WO2024090562A1 - Ｆａｄ型グルタミン酸デヒドロゲナーゼ - Google Patents

Ｆａｄ型グルタミン酸デヒドロゲナーゼ Download PDF

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WO2024090562A1
WO2024090562A1 PCT/JP2023/038908 JP2023038908W WO2024090562A1 WO 2024090562 A1 WO2024090562 A1 WO 2024090562A1 JP 2023038908 W JP2023038908 W JP 2023038908W WO 2024090562 A1 WO2024090562 A1 WO 2024090562A1
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amino acid
fad
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圭太朗 久世
啓太 戸田
敦 一柳
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Kikkoman Corp
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Definitions

  • the present invention relates to FAD-type glutamate dehydrogenase, a method for producing the same, a method for using the same, and a composition or kit containing FAD-type glutamate dehydrogenase.
  • L-glutamate measurement was traditionally performed using L-glutamate decarboxylase and L-glutamate dehydrogenase. All glutamate dehydrogenases known to date are enzymes that use nicotinamide adenine dinucleotide (NAD) as a coenzyme and generate ammonia.
  • NAD nicotinamide adenine dinucleotide
  • Conventional glutamate dehydrogenase is generally referred to as GLDH in academic literature. Since NAD is used as a coenzyme, in this specification, such conventional glutamate dehydrogenase (so-called GLDH) is sometimes referred to as NAD-type glutamate dehydrogenase or NAD-GLDH for convenience.
  • NAD-GLDH is generally expressed as an apoenzyme. Therefore, NAD is usually added from the outside to the expressed NAD-GLDH.
  • L-glutamate oxidase acting only on L-glutamic acid was reported by solid culture of actinomycetes (Non-Patent Document 1).
  • L-glutamate oxidase is generally written as GLOD.
  • L-glutamate oxidase is encoded as a single polypeptide having an ⁇ chain, a ⁇ chain, and a ⁇ chain, and two polypeptides form a homodimer. This homodimer is cleaved by a protease to become the mature form.
  • the mature form has a heterohexamer structure composed of ⁇ 2 ⁇ 2 ⁇ 2 .
  • the recombinantly expressed homodimer had weak L-glutamate oxidase activity, inferior substrate affinity compared to the original actinomycete L-glutamate oxidase, and was thermally unstable.
  • this recombinantly expressed homodimer enzyme was treated with protease, it had the same structure as the original actinomycete L-glutamate oxidase and the same enzymatic properties (Non-Patent Document 2).
  • a method is being used in which this precursor is mass-produced using recombinant Escherichia coli and then matured by protease treatment.
  • Patent Document 1 describes an L-glutamate oxidase mutant. There is no description in Patent Document 1 regarding the thermal stability of the L-glutamate oxidase mutant that has been created.
  • Patent document 2 describes a dry L-glutamate oxidase composition that contains the disaccharide lactose.
  • L-glutamic acid When detecting L-glutamic acid using L-glutamate oxidase, there are problems such as the need for protease treatment to express L-glutamate oxidase, the stability of L-glutamate oxidase, for example its thermal stability, being insufficient, and the possibility of it being affected by dissolved oxygen present in the system. In addition, L-glutamate oxidase transfers electrons to oxygen molecules to generate hydrogen peroxide, but electron transfer at a potential lower than the redox potential of hydrogen peroxide is difficult.
  • L-glutamate oxidase uses oxygen as an electron acceptor, and is therefore affected by dissolved oxygen. Therefore, there is a need for a method of measuring L-glutamic acid that is not or is less affected by dissolved oxygen. There is also a need for an enzyme for measuring L-glutamic acid that is highly thermostable. There is also a need for a simple method of producing an enzyme for measuring L-glutamic acid without protease treatment. There is also a need for an enzyme for measuring L-glutamic acid that is capable of electron transfer at a potential lower than the redox potential of hydrogen peroxide.
  • the present disclosure aims to at least partially solve the above problems.
  • the inventors have produced, as an example, glutamate dehydrogenase by introducing the amino acid substitutions disclosed herein into glutamate oxidase, and have completed the present invention, which includes this as one embodiment.
  • the inventors have produced, as an example, a freeze-dried composition containing GLOD or FAD-glutamate dehydrogenase and a trisaccharide, and have completed the present invention, which includes this as one embodiment.
  • a flavin adenine dinucleotide (FAD)-type glutamate dehydrogenase An FAD-type glutamate dehydrogenase in which amino acids at positions corresponding to 109 and/or 545 of SEQ ID NO:58 have been substituted, and the ratio of oxidase activity (OD) to dehydrogenase activity (DH) (OD/DH) of the polypeptide after amino acid substitution is reduced compared to the polypeptide before the amino acid substitution.
  • FAD flavin adenine dinucleotide
  • DH dehydrogenase activity
  • the amino acid at the position corresponding to position 109 of SEQ ID NO:58 is substituted with an amino acid residue selected from the group consisting of serine, tryptophan, glutamine, alanine, aspartic acid, glutamic acid, phenylalanine, arginine, histidine, lysine, threonine, asparagine, cysteine, glycine, proline, valine, leucine, isoleucine, and tyrosine, and/or
  • the amino acid at the position corresponding to position 545 of SEQ ID NO:58 is substituted with an amino acid residue selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, methionine, tyrosine and tryptophan.
  • the FAD-type glutamate dehydrogenase according to embodiment 1 or 2 further comprising an amino acid residue selected from the group consisting of glutamine, alanine, aspartic acid, glutamic acid, phenylalanine, arginine, histidine, lysine, serine, threonine, asparagine, cysteine, glycine, proline, valine, isoleucine, methionine, tyrosine, and tryptophan, at a position corresponding to position 425 of SEQ ID NO:58.
  • the FAD-type glutamate dehydrogenase according to any one of embodiments 1 to 5, having an amino acid substitution compared to the amino acid sequence of SEQ ID
  • the amino acid substitution at the position corresponding to position 297 of SEQ ID NO:58 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:58 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:58 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:58 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:58 is phenylalanine;
  • the amino acid substitution at the position corresponding to position 566 of SEQ ID NO:58 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:58 is selected from the group consisting of isoleucine and methionine.
  • the amino acid substitution at position corresponding to position 585 of SEQ ID NO:58 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:58 is leucine; 7.
  • the FAD-type glutamate dehydrogenase according to embodiment 6. [8] having 90% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 60; And, the amino acid sequence of the region corresponding to positions 459 to 507 of SEQ ID NO: 58 is deleted, and A position corresponding to position 186 of SEQ ID NO:58; A position corresponding to position 87 of SEQ ID NO:58; A position corresponding to position 103 of SEQ ID NO:58; A position corresponding to position 133 of SEQ ID NO:58; A position corresponding to position 297 of SEQ ID NO:58; A position corresponding to position 376 of SEQ ID NO:58; A position corresponding to position 393 of SEQ ID NO:58; A position corresponding to position 428 of SEQ ID NO:58; A position corresponding to position 516 of SEQ ID NO:58; A position corresponding to position 566 of SEQ ID NO:58; A position corresponding to position 568 of SEQ ID NO:58; A position corresponding to position
  • a composition, a reagent, an electrode, a sensor, or a kit comprising the glutamate dehydrogenase according to any one of embodiments 1 to 9.
  • a vector comprising the polynucleotide described in embodiment 11.
  • a host cell comprising the vector described in embodiment 12.
  • the method for producing glutamate dehydrogenase comprises: [15] A method for oxidizing glutamic acid contained in a sample by contacting the glutamate dehydrogenase according to any one of embodiments 1 to 9, or the composition, reagent, electrode, sensor, or kit according to embodiment 10 with the sample. [16] The method of embodiment 15, wherein glutamate is detected.
  • FAD-type glutamate dehydrogenase in which amino acids at positions corresponding to 109, 545, and/or 425 of SEQ ID NO:58 have been substituted, and the ratio of oxidase activity (OD) to dehydrogenase activity (DH) (OD/DH) of the polypeptide after amino acid substitution is reduced compared to the polypeptide before the amino acid substitution
  • the FAD-type glutamate dehydrogenase further comprises a thermostability-improving mutation, whereby the thermostability of the polypeptide after the amino acid substitution is improved compared to the polypeptide before the substitution; (i) when aligned with the amino acid sequence set forth in SEQ ID NO:58, an amino acid at a position corresponding to a position selected from the group consisting of positions 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO:58 is substituted; (ii)
  • the full-length amino acid sequence of the glutamate dehydrogenase has 70% or more, 80% or more, or 90% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:58 or SEQ ID NO:1; or (iv) In the above (i) or (ii), the full-length amino acid sequence of the glutamate dehydrogenase has 70% or more, 80% or more, or 90% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:58 or SEQ ID NO:1, the amino acid at the position corresponding to position 291 of SEQ ID NO:58 in the glutamate dehydrogenase is arginine, and the amino acid sequence at the positions corresponding to positions 51 to 56 of SEQ ID NO:58 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid).
  • the FAD-type glutamate dehydrogenase is selected from the group consisting of: [18]
  • the amino acid at the position corresponding to position 109 of SEQ ID NO:58 is substituted with an amino acid residue selected from the group consisting of serine, tryptophan, glutamine, alanine, aspartic acid, glutamic acid, phenylalanine, arginine, histidine, lysine, threonine, asparagine, cysteine, glycine, proline, valine, leucine, isoleucine, and tyrosine, and/or
  • the amino acid at the position corresponding to position 545 of SEQ ID NO:58 is substituted with an amino acid residue selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, glycine, pro
  • the amino acid substitution at the position corresponding to position 186 of SEQ ID NO:58 is selected from the group consisting of glycine, alanine, cysteine, leucine, methionine, phenylalanine, tyrosine, serine, threonine, asparagine, glutamine, histidine, aspartic acid, and glutamic acid;
  • the amino acid substitution at the position corresponding to position 87 of SEQ ID NO:58 is tyrosine;
  • the amino acid substitution at the position corresponding to position 103 of SEQ ID NO:58 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:58 is selected from the group consisting of leucine and tyrosine.
  • the amino acid substitution at the position corresponding to position 297 of SEQ ID NO:58 is selected from the group consisting of methionine, leucine, valine, and isoleucine;
  • the amino acid substitution at the position corresponding to position 376 of SEQ ID NO:58 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:58 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:58 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:58 is phenylalanine;
  • the amino acid substitution at the position corresponding to position 566 of SEQ ID NO:58 is selected from the group consisting of methionine, phenylalanine, leucine, and valine;
  • the amino acid substitution at the position corresponding to position 568 of SEQ ID NO:58 is selected from the group consisting of isoleucine and methionine.
  • the amino acid substitution at position corresponding to position 585 of SEQ ID NO:58 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:58 is leucine; 20.
  • a dry composition comprising FAD-type glutamate dehydrogenase or GLOD and a trisaccharide.
  • the effect of the present invention is to obtain glutamate dehydrogenase that uses FAD as a coenzyme.
  • FIG. 1 shows an alignment of StGLOD of SEQ ID NO: 1 and M7GLOD of SEQ ID NO: 58.
  • FIG. 1 shows an alignment of StGLOD of SEQ ID NO: 1 and M7GLOD of SEQ ID NO: 58.
  • the oxidase activity of GLOD and the dehydrogenase activity of FAD-type GLDH are shown.
  • the amino acid sequence of GLOD of SEQ ID NO: 58 is shown. For convenience of explanation, each unit is separated by a hyphen.
  • the ⁇ chain (MDDK...ENEP)- ⁇ chain (SAEP...AEAA)-region 1 removed by protease (LTVP...STLR)- ⁇ chain (GGVR...RAEA)-region 2 removed by protease (PRERAGTASATRTREKAVTS) are linked in this order.
  • L-glutamate oxidase is encoded by the GLOD gene as a single polypeptide having an ⁇ chain, a ⁇ chain, and a ⁇ chain, and the two polypeptides form a homodimer. In nature, this homodimer is cleaved by a protease to produce the mature form.
  • the mature form has a heterohexamer structure composed of ⁇ 2 ⁇ 2 ⁇ 2 .
  • 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
  • GLOD uses flavin adenine dinucleotide (FAD) as a coenzyme. There is no need to add FAD from the outside in order for GLOD to exert its enzymatic activity; if FAD is present in the GLOD expression system, it is incorporated when the polypeptide is folded, producing active GLOD. For example, when GLOD is recombinantly expressed in E. coli, FAD derived from E. coli is incorporated to produce active GLOD.
  • FAD flavin adenine dinucleotide
  • Oxidoreductases that use FAD as a coenzyme are known to have a FAD-binding motif sequence, for example, a Gly-Xaa-Gly-Xaa-Xaa-Gly motif (where Xaa is any amino acid).
  • a FAD-binding motif sequence for example, a Gly-Xaa-Gly-Xaa-Xaa-Gly motif (where Xaa is any amino acid).
  • SEQ ID NO:58 when SEQ ID NO:58 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:58 corresponds to the FAD binding motif sequence Gly-Xaa-Gly-Xaa-Xaa-Gly.
  • the Gly at positions 51, 53, and 56, respectively, based on SEQ ID NO:58 may not be substituted with amino acids.
  • the Gly at positions 51, 53, and 56 of L-glutamate oxidase SEQ ID NO:58 is not substituted with amino acids.
  • GLOD may be referred to as FAD-GLOD for convenience. Unless otherwise specified, GLOD in this specification refers to GLOD that uses FAD as a coenzyme.
  • GLOD is known to recognize the substrate glutamic acid through an arginine residue.
  • Arg(R) at position 291 is an important position for glutamic acid recognition.
  • the position corresponding to position 291 of SEQ ID NO:58 does not require an amino acid substitution.
  • the amino acid at the position corresponding to position 291 of SEQ ID NO:58 is Arg(R)
  • the amino acid is not substituted.
  • each position of the enzyme is defined by using SEQ ID NO:58 as the reference sequence.
  • SEQ ID NO:58 is a putative glutamate oxidase (M7GLOD) derived from Streptomyces sp. MOE7.
  • M7GLOD putative glutamate oxidase
  • MOE7 the secretory signal sequence
  • SEQ ID NO:58 the first amino acid in the ⁇ -chain region immediately after the secretory signal sequence is cleaved in the wild type has been changed from Ala in the wild type to Met.
  • the position of this Met corresponds to the position 1 in SEQ ID NO:58.
  • SEQ ID NO: 58 is used as the reference sequence, and the ⁇ chain of GLOD is a polypeptide consisting of 375 amino acid residues from position 1 to position 375 (MDDK...ENEP).
  • SEQ ID NO: 58 is used as the reference sequence, and the ⁇ chain of GLOD is a polypeptide consisting of 91 amino acids from position 376 to position 466 (SAEP...AEAA).
  • SAEP...AEAA amino acids from position 507 to position 670
  • SEQ ID NO: 58 is used as the reference sequence, and the region from position 467 to position 506 is a region that is naturally removed by protease (LTVP...STLR). Furthermore, for SEQ ID NO: 58 as the reference sequence, the C-terminal sequence from position 671 to position 690 (PRERAGTASATRTREKAVTS) is a region that is removed by protease. See Figure 3.
  • glutamate dehydrogenase can be produced by introducing the amino acid substitutions of the present disclosure into glutamate oxidase (GLOD).
  • GLOD glutamate oxidase
  • Such glutamate dehydrogenase may be referred to herein as FAD-GLDH or FAD-type glutamate dehydrogenase.
  • FAD-GLDH catalyzes the following chemical reaction: [Chemical formula 2] L-glutamic acid + oxidized mediator + H 2 O ⁇ 2-oxoglutaric acid + NH 3 + reduced mediator
  • Mediators include quinones, anthraquinones, phenazines, viologens, cytochromes, phenoxazines, phenylenediamines, phenothiazines, ferricyanides, ferricyanides such as potassium ferricyanide, ferredoxins, metal complexes, ferrocene, ferrocenes, osmium complexes and derivatives thereof, etc.
  • phenazine compounds include, but are not limited to, phenazine methosulfate (PMS), methoxy PMS, PES (ethylphenazinium ethyl sulfate), thionines, naphthoquinones, benzoquinones, phthalocyanines, viologens, benzyl viologens, etc.
  • Phenylenediamines include, but are not limited to, N,N'-diphenyl-p-phenylenediamine (DPPD), 4-isopropylaminodiphenylamine (IPPD), (1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), N,N,N',N'-tetramethyl-1,4-phenylenediamine, 2,3,5,6-tetramethyl-1,4-phenylenediamine, and N,N-dimethyl-p-phenylenediamine.
  • DPPD N,N'-diphenyl-p-phenylenediamine
  • IPPD 4-isopropylaminodiphenylamine
  • IPPD 1-butyl
  • 6PPD 1-phenyl-phenyl-p-phenylenediamine
  • N,N,N',N'-tetramethyl-1,4-phenylenediamine 2,3,5,6-tetramethyl
  • Mediators include, but are not limited to, brilliant cresyl blue, gallocyanine, safranine-O, resorufin, alizarin brilliant blue, phenothiazinone, phenazine ethosulfate, dichlorophenol indophenol, ferrocene, benzoquinone, and phthalocyanine.
  • the substrate can be quantified by adding, for example, 2,6-dichlorophenol indophenol (DCPIP) as a mediator as an electron acceptor and monitoring the decrease in absorbance at 600 nm.
  • DCPIP 2,6-dichlorophenol indophenol
  • the mediator may be called an artificial electron mediator, an artificial electron acceptor, or an electron mediator.
  • FAD-GLDH can be produced based on GLOD.
  • GLOD conventional GLOD or its mutants, such as amino acid sequence deleted GLOD mutants and thermostable mutants, can be used.
  • amino acid substitutions of the present disclosure includes substituting an amino acid residue at a position corresponding to position 545 of SEQ ID NO:58.
  • the position corresponding to position 545 of SEQ ID NO:58 may be substituted with alanine, aspartic acid, glutamic acid, phenylalanine, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, methionine, tyrosine, or tryptophan. Since position 545 in SEQ ID NO:58 is L, the substitution of this position with A may be conveniently described as L545A for M7GLOD. Position 545 of SEQ ID NO:58 corresponds to position 545 of SEQ ID NO:1.
  • the amino acid substitution of the present disclosure includes substituting an amino acid residue at a position corresponding to position 425 of SEQ ID NO:58.
  • the position corresponding to position 425 of SEQ ID NO:58 may be substituted with glutamine, alanine, aspartic acid, glutamic acid, phenylalanine, arginine, histidine, lysine, serine, threonine, asparagine, cysteine, glycine, proline, valine, isoleucine, methionine, tyrosine, or tryptophan. Since position 425 in SEQ ID NO:58 is L, the substitution of this position with Q may be conveniently written as L425Q for M7GLOD. Position 425 of SEQ ID NO:58 corresponds to position 425 of SEQ ID NO:1.
  • the amino acid substitution of the present disclosure includes substituting an amino acid residue at a position corresponding to position 109 of SEQ ID NO:58.
  • the position corresponding to position 109 of SEQ ID NO:58 may be substituted with serine, tryptophan, glutamine, alanine, aspartic acid, glutamic acid, phenylalanine, arginine, histidine, lysine, threonine, asparagine, cysteine, glycine, proline, valine, leucine, isoleucine, or tyrosine. Since position 109 in SEQ ID NO:58 is M, the substitution of S at this position may be written as M109S for M7GLOD for convenience. Position 109 of SEQ ID NO:58 corresponds to position 109 of SEQ ID NO:1.
  • FAD-GLDH may have an increased ratio of dehydrogenase activity (DH) to oxidase activity (OD) (DH/OD) compared to the polypeptide before introduction of the amino acid substitutions of the present disclosure.
  • a 5% increase means that the DH/OD of the polypeptide after the amino acid substitution is 105% when the DH/OD of the polypeptide before the amino acid substitution is 100%.
  • the ratio (OD/DH) of oxidase activity (OD) to dehydrogenase activity (DH) of FAD-GLDH may be reduced compared to the polypeptide before the amino acid substitution of the present disclosure is introduced.
  • “reduced to 1% or less” means that the OD/DH of the polypeptide after amino acid substitution is 1% when the OD/DH of the polypeptide before amino acid substitution is taken as 100%.
  • GLOD mutant with deletion of amino acid sequence It was thought that the region of the amino acid sequence of GLOD that is removed by protease hinders the functional expression of GLOD. Therefore, a GLOD mutant sequence was prepared by deleting the amino acid sequence between the ⁇ region and the ⁇ region, and this was expressed. For example, when deleting the region that is removed by protease from GLOD of SEQ ID NO: 58, the last 8 amino acids of the ⁇ region (GEDDAEAA, from position 459 to position 466) were also deleted from the mutant. In addition, the first amino acid residue of the ⁇ chain, G at position 507, was also deleted from the mutant.
  • the boundary between the ⁇ chain region and the ⁇ chain region of the prepared mutant was a sequence of ⁇ chain region...QRW-GVRP... ⁇ chain region, in which the C-terminus QRW of the ⁇ chain and the N-terminus GVRP of the ⁇ chain are linked.
  • a mutant exhibiting GLOD activity was obtained.
  • This amino acid sequence-deleted GLOD mutant does not require treatment with a protease to express activity.
  • the amino acid sequence deleted GLOD mutant is not treated with a protease when it is recombinantly expressed, and such GLOD can be used to produce FAD-GLDH.
  • the present inventors have prepared linkage region replacement type mutants in which the border sequence ( ⁇ -chain region...QRW-GVRP... ⁇ -chain region) has been replaced with various amino acid sequences.
  • the inventors have prepared mutants M7GLOD ⁇ 49C having the amino acid sequence of SEQ ID NO:59, mutant StGLOD ⁇ 49Ai having the amino acid sequence of SEQ ID NO:12, and mutant StGLOD ⁇ 49C having the amino acid sequence of SEQ ID NO:13 as mutants in which the border sequence ( ⁇ -chain region...QRW-GVRP... ⁇ -chain region) has been modified, and recombinant expression was performed to confirm the activity of the mutants.
  • These amino acid sequence deletion type GLOD mutants also do not require treatment with protease to express activity.
  • FAD-GLDH can be prepared using such GLOD.
  • the present disclosure provides a glutamate dehydrogenase that is missing a region corresponding to positions 459-507 of SEQ ID NO:58 and has amino acid substitutions at positions corresponding to positions 545, 425, and/or 109 of SEQ ID NO:58.
  • the FAD-GLDH may further be missing a region corresponding to positions 671-690 of SEQ ID NO:58.
  • the residual activity can be improved compared to the corresponding enzymes before the amino acid sequence is deleted.
  • the residual activity indicates the value of the activity of the FAD-GLDH sample after heat treatment, when the activity of the FAD-GLDH sample stored in a refrigerator (for example, at 4°C) is taken as 1.
  • the residual activity can be a value of 0 or more and 1 or less, but when FAD-GLDH is activated by heat treatment, the residual activity can be a value exceeding 1.
  • a mutant having the mutation of the present disclosure and having the amino acid sequence between the ⁇ -chain and the ⁇ -chain deleted may be referred to as a ⁇ -chain- ⁇ -chain amino acid sequence deleted FAD-GLDH mutant, or simply an amino acid sequence deleted FAD-GLDH mutant.
  • the amino acid sequence deleted FAD-GLDH mutant may also be referred to as "FAD-GLDH ⁇ ".
  • the number of deleted amino acid residues, XX may be written after FAD-GLDH ⁇ (FAD-GLDH ⁇ XX).
  • a FAD-GLDH mutant with 49 amino acid residues deleted may be written as FAD-GLDH ⁇ 49.
  • amino acid sequence deleted FAD-GLDH mutant includes not only StFAD-GLDH and M7FAD-GLDH, but also their modified forms and FAD-GLDH based on GLOD of other origins, which is a FAD-GLDH mutant based on GLOD in which a region corresponding to the deleted region of SEQ ID NO: 1, SEQ ID NO: 58, SEQ ID NO: 12, and SEQ ID NO: 13 is deleted.
  • a GLOD mutant with an amino acid sequence deleted may be written as "GLOD ⁇ ".
  • the number of deleted amino acid residues, XX may be written after GLOD ⁇ (GLOD ⁇ XX).
  • GLOD ⁇ 49 a GLOD mutant in which 49 amino acid residues have been deleted
  • the term "GLOD mutant with deletion of amino acid sequence” includes not only StGLOD and M7GLOD, but also their modified forms and GLOD of other origins in which the region corresponding to the deleted region in SEQ ID NO: 1, SEQ ID NO: 58, SEQ ID NO: 12, and SEQ ID NO: 13 has been deleted.
  • the residual activity of the modified FAD-GLDH 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, for example, 400% or more, when the residual activity of the enzyme before modification after heat treatment (dehydrogenase activity) is taken as 100%, and the residual activity of the modified enzyme after heat treatment (dehydrogenase activity) can be improved by, for example, 10% or more.
  • a 10% improvement in residual activity means that, when the residual activity of the enzyme before modification after heat treatment (dehydrogenase activity) is taken as 100%, the residual activity of the modified enzyme after heat treatment (dehydrogenase activity) is 110%.
  • the residual activity of GLOD after heat treatment of the enzyme before modification when taken as 100%, the residual activity of the modified GLOD 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, for example, 400% or more.
  • a 10% improvement in residual activity means that when the residual activity (oxidase activity) of the enzyme before modification after heat treatment is taken as 100%, the residual activity (oxidase activity) of the modified enzyme after heat treatment is 110%.
  • the thermal stability of the enzyme may be evaluated based on the residual activity after heat treatment for 30 or 35 minutes at 45°C, 50°C, 60°C, 65°C, or 70°C.
  • FAD-GLDH ⁇ lacking a region corresponding to positions 459 to 507 of SEQ ID NO:58 may have a residual activity (dehydrogenase 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 FAD-GLDH not lacking all or a portion of positions 459 to 507 of SEQ ID NO:58 after heat treatment for 30 or 35 minutes at 45°C, 50°C, 60°C, 65°C, or 70°C is taken as 100%.
  • FAD-GLDH ⁇ lacking a region corresponding to positions 459 to 507 of SEQ ID NO:58 may have a residual activity (dehydrogenase 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 FAD-GLDH not lacking the amino acid sequence of positions 459 to 466 of SEQ ID NO:58 after heat treatment at 45°C, 50°C, 60°C, 65°C or 70°C for 30 or 35 minutes is taken as 100%.
  • the mutant may further lack a region corresponding to positions 671 to 690 of SEQ ID NO:58.
  • the FAD-GLDH ⁇ mutant lacking the region corresponding to positions 459 to 507 of SEQ ID NO:58 may have improved thermal stability compared to the enzyme before modification.
  • ⁇ mutants lacking the amino acid sequence corresponding to positions 459 to 507 of SEQ ID NO:58 will also have improved thermal stability and can be used in various reactions. The same applies to the region corresponding to positions 671 to 690 of SEQ ID NO:58.
  • the present disclosure provides FAD-GLDH ⁇ having 90% or more, e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more, amino acid sequence identity to the amino acid sequence of SEQ ID NO:59, 12, or 13, lacking a region corresponding to positions 459 to 507 of SEQ ID NO:58, having amino acid substitutions at positions corresponding to positions 545, 425, and/or 109 of SEQ ID NO:58, and having glutamate dehydrogenase activity.
  • This FAD-GLDH ⁇ may further lack a region corresponding to positions 671 to 690 of SEQ ID NO:58.
  • FAD-GLDH can be produced based on GLOD.
  • GLOD is widely distributed in nature and can be obtained by searching for enzymes of microbial, animal or plant origin. In terms of microorganisms, it can be obtained, for example, from actinomycetes, filamentous fungi, yeast, or bacteria.
  • the origin of GLOD is not particularly limited, and means GLOD derived from a microorganism of the genus Streptomyces, for example 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.
  • GLOD gene a gene encoding GLOD (hereinafter, simply referred to as "GLOD gene")
  • a commonly used gene cloning method is used.
  • chromosomal DNA or mRNA can be extracted by a conventional method from microbial cells or various cells capable of producing GLOD.
  • cDNA can be synthesized using the mRNA as a template.
  • a chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA obtained in this manner.
  • a suitable probe DNA is synthesized based on the amino acid sequence of GLOD, and the GLOD gene is selected from a chromosomal DNA or cDNA library using this.
  • a suitable primer DNA is prepared based on the amino acid sequence, and DNA containing the 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, and these DNA fragments are linked to obtain DNA containing the full length of the desired GLOD gene.
  • PCR polymerase chain reaction
  • GLOD genes 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.
  • the GLOD gene may be linked to a vector.
  • vectors include any vector such as a plasmid, bacteriophage, or cosmid, for example, pBluescriptII SK+ (Stratagene).
  • the plasmid may be obtained according to a conventional method. For example, a plasmid containing the GLOD gene may be extracted and purified using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich). The obtained GLOD gene may be manipulated to produce a GLOD mutant gene or to obtain a purified enzyme.
  • the FAD-GLDH gene may be obtained by manipulating the GLOD gene.
  • the FAD-GLDH gene may be created by manipulating the GLOD gene to introduce an amino acid substitution (e.g., but not limited to, an amino acid substitution of Met to Ser) at a position corresponding to position 109 of SEQ ID NO:58.
  • the FAD-GLDH gene may be created by manipulating the GLOD gene to introduce an amino acid substitution (e.g., but not limited to, an amino acid substitution of Leu to Ala) at a position corresponding to position 545 of SEQ ID NO:58.
  • the FAD-GLDH gene may be created by manipulating the GLOD gene to introduce an amino acid substitution (e.g., but not limited to, an amino acid substitution of Leu to Gln) at a position corresponding to position 425 of SEQ ID NO:58.
  • an amino acid substitution e.g., but not limited to, an amino acid substitution of Leu to Gln
  • Mutation of the FAD-GLDH gene can be performed by any known method according to the intended form of mutation, including a method of contacting and reacting the FAD-GLDH gene or a recombinant DNA incorporating the gene with a mutagenic agent, ultraviolet irradiation, genetic engineering techniques, or protein engineering techniques.
  • 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.
  • the conditions for this contact and action can be varied according to the type of drug used, and are not particularly limited as long as the desired mutation can be actually induced in the FAD-GLDH gene.
  • the desired mutation can be induced by contacting and acting the drug at a 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.
  • ultraviolet irradiation it can also be performed according to the usual method as described above.
  • Methods that make full use of protein engineering techniques include the method generally known as Site-Specific Mutagenesis. Examples include 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. Acids Sci. U.S.A., 82, 488-492 (1985)).
  • the desired modified FAD-GLDH gene can also be directly synthesized by organic synthesis or enzymatic synthesis.
  • the base sequence of the FAD-GLDH gene can be confirmed, for example, using a multi-capillary DNA analysis system such as the Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific).
  • the FAD-GLDH gene can be incorporated into a vector, such as a bacteriophage, cosmid, or a plasmid used for transformation of prokaryotic or eukaryotic cells, by standard methods, which can then be used to transform or transduce a host corresponding to each vector by standard methods.
  • a vector such as a bacteriophage, cosmid, or a plasmid used for transformation of prokaryotic or eukaryotic cells
  • FAD-GLDH may 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, and Streptomyces microorganisms, e.g., Streptomyces violaceoruber.
  • E. coli hosts include, but are not limited to, various E.
  • coli strains e.g., K-12, JM109, DH5 ⁇ , BL21, JM109(DE3), DH5 ⁇ (DE3), BL21(DE3), TG1, 1100, W3110, C600, etc.
  • the host is transformed or transduced to obtain a host cell (transformant) into which the FAD-GLDH gene has been introduced.
  • a method for transferring a recombinant vector into such a host cell 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 adopted, and electroporation may also be used.
  • commercially available competent cells e.g., ECOS Competent Escherichia coli BL21(DE3); manufactured by Nippon Gene
  • the GLOD gene may be codon-optimized according to the expression host.
  • FAD-GLDH may be expressed using eukaryotic cells.
  • a eukaryotic host cell is yeast.
  • yeasts classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Schizosaccharomyces, Saccharomyces, Pichia, and Candida.
  • the inserted gene may contain a marker gene that allows the selection of transformed cells. Examples of marker genes include genes that complement the auxotrophy of the host, such as URA3 and TRP1. It is also desirable for the inserted gene to contain a promoter or other control sequence (e.g., an enhancer sequence, a terminator sequence, a polyadenylation sequence, etc.) that can express the target gene in the host cell.
  • a promoter or other control sequence e.g., an enhancer sequence, a terminator sequence, a polyadenylation sequence, etc.
  • promoters include the GAL1 promoter and the ADH1 promoter.
  • known methods such as a method using lithium acetate or electroporation can be suitably used, but are not limited thereto, and transformation can be performed using any method including the spheroplast method, the glass bead method, etc.
  • eukaryotic host cells include fungal cells (including filamentous fungi) such as those of the genus Aspergillus and Trichoderma.
  • fungal cells including filamentous fungi
  • filamentous fungi such as those of the genus Aspergillus and Trichoderma.
  • Method for producing a fungal cell transformant examples include a method in which a gene encoding FAD-GLDH is inserted into a host filamentous fungus in a manner that allows it to be expressed, according to standard methods.
  • a DNA construct is produced in which a gene encoding FAD-GLDH 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 FAD-GLDH to obtain a transformant that overexpresses the gene encoding FAD-GLDH.
  • the method for inserting the gene encoding FAD-GLDH into the host filamentous fungus in such a manner that it is expressed is not particularly limited, but examples include a method of directly inserting it into the chromosome of the host organism using homologous recombination, and a method of introducing it into the host filamentous fungus by linking it onto a plasmid vector.
  • a DNA construct can be ligated between sequences homologous to the upstream and downstream regions of the recombination site on the chromosome, and inserted into the genome of the host filamentous fungus.
  • a transformant can be obtained by self-cloning by overexpressing the gene in the host filamentous fungus under the control of its own high-expression promoter.
  • the high-expression promoter include the promoter region of the TEF1 gene (tef1), which is a translation elongation factor, the promoter region of the ⁇ -amylase gene (amy), and the promoter region of the alkaline protease gene (alp).
  • the DNA construct can be inserted into a plasmid vector used for transforming filamentous fungi in a conventional manner, and the corresponding host filamentous fungus can be transformed in a conventional manner.
  • Such suitable vector-host systems are not particularly limited as long as they are capable of producing FAD-GLDH 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.
  • the DNA construct is preferably introduced into the chromosome of the host filamentous fungus for use, but as an alternative, 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.
  • an autonomously replicating vector Zeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)
  • the DNA construct may contain a marker gene that allows the selection of transformed cells.
  • the marker gene is not particularly limited, and examples thereof include genes that complement the auxotrophy of the host, such as pyrG, niaD, and adeA; and drug resistance genes against drugs such as pyrithiamine, hygromycin B, and oligomycin.
  • the DNA construct also preferably contains a promoter, terminator, and other control sequences (e.g., enhancers, polyadenylation sequences, etc.) that allow the gene encoding FAD-GLDH to be overexpressed in the host cell.
  • the promoter is not particularly limited, and examples thereof include appropriate expression-inducing promoters and constitutive promoters, such as the tef1 promoter, the alp promoter, and the amy promoter.
  • the terminator is also not particularly limited, and examples thereof include the alp terminator, the amy terminator, and the tef1 terminator.
  • the expression control sequence of the gene encoding FAD-GLDH is not necessarily required if the DNA fragment containing the gene encoding FAD-GLDH to be inserted contains a sequence with an expression control function.
  • the DNA construct may not need to have a marker gene.
  • DNA construct is, for example, a DNA construct in which the tef1 gene promoter, the gene encoding FAD-GLDH, the alp gene terminator, and the pyrG marker gene are linked to an In-Fusion Cloning Site in the multiple cloning site of pUC19.
  • a method for transforming a filamentous fungus may be appropriately selected from methods known to those skilled in the art.
  • a protoplast PEG method using polyethylene glycol and calcium chloride after preparing a protoplast of the host filamentous fungus may be used.
  • a medium for regenerating the transformed filamentous fungus is used that is appropriate depending on the host filamentous fungus and transformation marker gene used.
  • the transformed filamentous fungus may be regenerated in, for example, Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2 M sorbitol.
  • Czapek-Dox minimal medium Difco
  • the promoter of the gene encoding FAD-GLDH originally present on the chromosome of the host filamentous fungus may be replaced with a high expression promoter such as tef1 using homologous recombination.
  • a transformation marker gene such as pyrG in addition to the high expression promoter.
  • a transformation cassette consisting of the upstream region of the gene encoding FAD-GLDH-transformation marker gene-high expression promoter-all or part of the gene encoding FAD-GLDH can be used.
  • the upstream region of the gene encoding FAD-GLDH and all or part of the gene encoding FAD-GLDH are used for homologous recombination. All or part of the gene encoding FAD-GLDH can be used including the region from the start codon to the middle.
  • the length of the region suitable for homologous recombination is preferably 0.5 kb or more.
  • 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 enzyme activity of FAD-GLDH is observed, and then confirming the activity of FAD-GLDH in the culture obtained after culturing.
  • confirmation that the transformed filamentous fungus of the present invention has been produced may be performed 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.
  • PCR is performed using a forward primer for the base sequence of the promoter used and a reverse primer for the base sequence of the transformation marker gene, and it is confirmed that a product of the expected length is produced.
  • the host may be a known microorganism, a known strain, or an equivalent of a known microorganism or strain described herein.
  • An equivalent refers to a host that exhibits an equivalent function with respect to recombinant expression of a protein.
  • An equivalent is a host that has been created and modified based on a host that was known at the time of filing this application, and includes a host that was developed after the filing of this application, and a host that has similar properties to a host that was known at the time of filing this application and was discovered after the filing of this application.
  • FAD-GLDH can further be subjected to high-throughput screening to obtain functional FAD-GLDH mutants.
  • a library of transformed or transduced strains having mutated FAD-GLDH genes can be prepared and subjected to microtiter plate-based high-throughput screening or droplet microfluidic-based ultra-high-throughput screening. Examples include constructing a combinatorial library of mutant genes encoding variants and then screening a large population of mutant FAD-GLDHs using phage display (e.g., Chem. Rev. 105 (11): 4056-72, 2005), yeast display (e.g., Comb Chem High Throughput Screen.
  • phage display e.g., Chem. Rev. 105 (11): 4056-72, 2005
  • yeast display e.g., Comb Chem High Throughput Screen.
  • Libraries may be transformed into suitable cells, such as electrocompetent EBY-100 cells, to obtain approximately 10 to the power of 7 mutants (10 million).
  • Yeast cells transformed with the libraries may then be subjected to cell sorting.
  • Polydimethoxylsiloxane (PDMS) microfluidic devices fabricated using standard soft lithography techniques may also be used.
  • Flow focus devices may be used to form monodisperse droplets.
  • the droplets formed containing the individual mutants may be subjected to an appropriate sorting device.
  • the presence or absence of FAD-GLDH activity may be utilized to select cells.
  • a reaction solution having a composition that produces color when the above-mentioned FAD-GLDH acts may be used.
  • absorbance at 600 nm may be measured using a 96-well plate, 192-well plate, 384-well plate, 9600-well plate, etc., and a plate reader.
  • Mutation introduction and selection may be repeated multiple times. Mutations as used herein include amino acid substitutions, insertions, deletions, and/or additions.
  • 1 to 10 mutations may be introduced into FAD-GLDH, and FAD-GLDH activity may be confirmed.
  • FAD-GLDH mutant confirmed to have activity 1 to 10 more mutations may be introduced, and activity may be confirmed.
  • a series of high-throughput screening (for example, the above-mentioned method of obtaining and screening approximately 10 to the power of 7 mutants) may 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.
  • mutants having 10 or more mutations, 50 or more mutations, for example 100 or more mutations, and still having activity may be rapidly obtained.
  • mutants having 20 or more mutations, 100 or more mutations, for example 200 or more mutations, and still having activity may be rapidly obtained.
  • Such operations may be performed by an automatic device or by repeating a routine process.
  • Mutations may be introduced at one or more positions from the first amino acid to the last amino acid in the full-length amino acid sequence of FAD-GLDH.
  • regions important for the function of the enzyme such as the active center, substrate recognition site, coenzyme recognition motif, and the vicinity thereof, are excluded.
  • FAD-GLDH has been known for a long time, and those skilled in the art are familiar with the regions important for the function of the enzyme, including the active center, substrate recognition site, and coenzyme recognition motif. These findings may also be applied to the FAD-GLDH of the present disclosure.
  • one or more mutations may be introduced first into the portion of positions 1 to 10 of the full-length sequence of FAD-GLDH.
  • one or more mutations may be further introduced into positions 11 to 20, and the activity may be confirmed. This may be repeated n times (n ⁇ 68). For example, one or more mutations may be introduced into positions 680 to 687 in the 68th round. Along the way, regions important for the function of the enzyme or regions not intended to be modified may be skipped as appropriate. For example, positions 51 to 56 of SEQ ID NO: 58 can be skipped.
  • any mutation to be introduced at any position in the full-length sequence, except for regions important to the function of the enzyme, and also allows for rapid acquisition of active FAD-GLDH 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.
  • Mutations may be introduced randomly or by rational design.
  • the 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 the substitution have similar chemical properties (e.g., Stryer et al., Biochemistry, 5th ed., 2002, pp. 44-49).
  • conservative amino acid substitutions may be selected from the group consisting of (i) a basic amino acid with a different kind of basic amino acid; (ii) an acidic amino acid with a different kind of acidic amino acid; (iii) an aromatic amino acid with a different kind of aromatic amino acid; (iv) a non-polar aliphatic amino acid with a different kind of non-polar aliphatic amino acid; and (v) a polar uncharged amino acid with a different kind of polar uncharged amino acid.
  • the basic amino acid may be selected from, for example, arginine, histidine, and lysine.
  • the acidic amino acid 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, proline, and isoleucine.
  • Polar uncharged amino acids may be selected from, for example, serine, threonine, cysteine, asparagine, and glutamine.
  • Conservative amino acid substitutions are highly likely to maintain the tertiary structure and have activity because the chemical properties of the amino acid residue before and after substitution are similar, and the position at which the conservative amino acid substitution is made is the same position in the tertiary structure of the protein.
  • the mutations introduced by rational design or randomly include substitutions with functionally similar amino acids.
  • Tables of functionally similar amino acids are widely known in the art.
  • the substitutions with functionally similar amino acids may be of 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), histidine (H); 5) Isoleucine (I), Leucine (L), Valine (V), Proline (P); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), Methionine (M).
  • non-conservative amino acid substitutions are substitutions of an amino acid with any amino acid that does not fall within the conservative substitutions (i) to (v) outlined above.
  • the amino acid substitution may be a non-conservative amino acid substitution. In this case, for example, it may be confirmed whether dehydrogenase activity is maintained before and after the non-conservative amino acid substitution is introduced. If activity is confirmed, the non-conservative amino acid substitution may be adopted.
  • the amino acid substitution may be a substitution with a similar amino acid (similarity substitution).
  • a substitution with a similar amino acid refers to a substitution with an amino acid that is evaluated as a positive value or a neutral value (zero) in the amino acid substitution matrix used in the ClustalW software and the Blosum62 algorithm (see, e.g., S. Heinkoff and J. G. Henikoff, Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 10915-10919, 1992, especially FIG. 2 therein, and Thompson, Nucleic Acid Research, 1994, Vol. 22, No. 22, pp. 4673-4680).
  • the matrix table was generated from aligned sequence segments of approximately 2000 blocks of over 500 related proteins. Moreover, whether starting from a single matrix or using a subset of proteins, repeated application leads to approximately the same set of scores. Therefore, the substitution matrix is considered versatile. It is the most widely used approach, taking advantage of the evolutionary relatedness of homologous sequences. Therefore, variants with similar substitutions in a glutamate oxidase or glutamate dehydrogenase are likely to be active. For example, in the substitution matrix, a serine to threonine substitution is scored as a positive value of "1", so for example, a serine to threonine substitution at position 212 of SEQ ID NO:58 corresponds to a similar amino acid substitution.
  • This position is threonine in the Streptomyces sp. X-119-6 GLOD of SEQ ID NO:1, and since both enzymes are active, the S212T mutant of SEQ ID NO:58 is likely to act on glutamate.
  • the S212T mutant is highly likely to act on glutamic acid. The same is true for a mutant in which T212S has been introduced into FAD-GLDH in which amino acid substitutions have been introduced at positions 545, 425, and/or 109 of SEQ ID NO:1. The same is true for other similar substitution variants.
  • the conservative amino acid substitution or the substitution with a functionally similar amino acid is not present in an area important to the function of the enzyme, such as the active center, substrate recognition site, coenzyme recognition motif, or the vicinity thereof, and therefore does not significantly affect the activity of the enzyme.
  • the conservative amino acid substitution or the substitution with a functionally similar amino acid is present in the active center, substrate recognition site, coenzyme recognition motif, or the vicinity thereof, but does not substantially affect the activity of the enzyme.
  • FAD-GLDH variants may also include those in which additional amino acids have been inserted compared to the pre-mutation sequence.
  • the amino acid insertion is not in an area critical to the function of the enzyme, such as the active center, the substrate recognition site, the coenzyme recognition motif, or nearby areas, and therefore does not significantly affect the activity of the enzyme.
  • FAD-GLDH variants may also include those in which additional amino acids have been added compared to the pre-mutation sequence.
  • the amino acid addition is made to the N-terminus or C-terminus of FAD-GLDH and does not significantly affect the activity of the enzyme.
  • additions include, but are not limited to, a short stretch of histidine residues (e.g., 2-6 histidine residues) to aid in purification of FAD-GLDH.
  • additions also include, but are not limited to, the addition of a signal peptide to aid in expression of FAD-GLDH.
  • Signal peptides include known signal sequences or functional equivalents thereof.
  • FAD-GLDH mutants may also contain deletions of amino acids compared to the sequence prior to mutation.
  • the deletions are not in regions critical to the function of the enzyme and therefore do not significantly affect the activity of the enzyme.
  • the deletions may be short, such as deletions of 1-2 amino acids.
  • the amino acid sequence of one FAD-GLDH may be compared to that of another FAD-GLDH, and if an amino acid is missing in one sequence, the deletion may be introduced into the other FAD-GLDH. Since both FAD-GLDHs exhibit activity, such deletions are unlikely to significantly affect the activity of the enzyme.
  • SEQ ID NO:58 when SEQ ID NO:58 is aligned with SEQ ID NO:1, positions 661, 671, and 672 of SEQ ID NO:58 are deleted in SEQ ID NO:1. Therefore, it is believed that deletion of positions 661, 671, and/or 672 from SEQ ID NO:58 is unlikely to significantly affect the activity of the enzyme.
  • Mutations can be introduced into FAD-GLDH in a manner that does not destroy secondary structures or structural motifs, such as ⁇ -helical structures or ⁇ -sheet structures. Regions of secondary structure can be identified, for example, by using secondary structure prediction algorithms. Examples of prediction algorithms include, but are not limited to, NetSurfP-2.0. The same applies to other structural motifs, such as nests and niches.
  • amino acid residues or amino acid sequence motifs essential for glutamate dehydrogenase activity are not substituted.
  • conservative amino acid substitutions or similar substitutions can be made at these positions, but the activity of the variants after the substitutions is confirmed.
  • no amino acid deletions or insertions are made before or after the amino acid residues essential for glutamate dehydrogenase activity.
  • the positions before or after the amino acid residues essential for glutamate dehydrogenase activity refer to positions one or two positions N-terminal or one or two positions C-terminal to the amino acid residues essential for the activity.
  • amino acid deletions or insertions can be made before or after the amino acid residues essential for glutamate dehydrogenase activity, but the activity of the variants after the substitutions is confirmed. Whether or not a variant has activity can be routinely confirmed, for example, by high-throughput screening.
  • the present disclosure provides an FAD-GLDH mutant having a mutation that improves thermostability.
  • the mutant has improved thermostability compared to FAD-GLDH before the mutation is introduced.
  • improved thermostability refers to the fact that, when FAD-GLDH is subjected to heat treatment at a specified temperature for a specified time, the residual activity of the FAD-GLDH mutant after heat treatment is improved compared to the residual activity of FAD-GLDH after heat treatment before the mutation is introduced.
  • the FAD-GLDH mutant of the present disclosure comprises: A position corresponding to position 186 of SEQ ID NO:58; A position corresponding to position 87 of SEQ ID NO:58; A position corresponding to position 103 of SEQ ID NO:58; A position corresponding to position 133 of SEQ ID NO:58; A position corresponding to position 297 of SEQ ID NO:58; A position corresponding to position 376 of SEQ ID NO:58; A position corresponding to position 393 of SEQ ID NO:58; A position corresponding to position 428 of SEQ ID NO:58; A position corresponding to position 516 of SEQ ID NO:58; A position corresponding to position 566 of SEQ ID NO:58; A position corresponding to position 568 of SEQ ID NO:58; A position corresponding to position 585 of SEQ ID NO:58, and a position corresponding to position 615 of SEQ ID NO:58; and has an amino acid substitution compared to SEQ ID NO: 58 at one or more positions selected from the group consisting
  • the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 186 of SEQ ID NO:58 may be selected from the group consisting of glycine, alanine, cysteine, leucine, methionine, phenylalanine, tyrosine, serine, threonine, asparagine, glutamine, histidine, aspartic acid, and glutamic acid.
  • the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 87 of SEQ ID NO:58 may be tyrosine.
  • the substituted amino acid introduced at the position corresponding to position 103 of SEQ ID NO:58 may be selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine.
  • the substituted amino acid introduced at the position corresponding to position 133 of SEQ ID NO:58 may be selected from the group consisting of leucine and tyrosine.
  • the substituted amino acid introduced at the position corresponding to position 297 of SEQ ID NO:58 may be selected from the group consisting of leucine, valine, isoleucine, and methionine.
  • the substituted amino acid introduced at the position corresponding to position 376 of SEQ ID NO:58 may be selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine.
  • the substituted amino acid introduced at the position corresponding to position 393 of SEQ ID NO:58 may be selected from the group consisting of leucine, valine, isoleucine, and methionine.
  • the substituted amino acid introduced at the position corresponding to position 428 of SEQ ID NO:58 may be selected from the group consisting of tyrosine and methionine.
  • the substituted amino acid introduced at the position corresponding to position 516 of SEQ ID NO:58 may be phenylalanine.
  • the substituted amino acid introduced at the position corresponding to position 566 of SEQ ID NO:58 may be selected from the group consisting of phenylalanine, leucine, valine, and methionine.
  • the substituted amino acid introduced at the position corresponding to position 568 of SEQ ID NO:58 may be selected from the group consisting of isoleucine and methionine.
  • the substituted amino acid introduced at the position corresponding to position 585 of SEQ ID NO:58 may be selected from the group consisting of leucine and methionine.
  • the substituted amino acid introduced at the position corresponding to position 615 of SEQ ID NO:58 can be leucine. The corresponding position is described below.
  • thermostability-improving mutation may be introduced into wild-type GLOD or conventional GLOD.
  • Conventional GLOD here refers to conventional GLOD that requires treatment with protease to express active protein.
  • GLOD into which such a thermostability-improving mutation has been introduced is not only active after treatment with protease like wild-type GLOD or conventional GLOD, but is also thought to have improved thermostability compared to GLOD before the introduction of the mutation.
  • such a mutant may be referred to as GLOD-T in this specification.
  • multiple such thermostability-improving mutations may be introduced.
  • GLOD into which one of the above-mentioned thermostability-improving mutations has been introduced may be referred to as GLOD-T1 in this specification.
  • mutants with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations 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.
  • the amino acid substitutions of the present disclosure are introduced into the above GLOD-T, thereby obtaining FAD-GLDH-T with improved glutamate dehydrogenase activity.
  • the amino acid substitutions of the present disclosure for example, amino acid substitutions at positions corresponding to positions 545, 425, and/or 109 of SEQ ID NO:58, are introduced into wild-type GLOD or conventional GLOD to obtain FAD-GLDH, and then one or more thermostability-improving mutations are introduced therein to obtain FAD-GLDH-T.
  • FAD-GLDH-T into which a thermostability-improving mutation has been introduced is considered to have improved thermostability compared to FAD-GLDH before the mutation is introduced.
  • FAD-GLDH into which one of the above thermostability-improving mutations has been introduced may be referred to as FAD-GLDH-T1.
  • mutants with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations may be referred to as FAD-GLDH-T2, FAD-GLDH-T3, FAD-GLDH-T4, FAD-GLDH-T5, FAD-GLDH-T6, FAD-GLDH-T7, FAD-GLDH-T8, FAD-GLDH-T9, FAD-GLDH-T10, FAD-GLDH-T11, FAD-GLDH-T12, or FAD-GLDH-T13, respectively.
  • mutants with amino acid substitutions at positions corresponding to 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585, or 615 of SEQ ID NO:58 have improved thermal stability compared to the enzyme before modification.
  • FAD-GLDH having the mutations disclosed herein based on GLOD of other origins in which similar amino acid substitutions have been introduced at positions corresponding to 186, 87, etc. of SEQ ID NO:58 also has improved thermal stability and can be used in various reactions.
  • the above-mentioned thermostability-improving mutations may be introduced into an amino acid sequence-deleted GLOD mutant (GLOD ⁇ ).
  • the amino acid sequence-deleted GLOD mutant does not require treatment with a protease to express an active protein.
  • the amino acid sequence-deleted GLOD mutant may have improved thermostability compared to the corresponding GLOD before the amino acid sequence deletion.
  • the above-mentioned thermostability-improving mutations may be further introduced into such a GLOD ⁇ .
  • such a mutant may be referred to herein as GLOD ⁇ -T.
  • GLOD ⁇ into which one thermostability-improving mutation has been introduced may be referred to herein as GLOD ⁇ -T1.
  • GLOD ⁇ 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.
  • FAD-GLDH can be obtained by introducing the amino acid substitutions of the present disclosure into these enzymes. Note that the thermostability-improving mutations, amino acid deletions, and amino acid substitutions of the present disclosure may be introduced into the enzyme in any order.
  • FAD-GLDH ⁇ containing one thermostability-improving mutation may be referred to as FAD-GLDH ⁇ -T1.
  • FAD-GLDH ⁇ mutants with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations may be referred to as FAD-GLDH ⁇ -T2, FAD-GLDH ⁇ -T3, FAD-GLDH ⁇ -T4, FAD-GLDH ⁇ -T5, FAD-GLDH ⁇ -T6, FAD-GLDH ⁇ -T7, FAD-GLDH ⁇ -T8, FAD-GLDH ⁇ -T9, FAD-GLDH ⁇ -T10, FAD-GLDH ⁇ -T11, FAD-GLDH ⁇ -T12, or FAD-GLDH ⁇ -T13, respectively.
  • FAD-GLDH such as a thermostability-improved mutant such as FAD-GLDH-T or FAD-GLDH-T1 to FAD-GLDH-T13, or an amino acid sequence-deleted FAD-GLDH mutant, such as FAD-GLDH ⁇ -T or FAD-GLDH ⁇ -T1 to FAD-GLDH ⁇ -T13, such as SEQ ID NO: 12, 13, 58, 59 or 60, or a FAD-GLDH mutant having 70% or more, 80% or more, or 90% or more amino acid sequence identity with any of these, may optionally have an amino acid substitution as described in WO 2021/193598.
  • Ala at position 106 may be replaced with Ser based on SEQ ID NO: 58 herein.
  • This amino acid substitution is referred to herein as A106S.
  • the FAD-GLDH mutant 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, L545I, P598A, C601S, and P609A. The same applies to positions corresponding to these positions.
  • the amino acid substitutions described in WO 2021/193598 may be represented as J. That is, J is a set of amino acid substitutions described in WO 2021/193598, and the order of each component does not matter.
  • a mutant having one mutation in J is designated as J1
  • a mutant having n mutations in J is designated as Jn
  • so on up to J25 J25
  • the present disclosure provides the following combination mutants. Those skilled in the art can prepare these finite combinations of mutants one by one and confirm their activity and thermostability.
  • GLOD Mutant By introducing an amino acid sequence deletion into the GLOD gene, a GLOD ⁇ mutant can be prepared, and GLOD, for example, GLOD with high thermostability, can be produced without protease treatment.
  • a GLOD-T mutant By introducing the amino acid substitution T into the GLOD gene, a GLOD-T mutant can be prepared, and GLOD with high thermostability can be produced.
  • a GLOD ⁇ -T mutant By introducing an amino acid sequence deletion and an amino acid substitution into the GLOD gene, a GLOD ⁇ -T mutant can be prepared, and GLOD, for example, GLOD with high thermostability, can be produced without protease treatment.
  • FAD-GLDH mutant By introducing an amino acid sequence deletion into the FAD-GLDH gene, a FAD-GLDH ⁇ mutant can be prepared, and FAD-GLDH, for example, FAD-GLDH with high thermostability, can be produced without performing protease treatment.
  • FAD-GLDH-T mutant By introducing an amino acid substitution T into the FAD-GLDH gene, a FAD-GLDH-T mutant can be prepared, and FAD-GLDH with high thermostability can be produced.
  • FAD-GLDH ⁇ -T mutant By introducing an amino acid sequence deletion and an amino acid substitution into the FAD-GLDH gene, a FAD-GLDH ⁇ -T mutant can be prepared, and FAD-GLDH, for example, FAD-GLDH with high thermostability, can be produced without performing protease treatment.
  • a thermostable GLOD may be prepared first, and the amino acid substitution of the present disclosure may be introduced thereinto.
  • the substituted amino acid is excluded from back-mutation to an amino acid in the native GLOD sequence (natural amino acid).
  • the substituted amino acid at a position corresponding to, for example, position 87 of SEQ ID NO:58 may be identical to the amino acid at that position in the native GLOD sequence (natural amino acid).
  • a method for identifying "corresponding positions" in amino acid sequences can be, for example, to compare amino acid sequences using known algorithms such as the Lippmann-Parson method, and to assign maximum identity to conserved amino acid residues present in the amino acid sequences of each GLOD.
  • By aligning the amino acid sequences of GLOD in this manner it is possible to determine the positions in each GLOD sequence of homologous amino acid residues, regardless of insertions or deletions in the amino acid sequence.
  • Corresponding positions are considered to be at the same positions in the three-dimensional structure, and it can be presumed that they have similar effects on the specific function of the target GLOD.
  • a similar presumption can be made for FAD-GLDH, in which the amino acid substitutions disclosed herein have been introduced into GLOD.
  • a position corresponding to position 87 in the amino acid sequence of SEQ ID NO: 58 refers to a position corresponding to position 87 in SEQ ID NO: 58 when the amino acid sequence of a subject polypeptide is compared with the amino acid sequence of SEQ ID NO: 58.
  • position corresponding to position 87 of the amino acid sequence of SEQ ID NO: 58 is position 87 of SEQ ID NO: 1.
  • a “corresponding region” in an amino acid sequence is defined in the same manner as the above-mentioned "corresponding position.”
  • a region corresponding to positions 459 to 507 in SEQ ID NO:58 is positions 459 to 507 in SEQ ID NO:1.
  • a region corresponding to positions 459 to 466 in SEQ ID NO:58 is positions 459 to 466 in SEQ ID NO:1.
  • a region corresponding to positions 671 to 690 in SEQ ID NO:58 is positions 670 to 687 in SEQ ID NO:1.
  • Homology, identity or similarity of amino acid sequences can be calculated using programs such as maximum matching and search homology of GENETYX (GENETYX), maximum matching and multiple alignment of DNASIS Pro (Hitachi Solutions), or multiple alignment of CLUSTAL W.
  • GENETYX GENETYX
  • DNASIS Pro Hitachi Solutions
  • CLUSTAL W multiple alignment of CLUSTAL W.
  • positions of identical amino acids in two or more GLODs can be examined when the two or more GLODs are aligned. Based on such information, identical regions in amino acid sequences can be determined.
  • the percent identity refers to the percentage calculated by aligning two or more amino acid sequences using an algorithm such as Blosum62, with the total number of amino acids in the aligned regions as the denominator and the number of positions occupied by identical amino acids as the numerator. Therefore, typically, 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.
  • amino acids that are similar in two or more GLODs can be aligned using CLUSTALW, in which case the algorithm Blosum62 is used, and amino acids that are determined to be similar when multiple amino acid sequences are aligned are sometimes called similar amino acids.
  • amino acid substitutions may be due to substitutions between such similar amino acids.
  • Such alignments make it possible to examine 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.
  • the FAD-GLDH mutant of the present disclosure has an alignment ratio of 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more when aligned with GLOD having the amino acid sequence shown in SEQ ID NO:58.
  • the FAD-GLDH 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 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO:58, and has amino acid substitutions at positions corresponding to positions 545, 425, and/or 109 of SEQ ID NO:58, and has an improved ratio of dehydrogenase activity to oxidase activity compared to unmodified GLOD.
  • one or several amino acids refers to 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 4, for example 1 to 3, for example 1 or 2 amino acids.
  • FAD-GLDH ⁇ has amino acid substitutions at positions corresponding to positions 545, 425, and/or 109 of SEQ ID NO:58, and has an improved ratio of dehydrogenase activity to oxidase activity compared to the polypeptide before the amino acid substitutions are introduced; (i) When aligned with the amino acid sequence set forth in SEQ ID NO:58, the amino acid sequence of a region corresponding to positions 459 to 507 of SEQ ID NO:58 is deleted, and optionally the amino acid sequence of a region corresponding to positions 671 to 690 of SEQ ID NO:58 is deleted.
  • the amino acid sequence is one in which one or several amino acids are substituted, deleted or added at positions other than the region corresponding to positions 459 to 507 of SEQ ID NO: 58, or one or several amino acids are substituted, deleted or added at positions other than the region corresponding to positions 459 to 507 and positions 671 to 690 of SEQ ID NO: 58.
  • the full-length amino acid sequence of the FAD-GLDH ⁇ has a sequence identity of 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, for example, 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, to the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:59, or (iv) In the above (i) or (ii), the full-length amino acid sequence of the FAD-GLDH ⁇ has a sequence similar to that of
  • the FAD-GLDH ⁇ has a sequence identity of 0% 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%, and the amino acid at a position corresponding to position 291 of SEQ ID NO:58 in the FAD-GLDH ⁇ is arginine, and the amino acid sequence at positions corresponding to positions 51 to 56 of SEQ ID NO:58 is Gly-Xaa-Gly-Xaa-Xaa-Gly (wherein Xaa represents any amino acid).
  • FAD-GLDH-T has amino acid substitutions at positions corresponding to positions 545, 425, and/or 109 of SEQ ID NO:58, and has an improved ratio of dehydrogenase activity to oxidase activity compared to the polypeptide before the amino acid substitutions are introduced; (i) when aligned with the amino acid sequence set forth in SEQ ID NO:58, an amino acid at a position corresponding to a position selected from the group consisting of positions 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO:58 is substituted; (ii) In the above (i), the amino acid sequence has one or several amino acids substituted, deleted or added at positions other than those corresponding to positions 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585 and 615 of SEQ ID NO: 58.
  • the full-length amino acid sequence of the FAD-GLDH-T has sequence identity with SEQ ID NO:58 or the amino acid sequence of SEQ ID NO:58 of 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, for example, 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, or (iv) In the above (i) or (ii), the full-length amino acid sequence of the FAD-GLDH-T has a similarity to SEQ ID NO:58 or the amino acid
  • the replacement amino acid introduced at the position corresponding to position 186 of SEQ ID NO:58 may be selected from the group consisting of glycine, alanine, cysteine, leucine, methionine, phenylalanine, tyrosine, serine, threonine, asparagine, glutamine, histidine, aspartic acid, and glutamic acid.
  • the replacement amino acid introduced at the position corresponding to position 87 of SEQ ID NO:58 may be tyrosine.
  • the replacement amino acid introduced at the position corresponding to position 103 of SEQ ID NO:58 may be selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine. In certain embodiments, with respect to the FAD-GLDH-T mutant of the present disclosure, the replacement amino acid introduced at the position corresponding to position 133 of SEQ ID NO:58 may be selected from the group consisting of leucine and tyrosine.
  • the replacement amino acid introduced at a position corresponding to position 297 of SEQ ID NO:58 may be selected from the group consisting of leucine, valine, isoleucine, and methionine.
  • the replacement amino acid introduced at a position corresponding to position 376 of SEQ ID NO:58 may be selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine.
  • the replacement amino acid introduced at a position corresponding to position 393 of SEQ ID NO:58 may be selected from the group consisting of leucine, valine, isoleucine, and methionine.
  • the replacement amino acid introduced at a position corresponding to position 428 of SEQ ID NO:58 may be selected from the group consisting of tyrosine and methionine.
  • the replacement amino acid introduced at the position corresponding to position 516 of SEQ ID NO:58 may be phenylalanine.
  • the replacement amino acid introduced at the position corresponding to position 566 of SEQ ID NO:58 may be selected from the group consisting of phenylalanine, leucine, valine, and methionine.
  • the replacement amino acid introduced at the position corresponding to position 568 of SEQ ID NO:58 may be selected from the group consisting of isoleucine and methionine.
  • the replacement amino acid introduced at the position corresponding to position 585 of SEQ ID NO:58 may be selected from the group consisting of leucine and methionine.
  • the replacement amino acid introduced at the position corresponding to position 615 of SEQ ID NO:58 may be leucine.
  • the FAD-GLDH ⁇ -T of the present disclosure has amino acid substitutions at positions corresponding to positions 545, 425, and/or 109 of SEQ ID NO:58, and has an improved ratio of dehydrogenase activity to oxidase activity compared to the polypeptide before the amino acid substitutions are introduced; (i) When aligned with the amino acid sequence set forth in SEQ ID NO:58, the amino acid sequence of a region corresponding to positions 459 to 507 of SEQ ID NO:58 is deleted, and optionally the amino acid sequence of a region corresponding to positions 671 to 690 of SEQ ID NO:58 is deleted; and When aligned with the amino acid sequence of SEQ ID NO:58, an amino acid at a position corresponding to a position selected from the group consisting of positions 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO:58 has been substituted, for example, modified
  • the amino acid sequence has one or several amino acids substituted, deleted or added at positions corresponding to positions 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 58, and at positions other than the region corresponding to positions 459 to 507 of SEQ ID NO: 58, or the amino acid sequence has one or several amino acids substituted, deleted or added at positions corresponding to positions 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO: 58, and at positions other than the region corresponding to positions 459 to 507 and 671 to 690 of SEQ ID NO: 58.
  • the full-length amino acid sequence of the FAD-GLDH ⁇ -T has 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, to the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:59, or SEQ ID NO:60, or (iv) In the above (i) or (ii), the full-length amino acid sequence of the FAD-GLDH ⁇ -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, to the amino acid sequence of SEQ ID NO:12, SEQ ID NO
  • the present disclosure provides a polypeptide having 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, such as 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, such as 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO:60, having amino acid substitutions at positions corresponding to positions 545, 425 and/or 109 of SEQ ID NO:58, and lacking a region corresponding to positions 459 to 507 of SEQ ID NO:58, and A position corresponding to position 186 of SEQ ID NO:58; A position corresponding to amino acid sequence
  • the replacement amino acid introduced at the position corresponding to position 186 of SEQ ID NO:58 may be selected from the group consisting of glycine, alanine, cysteine, leucine, methionine, phenylalanine, tyrosine, serine, threonine, asparagine, glutamine, histidine, aspartic acid, and glutamic acid.
  • the replacement amino acid introduced at the position corresponding to position 87 of SEQ ID NO:58 may be tyrosine.
  • the replacement amino acid introduced at the position corresponding to position 103 of SEQ ID NO:58 may be selected from the group consisting of phenylalanine, leucine, valine, isoleucine, and methionine. In certain embodiments, with respect to the FAD-GLDH ⁇ -T mutant of the present disclosure, the replacement amino acid introduced at the position corresponding to position 133 of SEQ ID NO:58 may be selected from the group consisting of leucine and tyrosine.
  • the replacement amino acid introduced at a position corresponding to position 297 of SEQ ID NO:58 may be selected from the group consisting of leucine, valine, isoleucine, and methionine.
  • the replacement amino acid introduced at a position corresponding to position 376 of SEQ ID NO:58 may be selected from the group consisting of phenylalanine, leucine, isoleucine, and methionine.
  • the replacement amino acid introduced at a position corresponding to position 393 of SEQ ID NO:58 may be selected from the group consisting of leucine, valine, isoleucine, and methionine.
  • the replacement amino acid introduced at a position corresponding to position 428 of SEQ ID NO:58 may be selected from the group consisting of tyrosine and methionine.
  • the replacement amino acid introduced at the position corresponding to position 516 of SEQ ID NO:58 can be phenylalanine.
  • the replacement amino acid introduced at the position corresponding to position 566 of SEQ ID NO:58 can be selected from the group consisting of phenylalanine, leucine, valine, and methionine. In certain embodiments, for the FAD-GLDH ⁇ -T mutant of the present disclosure, the replacement amino acid introduced at the position corresponding to position 568 of SEQ ID NO:58 can be selected from the group consisting of isoleucine and methionine.
  • the replacement amino acid introduced at the position corresponding to position 585 of SEQ ID NO:58 can be selected from the group consisting of leucine and methionine. In certain embodiments, for the FAD-GLDH ⁇ -T mutant of the present disclosure, the replacement amino acid introduced at the position corresponding to position 615 of SEQ ID NO:58 can be leucine.
  • the present invention provides a method for producing FAD-GLDH, comprising the steps of culturing a strain capable of producing FAD-GLDH under conditions that allow expression of the FAD-GLDH, and isolating FAD-GLDH from the culture or culture solution.
  • a host cell transformed with a vector incorporating a gene encoding the FAD-GLDH of the present disclosure can be used.
  • the conditions that allow expression of FAD-GLDH refer to conditions in which the FAD-GLDH gene is transcribed and translated, and a polypeptide encoded by the gene is produced.
  • GLOD can also be produced in a similar manner.
  • the medium for culturing the above-mentioned strains may be, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran extract, 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 carbohydrate raw materials, vitamins, etc. may also be appropriately added as necessary.
  • nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran extract
  • inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate
  • carbohydrate raw materials, vitamins, etc. may also be appropriately
  • the production amount of the target enzyme can be improved by adding to the medium a substrate on which the FAD-GLDH can act or a similar compound, such as glycosylated amino acids, glycosylated peptides, glycosylated protein hydrolysates, or glycosylated proteins such as glycosylated hemoglobin or glycosylated albumin.
  • a substrate on which the FAD-GLDH can act or a similar compound, such as glycosylated amino acids, glycosylated peptides, glycosylated protein hydrolysates, or glycosylated proteins such as glycosylated hemoglobin or glycosylated albumin.
  • the initial pH of the medium should be adjusted to pH 7-9.
  • Cultivation is preferably carried out at a culture temperature of 20-42°C, preferably around 25-37°C for 4-24 hours, more preferably around 25-37°C for 8-16 hours, by aeration and agitation submerged culture, shaking culture, static culture, etc.
  • FAD-GLDH can be collected from the culture by using a conventional enzyme collection method.
  • the cells can be subjected to ultrasonic destruction, grinding, etc., or the enzyme can be extracted using a lytic enzyme such as lysozyme, or the cells can be shaken or left to stand in the presence of toluene, etc. to cause lysis and excrete the enzyme from the cells.
  • the solution can then be filtered, centrifuged, etc. to remove solids, and nucleic acids can be removed as necessary using streptomycin sulfate, protamine sulfate, manganese sulfate, etc., after which ammonium sulfate, alcohol, acetone, etc. can be added to fractionate, and the precipitate can be collected to obtain the crude enzyme.
  • a more purified enzyme preparation from the crude enzyme for example, gel filtration using Sephadex, Superdex, Ultrogel, etc., adsorption elution using ion exchange carriers, hydrophobic carriers, 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 FAD-GLDH enzyme preparation.
  • the FAD-GLDH mutant is, e.g. (i) FAD is a coenzyme; (ii) recognizes glutamate as a substrate; (iii) its action is to oxidize glutamic acid to produce 2-oxoglutarate and ammonia; It can be something.
  • the residual activity of FAD-GLDH after heat treatment at 30 to 40°C, for example 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, when the activity before heat treatment is taken as 100%.
  • the residual activity of FAD-GLDH after heat treatment at 60°C, for example, 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, when the activity before heat treatment is taken as 100%.
  • the residual activity of FAD-GLDH after heat treatment at 65°C for 30 or 35 minutes may be 50% or more, 60% or more, 65% or more, 70% or more, for example 75% or more, when the activity before heat treatment is taken as 100%.
  • the residual activity of FAD-GLDH after heat treatment at 70°C for 30 or 35 minutes may be 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, for example 35% or more, when the activity before heat treatment is taken as 100%.
  • the FAD-GLDH disclosed herein excludes FAD-GLDH that does not exhibit activity against glutamate.
  • the present invention provides a glutamate measurement reagent composition, measurement reagent, electrode, sensor, or kit containing FAD-GLDH.
  • the composition, reagent, electrode, sensor, or kit may contain a measurement reagent for reduced compounds, a measurement reagent for hydrogen peroxide, a buffer, a surfactant, salts, a preservative, and the like.
  • a solubilizing agent a stabilizer, a reactivity enhancer, a glycosylated hemoglobin denaturant, a reducing agent, bovine serum albumin, a sugar (glycerin, lactose, sucrose, etc.), and the like may be added.
  • the composition, reagent, electrode, sensor, or kit may contain other known stabilizers, a system for elimination of impurities, and the like, as necessary.
  • the 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.
  • Surfactants include nonionic surfactants, ionic surfactants, such as cationic surfactants, anionic surfactants, and amphoteric surfactants.
  • nonionic surfactants include polyoxyethylene alkyl ethers, fatty acid sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides, and alkyl monoglyceryl ethers.
  • 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.
  • the present disclosure provides a method for measuring glutamic acid.
  • the measurement of glutamic acid may be a qualitative or quantitative method.
  • the quantitative method may include a step of contacting a sample containing glutamic acid with the FAD-GLDH of the present disclosure, and a step of measuring a reaction product or a consumed product.
  • the contact in the quantitative method includes any manner in which the enzyme and the sample are physically brought together so that the FAD-GLDH can catalyze the dehydrogenation reaction of glutamic acid, and includes, for example, not only a case in which a free enzyme and glutamic acid are mixed in a solution, but also a case in which a solution sample containing glutamic acid is added or dropped to an enzyme supported on a solid phase carrier.
  • glutamic acid is measured using the dehydrogenase activity of FAD-GLDH, hydrogen peroxide is removed from the reaction product to be measured (i.e., the generated hydrogen peroxide is not measured).
  • the reaction product to be measured includes the reduced form of the mediator (i.e., the produced reduced form of the mediator is measured).
  • glutamate is measured using the dehydrogenase activity of FAD-GLDH
  • the consumed product to be measured includes the oxidized form of the mediator (i.e., the consumed oxidized form of the mediator is measured).
  • the sample used for the measurement can be any sample that may contain glutamic acid.
  • the sample may be appropriately processed.
  • the amount of enzyme used and reaction time are kept constant, and the amount of glutamic acid added is varied.
  • the lowest detectable glutamic acid concentration detection limit concentration
  • 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.
  • a calibration curve can be created in advance by performing regression analysis such as the least squares method on the measured values of absorbance of a control containing a known concentration of glutamic acid.
  • the glutamic acid concentration in the sample can be quantified by plotting the measured values of a sample with an unknown glutamic acid concentration against the created calibration curve.
  • the time for allowing FAD-GLDH to act on a sample containing glutamic acid can be, for example, 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, less than 60 minutes, less than 30 minutes, less than 10 minutes, for example, less than 5 minutes, for example, 0.5 minutes or more to less than 60 minutes, 1 minute or more to less than 30 minutes, 1 minute or more to less than 20 minutes, for example, 1 minute or more to less than 10 minutes, for example, 1 minute or more to less than 5 minutes.
  • the acting temperature depends on the optimal temperature of the enzyme used, but is, for example, 20 to 45°C, and can be appropriately selected from temperatures used in normal enzyme reactions.
  • the amount of FAD-GLDH enzyme used depends 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 FAD-GLDH can act, e.g., the optimal pH.
  • the reaction pH is, for example, 3 to 11, 5 to 9, e.g., 6 to 8.
  • GLOD activity Method for measuring glutamate oxidase activity (GLOD activity)
  • the following is an example of a method for measuring GLOD activity using glutamic acid as a substrate, but the measurement method is not limited thereto.
  • Glutamic acid may be commercially available.
  • the enzyme activity is defined as 1 U, which is the amount of enzyme that produces 1 ⁇ mol of hydrogen peroxide per minute when measured at 30° C. and pH 7.4 using glutamic acid as a substrate.
  • A Activity measurement reagent (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 ⁇ l of reagent 1, 12.5 ⁇ l of reagent 2, 25 ⁇ l of reagent 3, 12.5 ⁇ l of reagent 4, deionized water (375-V) ⁇ l, and V ⁇ l of GLOD solution, and incubate at 30° C.
  • Glutamate oxidase activity can be calculated based on the following formula:
  • "39.2" represents the millimolar extinction coefficient (mM -1 cm -1 ) of the quinoneimine dye formed by condensation of 4-AA with TOOS for light with a wavelength of 555 nm.
  • the wavelength and millimolar extinction coefficient at that wavelength according to the color reagent may be used.
  • the following is an example of a method for measuring glutamate dehydrogenase activity using glutamic acid as a substrate, but the measurement method is not limited thereto.
  • Glutamic acid may be commercially available.
  • the enzyme titer is defined as the amount of enzyme that reacts with 1 ⁇ mol of glutamic acid as a substrate per minute when measured at 30° C. and pH 7.4 using glutamic acid as a substrate.
  • A Activity measurement reagent (reagent 1) 250 mM potassium phosphate buffer pH 7.4 (Reagent 2) 3 mM dichloroindophenol (DCIP) solution (Reagent 3) 30 mM 1-Methoxy PMS (mPMS) solution (Reagent 4) 300 mM sodium hydrogen glutamate solution
  • B Activity measurement method 300 ⁇ l of Reagent 1, 25 ⁇ l of Reagent 2, 25 ⁇ l of Reagent 3, deionized water (375-V) ⁇ l, and V ⁇ l of GLOD solution were mixed and kept at 30° C. for 5 minutes.
  • the glutamate dehydrogenase activity (U/ml) can be calculated based on the following formula:
  • "21" represents the millimolar extinction coefficient (mM -1 cm -1 ) of DCIP at pH 7.4 for light with a wavelength of 600 nm.
  • the wavelength and millimolar extinction coefficient at that wavelength according to the color reagent may be used.
  • GLOD or FAD-GLDH By drying GLOD or FAD-GLDH in the presence of a trisaccharide, the enzyme activity can be maintained even if the dried product is stored for a long period of time.
  • trisaccharides used in the present invention include raffinose, kestose, maltotriose, maltotriulose, nigerotriose, melezitose, etc., and raffinose is particularly preferable.
  • drying may be performed in the presence of a plurality of trisaccharides.
  • the dry composition of the present invention does not include a simple homogeneous mixture of dried GLOD or FAD-GLDH powder and dried trisaccharide powder.
  • the dry composition of GLOD or FAD-GLDH may contain, in addition to the trisaccharide, further additives (buffers, stabilizers, excipients, solubilizers, preservatives, etc.), or may contain two or more types of additives.
  • the amount of GLOD or FAD-GLDH contained in the dry composition of the present invention is preferably 0.1% by mass or more and less than 80% by mass, more preferably 1% by mass or more and less than 75% by mass, and even more preferably 10% by mass or more and less than 70% by mass.
  • the amount of trisaccharides contained in the dry composition of the present invention is preferably 1% by mass or more and less than 90% by mass. Also, it is more preferably 10% by mass or more and less than 80% by mass, and even more preferably 20% by mass or more and less than 75% by mass.
  • the amount of trisaccharide contained in the dry composition can be 0.10 mg or more and less than 50 mg, 0.10 mg or more and less than 40 mg, 0.10 mg or more and less than 30 mg, 0.10 mg or more and less than 20 mg, for example, 0.10 mg or more and less than 10 mg, relative to 100 U of GLOD or FAD-GLDH. Furthermore, the amount of trisaccharide contained in the dry composition is preferably 0.10 mg or more and less than 5 mg, more preferably 0.10 mg or more and less than 2.5 mg, and even more preferably 0.10 mg or more and less than 0.50 mg, relative to 100 U of GLOD or FAD-GLDH.
  • the stabilizing effect of trisaccharides on GLOD or FAD-GLDH is greater than that of monosaccharides or sugar alcohols. Specifically, when D-glucose, a monosaccharide, was used in the GLOD dry composition, the activity value decreased after two weeks of storage at 37°C. When xylitol, a sugar alcohol, was used in the GLOD dry composition, approximately 49% of the activity value remained after two weeks of storage at 37°C.
  • Patent Document 2 discloses a disaccharide (lactose) as a compound that stabilizes a dried product of GLOD derived from Streptomyces sp. X-119-6.
  • lactose a disaccharide
  • the activity of a freeze-dried product of GLOD derived from Streptomyces sp. X-119-6 to which lactose had been added was 101.5% after storage at 37°C for two weeks.
  • the activity of a freeze-dried product of GLOD derived from Streptomyces sp. X-119-6 to which sugar alcohol (mannitol) had been added was 50.5% after storage at 37°C for two weeks.
  • the dried composition of GLOD or FAD-GLDH may be prepared by freeze-drying or spray-drying.
  • a dry composition of GLOD or FAD-GLDH can be prepared by drying an aqueous composition containing GLOD or FAD-GLDH and a trisaccharide.
  • the aqueous composition may have a pH adjusted with a buffer. Any buffer having a buffering capacity in the range of pH 5.0 to 9.0 can be suitably used.
  • buffers examples include various carboxylates (acetate, citrate, ethylenediaminetetraacetate, phthalate, fumarate, malate, maleate, glutarate, etc.), phosphate, tris(hydroxymethyl)aminomethane, borate, and various Good's buffer solutions (MES, Bis-Tris, ADA, PIEPS, ACES, MOPSO, BES, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, etc.). These buffers may be used alone or in combination of two or more. Phosphate is preferred as a buffer for adjusting the pH of an aqueous composition containing GLOD or FAD-GLDH and a trisaccharide.
  • carboxylates acetate, citrate, ethylenediaminetetraacetate, phthalate, fumarate, malate, maleate, glutarate, etc.
  • phosphate tris(hydroxymethyl)a
  • the concentration of the buffer solution is not particularly limited, but is, for example, 5 mM to 100 mM, and preferably 5 mM to 50 mM.
  • the content of the buffer solution component in the dry composition is preferably 1 to 80% by mass.
  • the content of GLOD or FAD-GLDH in the aqueous composition is preferably 10 U/mL or more and less than 500 U/mL, and more preferably 25 U/mL or more and less than 300 U/mL.
  • the content of trisaccharides in the aqueous composition is, for example, 0.1 mg/mL or more and less than 100 mg/mL, preferably 0.5 mg/mL or more and less than 50 mg/mL, and more preferably 1 mg/mL or more and less than 10 mg/mL.
  • the disclosure provides a polynucleotide encoding glutamate dehydrogenase.
  • the disclosure provides a vector having such a polynucleotide.
  • the disclosure provides a host cell transformed with such a vector, i.e., a host cell comprising such a vector.
  • the disclosure provides a method for producing glutamate dehydrogenase, comprising culturing such a host cell to produce glutamate dehydrogenase, and obtaining the produced glutamate dehydrogenase.
  • the disclosure provides a dry composition comprising glutamate oxidase or FAD-glutamate dehydrogenase, and a method for producing the same.
  • the disclosure provides a method for contacting glutamate dehydrogenase, or a composition, reagent, electrode, sensor, or kit comprising the same, with a sample containing glutamate, to oxidize glutamate contained in the sample.
  • the method can detect glutamate.
  • the method can measure glutamate.
  • the present inventors have prepared GLOD ⁇ by introducing a specific amino acid sequence deletion into GLOD.
  • GLOD ⁇ -T by introducing a specific amino acid substitution into GLOD ⁇ has been prepared.
  • FAD-GLDH ⁇ by introducing a specific amino acid sequence deletion into GLOD ⁇ .
  • FAD-GLDH ⁇ -T by introducing a specific amino acid substitution into FAD-GLDH ⁇ has been prepared.
  • Amino acid substitutions, deletions and mutations can be combined as appropriate. Further mutants can also be prepared based on the description herein.
  • the present invention provides a method for preparing FAD-GLDH, comprising the steps of: (i) obtaining the GLOD gene or the GLOD ⁇ gene; (ii) inserting the GLOD gene or GLOD ⁇ gene into a vector, transforming a host cell, expressing GLOD or GLOD ⁇ , 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; (v) modifying the GLOD gene or GLOD ⁇ gene so that, when the amino acid sequence of GLOD or GLOD ⁇ is aligned with the amino acid sequence set forth in SEQ ID NO:58, an amino acid at a position corresponding to a position selected from the group consisting of positions 186, 87, 103, 133, 297, 376, 393, 428, 516, 566, 568, 585, and 615 of SEQ ID NO:58 is substituted, for example, an amino acid selected from
  • a peptide linker may be inserted into the region of an FAD-GLDH mutant, such as FAD-GLDH ⁇ or FAD-GLDH ⁇ -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 FAD-GLDH.
  • 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, 5-16, 6-15, for example, 7-14 amino acid residues.
  • the amino acid residues that compose the peptide linker may be natural amino acids and glycine. Examples include Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Asp, Glu, Arg, His, and Lys. Examples include Gly, Ala, Ser, and Thr.
  • the peptide linker can be GGGGS or a repeat sequence thereof. The number of repeats can be 2, 3, or 4.
  • the FAD-GLDH mutant such as FAD-GLDH ⁇ or FAD-GLDH ⁇ -T, does not have a peptide linker.
  • FAD-GLDH is a type of enzyme that has never been seen before, and is clearly distinguished from NAD-GLDH that was known in the prior art.
  • the FAD-GLDH of the present disclosure does not have amino acid sequence identity with conventional NAD-GLDH, or has low amino acid sequence identity.
  • the FAD-GLDH of the present disclosure can measure glutamate without adding NAD from the outside. It is known that NAD-type GLDH releases NAD or NADH from the enzyme. In addition, when the concentration of released NADH increases, a reverse reaction may occur. In the case of FAD-type GLOD, FAD is not released from the enzyme, or is released very little.
  • FAD is not released from the enzyme, or is released very little, in the case of the FAD-GLDH of the present disclosure. Therefore, it is believed that the FAD-GLDH of the present disclosure is less likely to undergo a reverse reaction. This is advantageous in applications as a sensor. In addition, the FAD-GLDH of the present disclosure can reduce the effect on the measured value of dissolved oxygen. This is also advantageous for use as a sensor.
  • the present disclosure provides a method for measuring glutamic acid using FAD-GLDH.
  • This method does not require the addition of NAD from the outside.
  • this method does not require the addition of peroxidase.
  • pretreatment with protease is not required to prepare FAD-GLDH.
  • NAD is not liberated from the enzyme, so it can be combined with various mediators.
  • no NAD is added (aspects in which NAD is added are excluded).
  • no peroxidase is added (aspects in which peroxidase is added are excluded).
  • the method of measuring glutamate using FAD-GLDH of the present disclosure does not release NAD from the enzyme, so glutamate can be measured even if NAD or NADH is present in the measurement sample.
  • the present disclosure provides a glutamate measurement reagent containing FAD-GLDH.
  • the measurement reagent does not contain NAD.
  • the measurement reagent does not contain peroxidase.
  • glutamate may be measured in a sample containing NAD using this measurement reagent.
  • the FAD-GLDH of the present disclosure is further illustrated by the following examples. However, these are for illustrative purposes only, and the present disclosure is in no way limited thereto.
  • Example 1 First, glutamate oxidase (GLOD) was produced, and then mutants of GLOD were produced and their dehydrogenase activities were confirmed.
  • GLOD glutamate oxidase
  • 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 assembling the gene.
  • 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 assembling the gene.
  • 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). PCR was performed according to the instructions for use with KOD One PCR Master Mix (Toyobo).
  • the SEQ ID NO:6 or SEQ ID NO:7 primer has a 15-base sequence that overlaps with StGLOD-f1 or StGLOD-f3, respectively, added to the 5' end of the sequence that anneals to pKK223-3. After adding 1.0 ⁇ l of DpnI (New England BioLabs) to the PCR solution and treating at 37°C for 1 hour, the amplified fragment was purified using the GFX PCR DNA and Gel Band Purification Kit (Cytiva).
  • composition in Table 1 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 strain.
  • the resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed by DNA sequence analysis to be the desired sequence.
  • StGLOD deletion mutant 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 solution was prepared by mixing 10 ⁇ l of KOD One PCR Master Mix (manufactured by Toyobo), 3 ⁇ l of 2 ⁇ M Fw primer, 3 ⁇ l of 2 ⁇ M Rv primer, 0.5 ⁇ l of 40 ⁇ g/ml pKK223-3-StGLOD, and 3.5 ⁇ l of ion-exchanged water.
  • the amino acid sequences of StGLOD ⁇ 49Ai and StGLOD ⁇ 49C are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively.
  • the PCR reaction conditions were a cycle of "98°C 10 seconds ⁇ 55°C 5 seconds ⁇ 68°C 35 seconds" repeated 7 times.
  • DpnI 1 ⁇ l of DpnI was added to the solution after PCR and treated at 37°C for 1 hour to degrade the template pKK223-3-StGLOD ⁇ 49C.
  • the resulting DpnI-treated solution was used to transform E. coli JM109 strain.
  • the resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed by DNA sequence analysis to be the desired sequence.
  • the GLOD-producing strain was inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 ⁇ g/ml) 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 ⁇ g/ml) containing 0.1 mM IPTG in a Sakaguchi flask and cultured at 25° C. and 130 rpm for 16 hours.
  • 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 suspension was ultrasonically disrupted, and then centrifuged at 15,000 rpm for 15 minutes to obtain the supernatant, which was used as the GLOD crude enzyme solution.
  • the reagents used to measure GLOD activity were 4-aminoantipyrine (4-AA) (Fujifilm Wako Pure Chemical Industries, Ltd.), TOOS (Dojindo Laboratories, Ltd.), and horseradish peroxidase (POD) (Toyobo Co., Ltd.).
  • the composition of the reagent is shown in Table 2.
  • the GLOD solution was diluted with 10 mM PPB (pH 7.5) containing 0.15% bovine serum albumin (BSA, Sigma-Aldrich Co., Ltd.).
  • the oxidase activity (U/ml) was calculated based on the following formula, where "39.2" represents the millimolar absorption coefficient (mM -1 cm -1 ) of the quinoneimine dye formed by condensation of 4-AA with TOOS for light with a wavelength of 555 nm.
  • the GLOD crude enzyme solution was diluted with 10 mM PPB pH 6.0 so that the final concentration of GLOD was 0.05 U/ml. Then, 240 ⁇ l of 0.05 U/ml GLOD solution and 160 ⁇ l of 250 mM PPB pH 6.0 were mixed and heated for 30 or 35 minutes in a water bath kept at a predetermined temperature. After heating, the GLOD solution was quickly cooled on ice, and activity was measured using 375 ⁇ l of the GLOD solution. The activity of the sample that was not heated but cooled on ice was set as 1, and the residual activity of the sample after heating was calculated. The residual activity was calculated three times for each GLOD, and the thermal stability was evaluated using the average value.
  • the residual activity of the StGLOD deletion mutants shown in Table 3 was increased by 0.11 to 0.37 compared to StGLOD, and all StGLOD deletion mutants had improved thermal stability. In other words, the residual activity was improved by approximately 20% and approximately 65% compared to the wild type.
  • Example 2 Preparation of Modified StGLOD ⁇ 49C The expression and residual activity of StGLOD ⁇ 49C were confirmed. Therefore, a modified mutant based on StGLOD ⁇ 49C was further prepared.
  • Site-directed mutagenesis was performed using the StGLOD ⁇ 49C expression plasmid (pKK223-3-StGLOD ⁇ 49C) as a template to obtain a plasmid carrying a gene encoding modified StGLOD.
  • the PCR reaction solution was prepared by mixing 10 ⁇ l of KOD one PCR Master Mix (Toyobo), 3 ⁇ l of 2 ⁇ M FW primer, 3 ⁇ l of 2 ⁇ M RV primer, 0.5 ⁇ l of 40 ⁇ g/ml pKK223-3-StGLOD, and 3.5 ⁇ l 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 15 cycles of "98°C 10 seconds ⁇ 55°C 5 seconds ⁇ 68°C 35 seconds".
  • the expression plasmid for multiple mutant StGLOD ⁇ 49C was constructed by repeating single mutagenesis.
  • a plasmid for expressing the double mutant StGLOD ⁇ 49C/F87Y/Y103I was constructed by performing PCR using pKK223-3-StGLOD ⁇ 49C/F87Y as a template and primers of sequence numbers 20 and 16.
  • the residual activity of the modified StGLOD ⁇ 49C shown in Table 5 was increased by 0.02 to 0.72 compared to StGLOD ⁇ 49C, and the thermal stability of all modified StGLOD ⁇ 49C was improved. In other words, the residual activity of the modified enzymes produced was improved by approximately 9% or more to approximately 325% or more compared to the unmodified StGLOD ⁇ 49C.
  • An octuplet mutant was created by combining eight amino acid substitutions (F87Y, Y103I, F133Y, F297M, F393L, F428Y, Y517F, Y536L) that contributed to improving the thermal stability of StGLOD ⁇ 49C.
  • Table 6 shows the residual activity of the StGLOD deletion mutant after heating under heat treatment conditions that are more severe than those in Table 5 (60°C, 65°C, or 70°C for 35 minutes).
  • the 8-fold mutant of StGLOD ⁇ 49C shown in Table 6 was stable when heated at 60°C for 35 minutes, and retained more than one-third of its activity when heated at 70°C for 35 minutes. It was revealed that the thermal stability of StGLOD ⁇ 49C was dramatically improved by combining the amino acid substitutions shown in Table 5.
  • the M7GLOD ⁇ 49C-T8 expression plasmid (pET22b-M7GLOD ⁇ 49C-T8) was prepared using the In-Fusion HD Cloning Kit (Clontech).
  • the GLOD gene having the base sequence of SEQ ID NO:61 was divided into three fragments of SEQ ID NO:62 (M7GLOD ⁇ 49C-T8-f1), SEQ ID NO:63 (M7GLOD ⁇ 49C-T8-f2), and SEQ ID NO:64 (M7GLOD ⁇ 49C-T8-f3), and synthesis was entrusted to Integrated DNA Technologies.
  • the 15 bases on the 5' end of M7GLOD ⁇ 49C-T8-f1 are a duplicated sequence for assembly into pET-22b(+).
  • the 15 bases on the 3' end of M7GLOD ⁇ 49C-T8-f1 and the 15 bases on the 5' end of M7GLOD ⁇ 49C-T8-f2 are overlapping sequences for gene assembly.
  • the 15 bases on the 3' end of M7GLOD ⁇ 49C-T8-f2 and the 15 bases on the 5' end of M7GLOD ⁇ 49C-T8-f3 are overlapping sequences for gene assembly.
  • the 15 bases on the 3' end of M7GLOD ⁇ 49C-T8-f3 are overlapping sequences for assembly into pET-22b(+).
  • 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 ⁇ l of DpnI (New England BioLabs) was added to the PCR solution and treated at 37°C for 1 hour, after which the amplified fragment was purified using the GFX PCR DNA and Gel Band Purification Kit (Cytiva).
  • the composition in Table 7 was used for the reaction at 50°C for 15 minutes to obtain the M7GLOD ⁇ 49C-T8 expression plasmid (pET22b-M7GLOD ⁇ 49C-T8).
  • the resulting plasmid was transformed into E. coli JM109 strain.
  • the resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed by DNA sequence analysis to be the desired sequence.
  • pET22b-M7GLOD ⁇ 49C-T8 was used to transform E. coli BL21 (DE3) strain, and a strain producing M7GLOD ⁇ 49C-T8 was created.
  • M7GLOD ⁇ 49C-T8 The M7GLOD ⁇ 49C-T8 production strain was inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 ⁇ g/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 ⁇ g/ml) in a Sakaguchi flask and cultured at 37 ° C. and 130 rpm.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • 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 6.0.
  • the bacterial suspension was ultrasonically disrupted, and then centrifuged at 15,000 rpm for 15 minutes to obtain the supernatant, which was used as the M7GLOD ⁇ 49C-T8 crude enzyme solution.
  • M7GLOD ⁇ 49C-T8 was heated at 60°C or 65°C for 35 minutes, and its residual activity was calculated according to the method described above (Table 8).
  • M7GLOD ⁇ 49C-T8 was stable when heated at 60°C for 35 minutes, and more than half of its activity remained even when heated at 65°C for 35 minutes, demonstrating high thermal stability. It was demonstrated that the thermal stability of GLOD, not limited to StGLOD, can be improved by combining the amino acid substitutions shown in Table 4.
  • FAD-GLDH Based on GLOD
  • amino acid substitutions were introduced into GLOD to prepare FAD-GLDH.
  • a mutation replacing the leucine at position 545 of SEQ ID NO:58 with another amino acid was introduced into the gene encoding GLOD.
  • Site-specific mutagenesis was performed using the M7GLOD ⁇ 49C-T8 expression plasmid (pET22b-M7GLOD ⁇ 49C-T8) as a template to obtain a plasmid carrying a gene encoding mutant M7GLOD ⁇ 49C-T8.
  • the PCR reaction solution was prepared by mixing 10 ⁇ l of KOD One PCR Master Mix (manufactured by Toyobo), 3 ⁇ l of 2 ⁇ M Fw primer, 3 ⁇ l of 2 ⁇ M Rv primer, 0.5 ⁇ l of 40 ⁇ g/ml pET22b-M7GLOD ⁇ 49C-T8, and 3.5 ⁇ l of ion-exchanged water.
  • the names of the mutants and the combinations of Fw primer and Rv primer that were prepared are shown in Table 9.
  • the leucine at position 545 of SEQ ID NO:58 corresponds to the leucine at position 496 of M7GLOD ⁇ 49C-T8 (SEQ ID NO:60).
  • the PCR reaction conditions were 15 cycles of "98°C 10 seconds ⁇ 55°C 5 seconds ⁇ 68°C 35 seconds".
  • the expression plasmid for the multiple mutant M7GLOD ⁇ 49C was constructed by repeating single mutation introduction.
  • a double mutant pET22b-M7GLOD ⁇ 49C-T8/L496A/L425Q expression plasmid was constructed by performing PCR using pET22b-M7GLOD ⁇ 49C-T8/L496A as a template and primers of SEQ ID NO:75 and SEQ ID NO:77.
  • the mutant M7GLOD ⁇ 49C-T8 was produced recombinantly using the same method as the previously described M7GLOD ⁇ 49C-T8.
  • Oxidase activity (OD) and dehydrogenase activity (DH) were measured for the mutants after substitution. OD was evaluated in the same manner as for M7GLOD ⁇ 49C-T8 described above.
  • DH Dichloroindophenol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 1-Methoxy PMS (mPMS) (manufactured by Dojindo Laboratories, Ltd.) were used as GLDH activity measurement reagents.
  • the composition of the activity measurement reagent is shown in Table 10.
  • GLDH solution was diluted with 10 mM PPB (pH 7.4) containing 0.15% bovine serum albumin (BSA, manufactured by Sigma-Aldrich).
  • BSA bovine serum albumin
  • DH activity U/ml
  • mM -1 cm -1 the predicted millimolar extinction coefficient (mM -1 cm -1 ) of DCIP at pH 7.4 for light with a wavelength of 600 nm.
  • the ratio of OD to DH was calculated for each GLOD and is shown in Table 11 below.
  • the OD/DH of GLOD before mutation was 32.4, whereas the mutant with a substitution at the L545 position surprisingly had a reduced OD/DH and a significantly improved dehydrogenase activity per oxidase activity.
  • the L496A mutant showed an approximately 22-fold improvement
  • the L496G mutant showed an approximately 65-fold improvement.
  • the double mutant L496A/L425Q showed a significant improvement. It is reasonably believed that similar improvements are likely to be achieved when L425Q is combined with other mutations.
  • the mutants with a substitution at the M109 position showed a reduced OD/DH and a significant improvement in dehydrogenase activity per oxidase activity.
  • mutants in which M109 was replaced with F, C, A, V, Q, T, H, Y, or S showed an improvement of approximately 29, 77, 163, 195, 284, 364, 470, 1080, or 2310 times.
  • the M109W mutant completely lost oxidase activity and was converted into a mutant that only exhibited dehydrogenase activity.
  • the positions of homologous amino acid residues in each GLOD sequence can be determined regardless of insertions or deletions in the amino acid sequence.
  • Corresponding positions are considered to be at the same positions in the three-dimensional structure and to play similar roles in each GLOD.
  • the present inventors have prepared glutamate dehydrogenase using FAD as a coenzyme by substituting the amino acid at the position corresponding to position 545 of SEQ ID NO:58 for GLOD using FAD as a coenzyme.
  • glutamate dehydrogenase using FAD as a coenzyme was prepared by substituting the amino acid at the position corresponding to position 425 of SEQ ID NO:58.
  • glutamate dehydrogenase using FAD as a coenzyme was prepared by substituting the amino acid at the position corresponding to position 109 of SEQ ID NO:58.
  • plasmids for expressing various modified M7GLOD ⁇ 49C-T7 were constructed in the same manner as in "6. Preparation of modified StGLOD ⁇ 49C". The names of the mutants and the combinations of Fw and Rv primers that were prepared are shown in Table 12.
  • E. coli BL21(DE3) was transformed with pET22b-M7GLOD ⁇ 49C-T7 or the expression plasmids for various modified M7GLOD ⁇ 49C-T7 to create strains producing M7GLOD ⁇ 49C-T7 and various modified M7GLOD ⁇ 49C-T7.
  • M7GLOD ⁇ 49C-T7 and its mutants M7GLOD ⁇ 49C-T7 and modified M7GLOD ⁇ 49C-T7 producing strains were inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 ⁇ g/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 ⁇ g/ml) in a Sakaguchi flask and cultured at 37 ° C. and 130 rpm.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the culture medium was centrifuged at 8,000 rpm for 10 minutes, and the resulting pellet was resuspended in 4-25 ml of 10 mM potassium phosphate buffer (PPB) pH 6.0.
  • the bacterial cell suspension was ultrasonically disrupted, and then centrifuged at 15,000 rpm for 15 minutes to recover the supernatant, which was used as M7GLOD ⁇ 49C-T7 and modified M7GLOD ⁇ 49C-T7 crude enzyme solutions.
  • the crude enzyme solutions of M7GLOD ⁇ 49C-T7 and modified M7GLOD ⁇ 49C-T7 were diluted with 10 mM PPB pH 6.0 so that the final concentration of GLOD was 0.05 U/ml. Then, 240 ⁇ l of 0.05 U/ml GLOD solution was mixed with 160 ⁇ l of 250 mM PPB pH 6.0 and heated for 30 minutes in a water bath kept at a specified temperature. The residual activity was calculated according to the method described above (Table 13).
  • M7GLOD ⁇ 49C-T7 had a residual activity of 0.93 after heating at 60°C for 30 minutes and 0.69 after heating at 65°C for 30 minutes, demonstrating high thermal stability.
  • the residual activity of the various modified M7GLOD ⁇ 49C-T7s increased by 0.05 to 0.58 compared to M7GLOD ⁇ 49C-T7, and the thermal stability of all modified M7GLOD ⁇ 49C-T7s was improved.
  • the residual activity of the modified enzymes produced was improved by approximately 29% to approximately 341% compared to M7GLOD ⁇ 49C-T7 before modification.
  • the lyophilized product was stored at 37°C for 2 weeks, then dissolved in water to give an estimated concentration of 5 U/mL. It was then diluted with 10 mM potassium phosphate buffer, pH 7.4, containing 0.1% BSA to give an estimated concentration of 0.25 U/mL, and the activity of these was measured.
  • the present disclosure provides a novel type of glutamate dehydrogenase that can be used to detect glutamate and for dehydrogenation reactions. It can also be produced on a large scale.
  • SEQ ID NO: 1 Amino acid sequence of glutamate oxidase (StGLOD) derived from Streptomyces sp. X-119-6 MNEMTYEQLARELLLVGPAPTNEDLKLRYLDVLIDNGLNPPPGPPKRILIVGAGIAGLVAG DLLTRAGHDVTILEANANRVGGRIKTFHAKKGEPSPFADPAQYAEAGAMRLPSFHPLTLA LIDKLGLKRRLFFNVDIDPQTGNQDAPVPPVFYKSFKDGKTWTNGAPSPEFKEPDKRNHT WIRTNREQVRRAQYATDPSSINEGFHLTGCETRLTVSDMVNQALEPVRDYYSVKQDDGTR VNKPFKEWLAGWADVVRDFDGYSMGRFLREYAEFSDEAVEAIGTIENMTSRLHLAFFHSF LGRSDIDPRATYWEIEGGSRMLPETLAKDLRDQIVMGQRMVRLEYYDPGRDGHHGELTGP GGPA
  • SEQ ID NO: 2 Nucleotide sequence of glutamate oxidase (StGLOD) derived from Streptomyces sp. X-119-6
  • SEQ ID NO: 13 Amino acid sequence of StGLOD ⁇ 49C
  • SEQ ID NO: 58 Amino acid sequence of putative glutamate oxidase (M7GLOD) from Streptomyces sp. MOE7
  • SEQ ID NO: 59 Amino acid sequence of deleted M7GLOD (M7GLOD ⁇ 49C)
  • SEQ ID NO: 61 Nucleotide sequence of M7GLOD ⁇ 49C-T8

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