or more positions selected from the group consisting of: The mutant of claim 1, wherein the mutant has improved thermal stability after amino acid substitution compared to glutamate oxidase before substitution.

10. the amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 1 is tyrosine; The amino acid substitution at the position corresponding to position 103 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine. The amino acid substitution at the position corresponding to position 133 of SEQ ID NO: 1 is selected from the group consisting of leucine and tyrosine. The amino acid substitution at the position corresponding to position 186 of SEQ ID NO: 1 is selected from the group consisting of glutamic acid, aspartic acid, tyrosine, glutamine, asparagine, alanine, leucine, cysteine, methionine, phenylalanine, serine, histidine, and threonine. The amino acid substitution at the position corresponding to position 297 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, isoleucine, and methionine. The amino acid substitution at the position corresponding to position 376 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine. The amino acid substitution at the position corresponding to position 393 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, isoleucine, and methionine. The amino acid substitution at the position corresponding to position 428 of SEQ ID NO: 1 is selected from the group consisting of tyrosine and methionine. the amino acid substitution at the position corresponding to position 516 of SEQ ID NO: 1 is phenylalanine; The amino acid substitution at the position corresponding to position 566 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, valine, and methionine. The amino acid substitution at the position corresponding to position 568 of SEQ ID NO: 1 is selected from the group consisting of isoleucine and methionine. the amino acid substitution at the position corresponding to position 585 of SEQ ID NO: 1 is selected from the group consisting of leucine and methionine; or The amino acid substitution at the position corresponding to position 615 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, and phenylalanine; The mutant of claim 9.

11. having an amino acid sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 69; and the amino acid sequence is deleted, and a position corresponding to position 87 of SEQ ID NO: 1; a position corresponding to position 103 of SEQ ID NO: 1; a position corresponding to position 133 of SEQ ID NO: 1; a position corresponding to position 186 of SEQ ID NO: 1; a position corresponding to position 297 of SEQ ID NO: 1; a position corresponding to position 376 of SEQ ID NO: 1; a position corresponding to position 393 of SEQ ID NO: 1; a position corresponding to position 428 of SEQ ID NO: 1; a position corresponding to position 516 of SEQ ID NO: 1; a position corresponding to position 566 of SEQ ID NO: 1; a position corresponding to position 568 of SEQ ID NO: 1; a position corresponding to position 585 of SEQ ID NO: 1; and a position corresponding to position 615 of SEQ ID NO: 1; 11. The mutant according to claim 10, having an amino acid substitution compared to the amino acid sequence of SEQ ID NO: 58 at one or more positions selected from the group consisting of:

12. The glutamate oxidase mutant according to claim 1, further lacking the amino acid sequence of a region corresponding to positions 670 to 687 of SEQ ID NO:

1.

13. A composition, reagent, electrode, sensor or kit comprising the glutamate oxidase variant according to any one of claims 1 to 11.

14. A polynucleotide encoding the glutamate oxidase mutant according to any one of claims 1 to 12.

15. An expression vector comprising the polynucleotide of claim 14.

16. A host cell comprising the expression vector of claim 15.

17. Culturing the host cell of claim 16 to produce a glutamate oxidase variant expressed by the expression vector; and Obtaining the produced glutamate oxidase variant; A method for producing a glutamate oxidase mutant, comprising:

18. A method for oxidizing glutamic acid contained in a sample, comprising contacting the glutamate oxidase mutant according to any one of claims 1 to 12 with the sample.

19. 19. The method of claim 18, wherein glutamate is detected.

20. a glutamate oxidase variant comprising an amino acid substitution, wherein the glutamate oxidase variant after the amino acid substitution has improved thermostability compared to the glutamate oxidase before the substitution; (i) when aligned with the amino acid sequence set forth in SEQ ID NO: 1, an amino acid at a position corresponding to a position selected from the group consisting of positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 1 is substituted; (ii) In (i), the amino acid sequence has one or more amino acids substituted, deleted or added at positions other than those corresponding to positions 87, 103, 133, 186, 297, 376, 393, 428, 516, 566, 568, 585 and 615 of SEQ ID NO:

1. (iii) In the above (i) or (ii), the full-length amino acid sequence of the glutamate oxidase variant 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 or SEQ ID NO: 58; or (iv) In the above (i) or (ii), the full-length amino acid sequence of the glutamate oxidase variant 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 or SEQ ID NO: 58, and the amino acid at position 291 of SEQ ID NO: 1 is arginine, and the amino acid sequence at positions 51 to 56 of SEQ ID NO: 1 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid). Glutamate oxidase mutants selected from the group consisting of:

21. the amino acid substitution at the position corresponding to position 87 of SEQ ID NO: 1 is tyrosine; The amino acid substitution at the position corresponding to position 103 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine. The amino acid substitution at the position corresponding to position 133 of SEQ ID NO: 1 is selected from the group consisting of leucine and tyrosine. The amino acid substitution at the position corresponding to position 186 of SEQ ID NO: 1 is selected from the group consisting of glutamic acid, aspartic acid, tyrosine, asparagine, glutamine, alanine, leucine, cysteine, methionine, phenylalanine, serine, histidine, and threonine. The amino acid substitution at the position corresponding to position 297 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, isoleucine, and methionine. The amino acid substitution at the position corresponding to position 376 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine. The amino acid substitution at the position corresponding to position 393 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, isoleucine, and methionine. The amino acid substitution at the position corresponding to position 428 of SEQ ID NO: 1 is selected from the group consisting of tyrosine and methionine. the amino acid substitution at the position corresponding to position 516 of SEQ ID NO: 1 is phenylalanine; The amino acid substitution at the position corresponding to position 566 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, valine, and methionine. The amino acid substitution at the position corresponding to position 568 of SEQ ID NO: 1 is selected from the group consisting of isoleucine and methionine. the amino acid substitution at the position corresponding to position 585 of SEQ ID NO: 1 is selected from the group consisting of leucine and methionine; or The amino acid substitution at the position corresponding to position 615 of SEQ ID NO: 1 is selected from the group consisting of leucine, valine, and phenylalanine; The glutamate oxidase variant of claim 20.