NADH oxidase, variant thereof, freeze dried sample, kit including same, electrode, sensor comprising electrode, and NADH measurement method

Abstract

One embodiment of the present disclosure addresses the problem of providing a novel NADH oxidase.　Embodiments of the present disclosure relate to: an NADH oxidase having amino acid sequence identity of not less than 70% with any amino acid sequence from among SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, and 78; an N-terminal domain deleted variant of the same; an amino acid substituted variant of the same; and a freeze dried sample, a kit, and an electrode which include the same; a sensor comprising the electrode; and an NADH measurement method using the NADH oxidase.

Description

NADH oxidase, its mutant, freeze-dried preparation, kit or electrode containing the same, sensor equipped with electrode, and method for measuring NADH

[0001] The present invention relates to NADH oxidase, a mutant thereof, a freeze-dried preparation of NADH oxidase or a mutant thereof, a kit containing NADH oxidase or a mutant thereof, an electrode containing NADH oxidase or a mutant thereof, a sensor equipped with the electrode, and a method for measuring NADH using NADH oxidase or a mutant thereof.

[0002] NADH oxidase oxidizes NADH using oxygen as an electron acceptor, producing NAD ＋ It is an enzyme that oxidizes NADH and produces NAD ＋ When generating ２ O ２ and a type that generates H ２ It is known that there are two types of oxidizers: one that produces O and the other that produces H. ２ O ２ The type that produces NOX1 is called NOX1 type, and H ２ The type that produces O is called NOX2 type.

[0003] Patent Document 1 describes a NOX1-type NADH oxidase derived from Bacillus cereus. Although NADH oxidases derived from other sources are also disclosed, among the enzymes listed in Table 2 of Patent Document 1, the NADH oxidase derived from Bacillus cereus is the most active. Although this document characterizes the NADH oxidase, the gene encoding the NADH oxidase has not been isolated, and no nucleotide or amino acid sequence information is provided. Therefore, the NADH oxidase described in this document cannot be recombinantly expressed. Furthermore, the disclosed NADH oxidase derived from Bacillus cereus is stable up to 30°C when treated at pH 7.2 for 10 minutes (see page 493, lower right column). However, the thermostability of this NADH oxidase is not sufficient.

[0004] Non-Patent Document 1 reports an NADH oxidase derived from Bacillus megaterium. This enzyme has not been cloned, and the base sequence of the gene and the amino acid sequence of the enzyme are unknown. Furthermore, according to Patent Document 1, the amount of NADH oxidase produced by Bacillus megaterium is extremely small, making it difficult to put into practical use for enzyme analysis, etc.

[0005] Patent Document 2 describes NADH oxidase derived from Amphibacillus xylanus Ep01. This enzyme has an optimum temperature of 37° C. Patent Document 3 describes a gene encoding NADH oxidase derived from Amphibacillus xylanus Ep01.

[0006] Patent Document 4 describes the amino acid sequence and the nucleotide sequence of the gene for NADH oxidase derived from Brevibacterium microorganisms. This enzyme is said to be highly thermostable, retaining most of its activity even after incubation at 70°C for 1 hour (Figure 3 in Patent Document 4). However, enzymes with high thermostability at high temperatures can sometimes have low relative activity at room temperature or 37°C. The NADH oxidase described in Patent Document 4 exhibits a relative activity at 37°C of approximately 50% of its 100% relative activity at 70°C (Figure 4 in Patent Document 4). Therefore, it is difficult to say that it has sufficient activity at room temperature or 37°C. Other NADH oxidases derived from highly thermophilic bacteria are known, but while they all have high thermostability at high temperatures, their activity at room temperature or 37°C is not necessarily sufficient.

[0007] Industrial mass production of NADH oxidase requires culturing an NADH oxidase-producing microorganism in a medium and recovering the NADH oxidase from the culture. The enzyme produced by this method can be used for various applications, but the amount produced depends on the NADH oxidase-producing microorganism. Furthermore, the properties of the NADH oxidase produced are the same as those of conventionally known NADH oxidases.

[0008] NADH oxidase can recognize not only NADH but also NADPH as a substrate. When NADH oxidase recognizes NADPH as a substrate, NADP ＋Generally, a low reactivity to NADPH is preferable. Therefore, an NADH oxidase with a low reactivity to NADPH is needed.

[0009] From the viewpoint of industrial use, the optimum temperature of an enzyme may preferably be about 20°C to 70°C. For example, the optimum temperature of an implantable sensor is preferably about body temperature, 37°C. Furthermore, the optimum temperature of test reagents and experimental reagents is preferably about room temperature, for example, about 25°C.

[0010] JP 63-251082 A JP 02-107186 JP 05-344890 JP 2009-165417

[0011] J. Biochem. 98, 1433-1440 (1985)

[0012] An object of the present disclosure is to provide an NADH oxidase that at least partially solves the problems of the prior art.

[0013] In one embodiment, an object of the present disclosure is to provide an NADH oxidase that is highly thermostable. In one embodiment, an object of the present disclosure is to provide an NADH oxidase that is more thermostable when treated at pH 7.2 for 10 minutes than an NADH oxidase that is stable up to 30°C. In one embodiment, an object of the present disclosure is to provide an NADH oxidase that is highly thermostable and has sufficient residual activity even after heat treatment at 50°C or 75°C for 15 minutes.

[0014] In one embodiment, an object of the present disclosure is to provide an NADH oxidase having an optimum temperature in the range of 20° C. to 70° C. In one embodiment, an object of the present disclosure is to provide an NADH oxidase having an optimum temperature in the range of 20° C. to 40° C.

[0015] In one embodiment, an objective of the present disclosure is to provide an NADH oxidase that has high reactivity to NADP and low reactivity to NADPH.

[0016] In certain embodiments, the present disclosure aims to provide an NADH oxidase that can be recombinantly produced.

[0017] It should be noted that the above description is merely for the convenience of explaining the present disclosure more specifically, and does not mean that all of the above problems must be solved simultaneously in relation to the present disclosure.

[0018] As a result of extensive research, the present inventors have produced an NADH oxidase that at least partially solves the problems of the prior art, and have completed the present invention, which includes this as one embodiment.

[0019] Furthermore, the present inventors have surprisingly found that, as an example, by deleting the N-terminal domain of NADH oxidase, the properties of the enzyme can be modified while retaining the enzyme activity, and have completed the present invention, which includes this as one embodiment.

[0020] Furthermore, the present inventors have found that, as an example, the properties of an NADH oxidase can be modified by substituting amino acid residues at specific positions, and have completed the present invention, which includes this as one embodiment.

[0021] Furthermore, the present inventors have discovered that, as an example, a freeze-dried reagent that retains activity can be produced by contacting NADH oxidase with a specific stabilizer, and have completed the present invention, which includes this as one embodiment.

[0022] Furthermore, the present inventors have discovered that, as an example, a kit can be produced that contains NADH oxidase, its N-terminal domain deletion mutant, and amino acid substitution mutant as components, and have completed the present invention, which includes this as one embodiment.

[0023] Furthermore, the present inventors have discovered that, as an example, an electrode containing NADH oxidase, its N-terminal domain deletion mutant, or amino acid substitution mutant can be produced, and have completed the present invention, which includes this as one embodiment.

[0024] Furthermore, the present inventors have discovered that, as an example, it is possible to produce a sensor equipped with an electrode containing NADH oxidase, its N-terminal domain deletion mutant, or its amino acid substitution mutant, and have completed the present invention, which includes this as one embodiment.

[0025] Furthermore, the present inventors have discovered, as an example, a method for measuring NADH in a sample using NADH oxidase, its N-terminal domain deletion mutant, and amino acid substitution mutant, and have completed the present invention, which includes this method as one embodiment.

[0026] The present disclosure encompasses the following embodiments: [1] An NADH oxidase derived from Geobacillus species, having the following properties: (i) action: oxidizing NADH to produce NAD+, (ii) thermostability: having 65% or more of the remaining activity after heat treatment at 75°C for 15 minutes, (iii) having a flavin compound as a prosthetic group, and (iv) molecular weight: having a molecular weight of approximately 52 kDa when the lysate is measured by SDS-polyacrylamide electrophoresis. [2] An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 74, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO: 74 (excluding those having 100% amino acid sequence identity with SEQ ID NO: 74, those having 100% amino acid sequence identity with SEQ ID NO: 76, and those having 100% amino acid sequence identity with SEQ ID NO: 78). [3] An NADH oxidase derived from Geobacillus species, having the following properties: (i) activity: oxidizing NADH to produce NAD+; (ii) thermal stability: retaining 40% or more of its activity after heat treatment at 75°C for 15 minutes; (iii) a flavin compound as a prosthetic group; and (iv) molecular weight: the molecular weight of the lysate is approximately 52 kDa when measured by SDS-polyacrylamide gel electrophoresis. [4] An NADH oxidase having 90% or more amino acid sequence identity with SEQ ID NO: 76, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have 90% or more amino acid sequence identity with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has 70% or more amino acid sequence identity with SEQ ID NO: 76 (excluding those having 100% amino acid sequence identity with SEQ ID NO: 74, those having 100% amino acid sequence identity with SEQ ID NO: 76, and those having 100% amino acid sequence identity with SEQ ID NO: 78).[5] An NADH oxidase derived from Geobacillus species, having the following properties: (i) activity: oxidizing NADH to produce NAD+; (ii) thermal stability: retaining 50% or more of its activity after heat treatment at 75°C for 15 minutes; (iii) a flavin compound as a prosthetic group; and (iv) molecular weight: the molecular weight of the lysate is approximately 53 kDa when measured by SDS-polyacrylamide gel electrophoresis. [6] An NADH oxidase having 90% or more amino acid sequence identity with SEQ ID NO: 78, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have 90% or more amino acid sequence identity with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has 70% or more amino acid sequence identity with SEQ ID NO: 78 (excluding those having 100% amino acid sequence identity with SEQ ID NO: 74, those having 100% amino acid sequence identity with SEQ ID NO: 76, and those having 100% amino acid sequence identity with SEQ ID NO: 78). [7] An NADH oxidase derived from a Bacillus species, having the following properties (i) to (vii): (i) action: oxidizing NADH to produce NAD+; (ii) substrate specificity: lower reactivity with NADPH compared to reactivity with NADH; (iii) thermostability: retaining 60% or more of its activity after heat treatment at 50°C for 15 minutes; (iv) having a flavin compound as a prosthetic group; (v) optimum pH: 5.0 to 7.5; (vi) stable pH: 6.0 to 8.0; and (vii) molecular weight: having a molecular weight of approximately 54 kDa as measured by SDS-polyacrylamide gel electrophoresis. [8] An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 3, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO: 3.[9] An NADH oxidase derived from a Bacillus species, having the following properties (i) to (vii): (i) action: oxidizing NADH to produce NAD+; (ii) substrate specificity: lower reactivity with NADPH compared to reactivity with NADH; (iii) thermostability: retaining 80% or more of its activity after heat treatment at 50°C for 15 minutes; (iv) having a flavin compound as a prosthetic group; (v) optimum pH: 7.0-8.0; (vi) stable pH: 5.0-10.0; and (vii) molecular weight: having a molecular weight of approximately 51 kDa as measured by SDS-polyacrylamide gel electrophoresis.

[10] An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO:5, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO:1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO:5.

[11] An NADH oxidase derived from a Bacillus species, having the following properties (i) to (vii): (i) action: oxidizing NADH to produce NAD+; (ii) substrate specificity: lower reactivity with NADPH compared to reactivity with NADH; (iii) thermostability: retaining 30% or more of its activity after heat treatment at 50°C for 15 minutes; (iv) having a flavin compound as a prosthetic group; (v) optimum pH: 5.0 to 6.0; (vi) stable pH: 7.5 to 9.5; and (vii) molecular weight: having a molecular weight of approximately 52 kDa as measured by SDS-polyacrylamide gel electrophoresis.

[12] An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 11, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO: 11.

[13] An N-terminal domain deletion mutant of NADH oxidase, in which all or part of the N-terminal domain is deleted and which retains NADH oxidase activity.

[14] An N-terminal domain deletion mutant of NADH oxidase, in which a sequence consisting of 197 consecutive amino acids on the N-terminal side is deleted and which retains NADH oxidase activity.

[15] The sequence consisting of 197 consecutive amino acids on the N-terminal side is: positions 4 to 200 of SEQ ID NO:74, positions 5 to 201 of SEQ ID NO:74, positions 6 to 202 of SEQ ID NO:74, positions 7 to 203 of SEQ ID NO:74, positions 8 to 204 of SEQ ID NO:74, positions 9 to 205 of SEQ ID NO:74, positions 10 to 206 of SEQ ID NO:74, positions 11 to 207 of SEQ ID NO:74; positions 4 to 200 of SEQ ID NO:76, positions 5 to 201 of SEQ ID NO:76, positions 6 to 202 of SEQ ID NO:76, positions 7 to 203 of SEQ ID NO:76, positions 8 to 204 of SEQ ID NO:76, positions 9 to 205 of SEQ ID NO:76, positions 10 to 206 of SEQ ID NO:76, positions 11 to 207 of SEQ ID NO:76; The N-terminal domain deletion mutant of NADH oxidase according to embodiment 13 or 14, which has a sequence corresponding to a position selected from the group consisting of positions 4 to 200 of SEQ ID NO: 78, positions 5 to 201 of SEQ ID NO: 78, positions 6 to 202 of SEQ ID NO: 78, positions 7 to 203 of SEQ ID NO: 78, positions 8 to 204 of SEQ ID NO: 78, positions 9 to 205 of SEQ ID NO: 78, positions 10 to 206 of SEQ ID NO: 78, and positions 11 to 207 of SEQ ID NO: 78.

[16] An N-terminal domain deletion mutant of NADH oxidase, in which the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 1 is deleted and which has NADH oxidase activity.

[17] The N-terminal domain deletion mutant of NADH oxidase according to any one of embodiments 13 to 16, wherein the NADH oxidase is derived from the genus Bacillus, Priestia, Achydianus, Archaeoglobus, Brevibacterium, Methanocaldococcus, Pseudothermotoga, Pyrococcus, Saccharolobus, Streptococcus, Sulfolobus, Thermotoga, or Geobacillus.

[18] The N-terminal domain deletion mutant of NADH oxidase according to any one of embodiments 13 to 17, wherein the amino acid sequence before deletion of the N-terminal domain has 90% or more amino acid sequence identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, or the amino acid sequence after deletion of the N-terminal domain has 90% or more amino acid sequence identity with SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124.

[19] An N-terminal domain deletion mutant of NADH oxidase, in which the N-terminal domain of NADH oxidase is deleted and the FAD domain and NADH domain of NADH oxidase are present, wherein the amino acid sequences of the FAD domain and the NADH domain of NADH oxidase have 90% or more amino acid sequence identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, or have 90% or more amino acid sequence identity with SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124.

[20] An NADH oxidase mutant having amino acid substitutions at positions corresponding to positions 503, 203, 246, 283, 284, 329, 378, 383, 467, and / or 494 of SEQ ID NO: 1.

[21] The amino acid after substitution at the position corresponding to position 503 of SEQ ID NO: 1 is tyrosine (Y); The amino acid after substitution at the position corresponding to position 203 of SEQ ID NO: 1 is leucine (L); The amino acid after substitution at the position corresponding to position 246 of SEQ ID NO: 1 is arginine (R), lysine (K), or threonine (T); The amino acid after substitution at the position corresponding to position 283 of SEQ ID NO: 1 is glutamic acid (E), and / or The amino acid after substitution at the position corresponding to position 284 of SEQ ID NO: 1 is asparagine (N); The amino acid after substitution at the position corresponding to position 329 of SEQ ID NO: 1 is glutamic acid (E); The amino acid after substitution at the position corresponding to position 378 of SEQ ID NO: 1 is leucine (L), glutamic acid (E), or aspartic acid (D); The amino acid after substitution at the position corresponding to position 383 of SEQ ID NO: 1 is aspartic acid (D), asparagine (N), or glutamic acid (E). 21. The NADH oxidase variant according to embodiment 20, wherein the amino acid after substitution at the position corresponding to position 467 of SEQ ID NO: 1 is glutamine (Q), and / or the amino acid after substitution at the position corresponding to position 494 of SEQ ID NO: 1 is glutamine (Q) or glutamic acid (E).

[22] The NADH oxidase before amino acid substitution is an NADH oxidase having 90% or more amino acid sequence identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, or an NADH oxidase that lacks the N-terminal domain of NADH oxidase and has the FAD domain and NADH domain of NADH oxidase, wherein the amino acid sequences of the FAD domain and the NADH domain of NADH oxidase have 90% or more amino acid sequence identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, or have 90% or more amino acid sequence identity with SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124; 22. The NADH oxidase variant according to embodiment 20 or 21, which is an N-terminal domain deletion variant of NADH oxidase.

[23] A freeze-dried preparation comprising NADH oxidase and a stabilizer, wherein the stabilizer is selected from the group consisting of trehalose, raffinose, melezitose, maltotriose, sucrose, lactose, maltose, turanose, cellobiose, ribose, lyxose, xylose, arabinose, glucose, altrose, mannose, galactose, idose, allose, talose, gulose, psicose, fructose, sorbose, tagatose, deoxyribose, fucose, fuculose, rhamnose, ribulose, and xylulose, glycogen, starch, cellulose, dextrin, glucan, fructan, chitin, chitosan, glucomannan, peanut, and the like. A freeze-dried preparation comprising a polysaccharide selected from the group consisting of cutin, alginic acid, hyaluronic acid, chondroitin sulfate, and heparin; a sugar alcohol selected from the group consisting of erythritol, threitol, arabinitol, xylitol, ribitol, iditol, galactitol, sorbitol, and mannitol; an amino acid or a salt thereof selected from the group consisting of glutamic acid, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, asparagine, glutamine, aspartic acid, histidine, lysine, and arginine; or a phosphoric acid, a carboxylic acid, or a salt thereof.

[24] The freeze-dried preparation according to embodiment 23, wherein the NADH oxidase is the NADH oxidase according to any one of embodiments 1 to 22.

[25] A kit for measuring NADH, comprising the NADH oxidase according to any one of embodiments 1 to 22.

[26] An electrode comprising the NADH oxidase according to any one of embodiments 1 to 22.

[27] A sensor equipped with the electrode according to embodiment 26.

[28] A method for measuring NADH in a sample, comprising oxidizing NADH in the sample using the NADH oxidase according to any one of embodiments 1 to 22 and measuring the hydrogen peroxide produced or the oxygen consumed.

[29] A method for measuring NADH in a sample, comprising the step of contacting the electrode according to embodiment 26 with the sample.

[0027] This specification includes the disclosure of Japanese Patent Application No. 2024-025886, from which the present application claims priority.

[0028] As an effect of the present invention, NADH oxidase is provided.

[0029] Figure 1 shows an alignment of NADH oxidases from various sources. Figure 2 shows an alignment of NADH oxidases from various sources. Figure 3 shows an alignment of NADH oxidases from various sources. Figure 4 shows the molecular weight of purified NOX-b. Figure 5 shows the pH dependence of the NADH oxidation activity of NOX-b. Figure 6 shows the pH dependence of the NADH oxidation activity of NOX-c. Figure 7 shows the pH dependence of the NADH oxidation activity of NOX-f. Figure 8 shows the pH stability of NOX-b. Figure 9 shows the pH stability of NOX-c. Figure 10 shows the pH stability of NOX-f. Photographs of SDS-PAGE of various NOX mutants. The estimated molecular weight of NOX is approximately 55 kDa, but lane b (i.e., NOX-b) is approximately 10 kDa smaller. Figure 11 shows the pH dependence of the NADH oxidation activity of NOX-bΔ197. Figure 12 shows the pH dependence of the NADH oxidation activity of NOX-cΔ197. 1 shows the pH dependence of NADH oxidation activity of NOX-fΔ197, 2 shows the pH stability of NOX-bΔ197, 3 shows the pH stability of NOX-cΔ197, and 4 shows the pH stability of NOX-fΔ197.

[0030] H ２ O ２ NADH oxidase that produces the following is classified as EC 1.6.3.3. The NADH oxidase of the present disclosure catalyzes the following reaction: [Chemical Formula 1] NADH + H ＋ + O ２ ←→ NAD + H ２ O ２

[0031] In certain embodiments, the NADH oxidase of the present disclosure has the following properties: (i) Action: Oxidize NADH to NAD ＋(ii) substrate specificity: lower reactivity with NADPH compared to reactivity with NADH; (iii) thermostability: retaining 60% or more of the activity after heat treatment at 50°C for 15 minutes; (iv) using a flavin compound as the prosthetic group; (v) optimum pH: 5.0 to 7.5; (vi) stable pH: 6.0 to 8.0; (vii) molecular weight: the molecular weight as measured by SDS-polyacrylamide electrophoresis is approximately 54 kDa. The optimum pH was calculated from the pH range in which the relative activity was 80% or more. The stable pH was calculated from the pH range in which the residual activity was 80% or more after heat treatment at 40°C for 60 minutes.

[0032] In certain embodiments, the NADH oxidase may be derived from a Bacillus species. In certain embodiments, the NADH oxidase has 90% or greater amino acid sequence identity with SEQ ID NO: 3. In certain embodiments, the NADH oxidase has 70% or greater full-length amino acid sequence identity with SEQ ID NO: 3, and the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have 90% or greater amino acid sequence identity with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1. In certain embodiments, the reactivity of the NADH oxidase with NADPH may be 35% or less, for example, 31% or less, when the reactivity with NADH is taken as 100%.

[0033] In another embodiment, the NADH oxidase of the present disclosure has the following properties: (i) action: oxidizes NADH to produce NAD+; (ii) substrate specificity: lower reactivity with NADPH than with NADH; (iii) thermostability: retaining 80% or more of its activity after heat treatment at 50°C for 15 minutes; (iv) a flavin compound as a prosthetic group; (v) optimum pH: 7.0 to 8.0; (vi) stable pH: 5.0 to 10.0; (vii) molecular weight: a molecular weight of approximately 51 kDa as measured by SDS-polyacrylamide electrophoresis. The optimum pH was calculated from the pH range in which the relative activity was 80% or higher. The stable pH was calculated from the pH range in which the residual activity was 80% or higher after heat treatment at 40°C for 60 minutes.

[0034] In certain embodiments, the NADH oxidase may be derived from a Bacillus species. In certain embodiments, the NADH oxidase has 90% or greater amino acid sequence identity with SEQ ID NO: 5. In certain embodiments, the NADH oxidase has 70% or greater full-length amino acid sequence identity with SEQ ID NO: 5, and the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have 90% or greater amino acid sequence identity with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1. In certain embodiments, the reactivity of the NADH oxidase with NADPH may be 20% or less, for example, 15% or less, when the reactivity with NADH is taken as 100%.

[0035] In another embodiment, the NADH oxidase of the present disclosure has the following properties: (i) Action: oxidizes NADH to NAD ＋(ii) substrate specificity: lower reactivity with NADPH compared to reactivity with NADH; (iii) thermostability: retaining 30% or more of the activity after heat treatment at 50°C for 15 minutes; (iv) using a flavin compound as the prosthetic group; (v) optimum pH: 5.0 to 6.0; (vi) stable pH: 7.5 to 9.5; (vii) molecular weight: the molecular weight as measured by SDS-polyacrylamide electrophoresis is approximately 52 kDa. The optimum pH was calculated from the pH range in which the relative activity was 80% or higher. The stable pH was calculated from the pH range in which the residual activity was 80% or higher after heat treatment at 40°C for 60 minutes.

[0036] In certain embodiments, the NADH oxidase may be derived from a Bacillus species. In certain embodiments, the NADH oxidase has 90% or greater amino acid sequence identity with SEQ ID NO: 11. In certain embodiments, the NADH oxidase has 70% or greater full-length amino acid sequence identity with SEQ ID NO: 11, and the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have 90% or greater amino acid sequence identity with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1. In certain embodiments, the reactivity of the NADH oxidase with NADPH may be 10% or less, 5% or less, for example 1% or less, when the reactivity with NADH is taken as 100%.

[0037] In another embodiment, the NADH oxidase of the present disclosure has the following properties: (i) action: oxidizing NADH to NAD ＋(ii) thermostability: after heat treatment at 75°C for 15 minutes, the residual activity is 65% or more, for example, 66% or more, 67% or more, for example, 68% or more; (iii) a flavin compound is used as a prosthetic group; and (iv) molecular weight: the molecular weight of the lysate is about 54 kDa as measured by SDS-polyacrylamide electrophoresis. In certain embodiments, the NADH oxidase may be derived from Geobacillus thermoleovorans. In certain embodiments, this NADH oxidase may further have: (v) substrate specificity: its reactivity to NADPH may be lower than its reactivity to NADH; (vi) an optimal pH that corresponds to that of the NADH oxidase derived from Geobacillus thermoleovorans of SEQ ID NO: 74; and (vii) a stable pH that corresponds to that of the NADH oxidase derived from Geobacillus thermoleovorans of SEQ ID NO: 74. The optimal pH can be calculated from the pH range in which the relative activity is 80% or higher. The stable pH can be calculated from the pH range in which the residual activity after heat treatment at 40°C for 60 minutes is 80% or higher.

[0038] In some embodiments, the NADH oxidase may be derived from Geobacillus thermoleovorans. In some embodiments, the NADH oxidase has 90% or greater amino acid sequence identity to SEQ ID NO: 74. In some embodiments, the NADH oxidase has 70% or greater full-length amino acid sequence identity to SEQ ID NO: 74, and the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have 90% or greater amino acid sequence identity to the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1. In some embodiments, the NADH oxidase derived from Geobacillus thermoleovorans excludes those with 100% amino acid sequence identity to SEQ ID NO: 74, those with 100% amino acid sequence identity to SEQ ID NO: 76, and those with 100% amino acid sequence identity to SEQ ID NO: 78. In this embodiment, other known sequences (eg, sequences registered in databases, sequences described in public literature, etc.) are also excluded.

[0039] In another embodiment, the NADH oxidase of the present disclosure has the following properties: (i) action: oxidizing NADH to NAD ＋ (ii) thermostability: after heat treatment at 75°C for 15 minutes, the residual activity is 40% or more, for example, 41% or more, 42% or more, 43% or more, for example, 44% or more; (iii) a flavin compound is used as a prosthetic group; and (iv) molecular weight: the molecular weight of the lysate is about 54 kDa as measured by SDS-polyacrylamide electrophoresis. In certain embodiments, the NADH oxidase may be derived from Geobacillus stearothermophilus. In certain embodiments, this NADH oxidase may further have: (v) substrate specificity: its reactivity to NADPH may be lower than its reactivity to NADH; (vi) an optimum pH that corresponds to that of the NADH oxidase derived from Geobacillus stearothermophilus of SEQ ID NO: 76; and (vii) a stable pH that corresponds to that of the NADH oxidase derived from Geobacillus stearothermophilus of SEQ ID NO: 76. The optimum pH can be calculated from the pH range in which the relative activity is 80% or higher. The stable pH can be calculated from the pH range in which the residual activity after heat treatment at 40°C for 60 minutes is 80% or higher.

[0040] In some embodiments, the NADH oxidase may be derived from Geobacillus stearothermophilus. In some embodiments, the NADH oxidase has 90% or greater amino acid sequence identity to SEQ ID NO: 76. In some embodiments, the NADH oxidase has 70% or greater full-length amino acid sequence identity to SEQ ID NO: 76, and the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have 90% or greater amino acid sequence identity to the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1. In some embodiments, the NADH oxidases derived from Geobacillus species exclude those with 100% amino acid sequence identity to SEQ ID NO: 74, SEQ ID NO: 76, and SEQ ID NO: 78. In this embodiment, other known sequences (eg, sequences registered in databases, sequences described in public literature, etc.) are also excluded.

[0041] In another embodiment, the NADH oxidase of the present disclosure has the following properties: (i) action: oxidizing NADH to NAD ＋(ii) thermostability: after heat treatment at 75°C for 15 minutes, the residual activity is 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, for example, 51% or more; (iii) a flavin compound is used as a prosthetic group; (iv) molecular weight: the molecular weight of the lysate is about 54 kDa when measured by SDS-polyacrylamide electrophoresis. In certain embodiments, the NADH oxidase may be derived from Geobacillus thermocatenulatus. In certain embodiments, this NADH oxidase may further have: (v) substrate specificity: its reactivity to NADPH may be lower than its reactivity to NADH; (vi) an optimal pH that corresponds to that of the NADH oxidase derived from Geobacillus thermocatenulatus of SEQ ID NO: 78; and (vii) a stable pH that corresponds to that of the NADH oxidase derived from Geobacillus thermocatenulatus of SEQ ID NO: 78. The optimal pH can be calculated from the pH range in which the relative activity is 80% or higher. The stable pH can be calculated from the pH range in which the residual activity after heat treatment at 40°C for 60 minutes is 80% or higher.

[0042] In some embodiments, the NADH oxidase may be derived from Geobacillus thermocatenulatus. In some embodiments, the NADH oxidase has 90% or greater amino acid sequence identity to SEQ ID NO: 78. In some embodiments, the NADH oxidase has 70% or greater full-length amino acid sequence identity to SEQ ID NO: 74, and the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have 90% or greater amino acid sequence identity to the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1. In some embodiments, the NADH oxidases derived from Geobacillus species exclude those with 100% amino acid sequence identity to SEQ ID NO: 74, those with 100% amino acid sequence identity to SEQ ID NO: 76, and those with 100% amino acid sequence identity to SEQ ID NO: 78. In this embodiment, other known sequences (eg, sequences registered in databases, sequences described in public literature, etc.) are also excluded.

[0043] NADH oxidase recognizes NADH as a substrate, and therefore can measure various metabolites and compounds. For example, NADH oxidase can measure β-D-glucose, L-lactic acid, etc., when combined with NAD-dependent dehydrogenase. When measuring β-D-glucose, the following reaction is first catalyzed by NAD-dependent glucose dehydrogenase: [Chemical Formula 2] β-D-glucose + NAD ＋ ←→ D-glucono-δ-lactone + NADH + H ＋

[0044] Next, NADH oxidase is allowed to act on the produced NADH, ２ O ２ The generated H ２ O ２ can be determined by, for example, electrochemical measurement or colorimetry. Similarly, when measuring L-lactic acid, NADH is first generated by NAD-dependent lactate dehydrogenase. Then, NADH oxidase acts on the generated NADH to produce H ２ O ２Other metabolites and compounds may be measured as well.

[0045] NADH oxidase uses flavin adenine dinucleotide (FAD) as a prosthetic group. Oxidoreductases that use FAD as a prosthetic group are known to have an FAD-binding motif sequence (also referred to as an adenine dinucleotide-binding motif sequence), such as a Gly-Xaa-Gly-Xaa-Xaa-Gly motif (where Xaa represents any amino acid). For example, when SEQ ID NO: 1 is used as a reference sequence, the amino acid sequence "Gly-Gly-Gly-Pro-Ala-Gly" from positions 215 to 220 of SEQ ID NO: 1 corresponds to the FAD-binding motif sequence Gly-Xaa-Gly-Xaa-Xaa-Gly. In certain embodiments, with respect to the variants of the present disclosure, the Gly at positions 215, 217, and 220 of SEQ ID NO: 1 may not be substituted with amino acids. In one embodiment, the Gly at positions corresponding to positions 215, 217, and 220 in SEQ ID NO: 1 of the NADH oxidase are not substituted with amino acids.

[0046] NADH oxidase uses nicotinamide adenine dinucleotide (NAD) as a substrate. Therefore, it is known to have an NAD-binding motif sequence (also referred to as an adenine dinucleotide-binding motif sequence), such as a Gly-Xaa-Gly-Xaa-Xaa-Gly motif (where Xaa represents any amino acid). For example, when SEQ ID NO: 1 is used as a reference sequence, the amino acid sequence "Gly-Gly-Gly-Asn-Ser-Gly" from positions 354 to 359 of SEQ ID NO: 1 corresponds to the NAD-binding motif sequence Gly-Xaa-Gly-Xaa-Xaa-Gly. In certain embodiments, with respect to the variants of the present disclosure, the Gly at positions 354, 356, and 359 of SEQ ID NO: 1 may not be substituted with amino acids. In one embodiment, the Gly at positions corresponding to positions 354, 356, and 359 in SEQ ID NO: 1 of the NADH oxidase are not substituted with amino acids.

[0047] (Reference Sequence) For convenience, in this specification, each position of NADH oxidase is defined using SEQ ID NO: 1 as the reference sequence. SEQ ID NO: 1 is the amino acid sequence of NADH oxidase derived from the genus Bacillus sp.

[0048] In some embodiments, 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, 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, e.g., 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, and having NADH oxidase activity. The polypeptide may have an N-terminal domain deletion as described herein. The polypeptide may further have an amino acid substitution as described herein.

[0049] (NADH oxidase mutant with deleted amino acid sequence) The present inventors surprisingly found that the N-terminal domain of the amino acid sequence of NADH oxidase is not necessary for enzymatic activity. That is, the present inventors surprisingly confirmed that an NADH oxidase mutant lacking the N-terminal domain retains NADH oxidase activity. Therefore, the present disclosure provides an NADH oxidase mutant lacking the N-terminal domain (e.g., an NADH oxidase mutant lacking all or part of the N-terminal domain), a lyophilized preparation, kit, or electrode containing the same, a sensor equipped with the electrode, and a method for measuring NADH using an NADH oxidase mutant lacking the N-terminal domain.

[0050] In certain embodiments, the present disclosure provides an N-terminal domain-deleted NADH oxidase mutant that lacks the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 1 and has NADH oxidase activity. In certain embodiments, the N-terminal domain of an NADH oxidase refers to a domain consisting of amino acids corresponding to positions 1 to 198 of SEQ ID NO: 1. In certain embodiments, the first amino acid after deletion of the N-terminal domain (the first amino acid on the N-terminal side) of the mutant may be methionine. This methionine may be derived from the methionine corresponding to position 1 of SEQ ID NO: 1. In this case, the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 1 is deleted. Alternatively, this methionine may be a methionine newly added to the N-terminal side of the amino acid corresponding to position 199 of SEQ ID NO: 1 after deleting the N-terminal domain corresponding to positions 1 to 198 of SEQ ID NO: 1. Since the result is the same in either case, for convenience, these will be described herein as having a deletion of the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 1. The N-terminal domain-deleted NADH oxidase variant may be based on an NADH oxidase derived from a microorganism described herein. In certain embodiments, the N-terminal domain-deleted NADH oxidase variant may further have one or more amino acid substitutions, for example, amino acid substitutions that improve thermostability and / or amino acid substitutions that improve substrate specificity.

[0051] Even if the deletion extends to the sequence after the N-terminal domain, as long as the number of deleted amino acids is 197, the N-terminal domain can be considered to have been deleted. For example, when amino acids corresponding to positions 3 to 199 of SEQ ID NO: 1 are deleted, the amino acid at position 199 after the N-terminal domain is deleted. However, the amino acid at position 2 of SEQ ID NO: 1 complements the amino acid at position 199. Therefore, for convenience, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant herein. Similarly, when amino acids corresponding to positions 4 to 200 of SEQ ID NO: 1 are deleted, the amino acids at positions 199 to 200 after the N-terminal domain are deleted. However, the amino acids at positions 2 and 3 of SEQ ID NO: 1 complement the amino acids at positions 199 to 200. Therefore, for convenience, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant herein. Similarly, when the amino acids corresponding to positions 5 to 201 of SEQ ID NO: 1 are deleted, the amino acids at positions 199 to 201 after the N-terminal domain are deleted, but the amino acids at positions 2 to 4 of SEQ ID NO: 1 complement the amino acids at positions 199 to 201. Therefore, for convenience, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant in this specification. Similarly, when the amino acids corresponding to positions 6 to 202 of SEQ ID NO: 1 are deleted, the amino acids at positions 199 to 202 after the N-terminal domain are deleted, but the amino acids at positions 2 to 5 of SEQ ID NO: 1 complement the amino acids at positions 199 to 202. Therefore, for convenience, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant in this specification. Similarly, when the amino acids corresponding to positions 7 to 203 of SEQ ID NO: 1 are deleted, the amino acids at positions 199 to 203 after the N-terminal domain are deleted, but the amino acids at positions 2 to 6 of SEQ ID NO: 1 complement the amino acids at positions 199 to 203. Therefore, for convenience, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant herein. Similarly, when the amino acids corresponding to positions 8 to 204 of SEQ ID NO: 1 are deleted, the amino acids at positions 199 to 204 after the N-terminal domain are deleted, but the amino acids at positions 2 to 7 of SEQ ID NO: 1 complement the amino acids at positions 199 to 204. Therefore, for convenience, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant herein.Similarly, when the amino acids corresponding to positions 9 to 205 of SEQ ID NO: 1 are deleted, the amino acids at positions 199 to 205 after the N-terminal domain are deleted, but the amino acids at positions 2 to 8 of SEQ ID NO: 1 complement the amino acids at positions 199 to 205. Therefore, for convenience, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant herein. Similarly, when the amino acids corresponding to positions 10 to 206 of SEQ ID NO: 1 are deleted, the amino acids at positions 199 to 206 after the N-terminal domain are deleted, but the amino acids at positions 2 to 9 of SEQ ID NO: 1 complement the amino acids at positions 199 to 206. Therefore, for convenience, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant herein. Similarly, when the amino acids corresponding to positions 11 to 207 of SEQ ID NO: 1 are deleted, the amino acids at positions 199 to 207 after the N-terminal domain are deleted, but because the amino acids at positions 2 to 10 of SEQ ID NO: 1 complement the amino acids at positions 199 to 207, for convenience in this specification, this deletion mutant is also referred to as an N-terminal domain-deleted NADH oxidase mutant. In other words, these mutants can also be referred to as N-terminal domain deletion mutants of NADH oxidase, in which the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 1 is deleted and several amino acid substitutions are present on the N-terminal side after the deletion.

[0052] In one embodiment, the present disclosure provides an N-terminal domain-deleted NADH oxidase mutant having NADH oxidase activity, wherein the sequence consisting of 197 consecutive amino acids from the N-terminus is deleted. In one embodiment, the sequence consisting of 197 consecutive amino acids from the N-terminus is: positions 4 to 200 of SEQ ID NO:74, positions 5 to 201 of SEQ ID NO:74, positions 6 to 202 of SEQ ID NO:74, positions 7 to 203 of SEQ ID NO:74, positions 8 to 204 of SEQ ID NO:74, positions 9 to 205 of SEQ ID NO:74, positions 10 to 206 of SEQ ID NO:74, and positions 11 to 207 of SEQ ID NO:74; positions 4 to 200 of SEQ ID NO:76, positions 5 to 201 of SEQ ID NO:76, positions 6 to 202 of SEQ ID NO:76, positions 7 to 203 of SEQ ID NO:76, positions 8 to 204 of SEQ ID NO:76, positions 9 to 205 of SEQ ID NO:76, positions 10 to 206 of SEQ ID NO:76, and positions 11 to 207 of SEQ ID NO:76; The sequence may be a sequence corresponding to a position selected from the group consisting of positions 4 to 200 of SEQ ID NO:78, positions 5 to 201 of SEQ ID NO:78, positions 6 to 202 of SEQ ID NO:78, positions 7 to 203 of SEQ ID NO:78, positions 8 to 204 of SEQ ID NO:78, positions 9 to 205 of SEQ ID NO:78, positions 10 to 206 of SEQ ID NO:78, and positions 11 to 207 of SEQ ID NO:78. In one embodiment, the sequence consisting of 197 consecutive amino acids on the N-terminal side is: positions 4 to 200 of SEQ ID NO:74, positions 5 to 201 of SEQ ID NO:74, positions 7 to 203 of SEQ ID NO:74, positions 8 to 204 of SEQ ID NO:74, positions 9 to 205 of SEQ ID NO:74, positions 10 to 206 of SEQ ID NO:74, positions 11 to 207 of SEQ ID NO:74; positions 4 to 200 of SEQ ID NO:76, positions 5 to 201 of SEQ ID NO:76, positions 6 to 202 of SEQ ID NO:76, positions 8 to 204 of SEQ ID NO:76, positions 9 to 205 of SEQ ID NO:76, positions 10 to 206 of SEQ ID NO:76, positions 11 to 207 of SEQ ID NO:76; positions 4 to 200 of SEQ ID NO:78, positions 5 to 201 of SEQ ID NO:78, positions 8 to 204 of SEQ ID NO:78, positions 9 to 205 of SEQ ID NO:78, positions 10 to 206 of SEQ ID NO:78, and positions 11 to 207 of SEQ ID NO:78. In this specification, the "N-terminal side" of NADH oxidase refers not only to the N-terminal domain of NADH oxidase (the domain consisting of amino acids at positions 1 to 198 of SEQ ID NO: 1), but also to the range extending beyond this to positions 1 to 207 of SEQ ID NO: 1.

[0053] Furthermore, the present inventors surprisingly found that the specific activity (U / mg) of the N-terminal domain-deleted NADH oxidase mutant was improved compared to the full-length NADH oxidase before deletion of the N-terminal domain. To the best of the present inventors' knowledge, there have been no reported examples of mutants in which the N-terminal domain has been deleted from NADH oxidase. Furthermore, it was surprising that NADH oxidase activity was maintained even when positions 2-198 of the 509 amino acids in SEQ ID NO: 1 near the N-terminal domain were deleted. This corresponds to a deletion of 38% of the full-length sequence. Furthermore, it was unexpected that not only was NADH oxidase activity maintained even after deletion of the N-terminal domain, but the specific activity (U / mg) was also improved compared to the full-length NADH oxidase before deletion of the N-terminal domain.

[0054] This finding applies not only to the NADH oxidase of SEQ ID NO: 1, but also to the NADH oxidases of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, and 78, and it was confirmed that the enzymatic activity was improved by deleting the N-terminal domain from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, and 78. Therefore, those skilled in the art will reasonably understand that it is highly likely that NADH oxidase mutants with improved enzymatic activity can be obtained by similarly deleting the N-terminal domain from NADH oxidases of other origins that share amino acid sequence identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, and 78.

[0055] In certain embodiments, the N-terminal domain-deleted NADH oxidase mutant of the present disclosure has an enzymatic activity that is, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, or 3.0-fold higher than the enzymatic activity of the full-length NADH oxidase before deletion of the N-terminal domain, assuming that the enzymatic activity is 100%. The enzymatic activity can be improved by 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5-fold, 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, for example, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, for example, 10-fold or more. Here, "a 1.1-fold improvement in enzymatic activity" means that, when the enzymatic activity of the full-length NADH oxidase before deletion of the N-terminal domain is set to 1, the enzymatic activity of the NADH oxidase mutant after deletion of the N-terminal domain under the same conditions is 1.1.

[0056] Furthermore, the present inventors have surprisingly found that, in certain embodiments, the substrate specificity of an N-terminal domain-deleted NADH oxidase mutant is improved compared to the full-length NADH oxidase before the N-terminal domain deletion. Here, substrate specificity refers to the ratio of the enzyme activity when NADPH is used as a substrate divided by the enzyme activity when NADH is used as a substrate (NADPH / NADH ratio). Furthermore, "improved substrate specificity" refers to a reduced NADPH / NADH ratio of the N-terminal domain-deleted NADH oxidase mutant compared to the full-length NADH oxidase before the N-terminal domain deletion.

[0057] In certain embodiments, when the NADPH / NADH ratio of the full-length NADH oxidase before deletion of the N-terminal domain is taken as 100%, the enzymatic activity of the N-terminal domain-deleted NADH oxidase mutant of the present disclosure can be reduced to, for example, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or for example, 5% or less (substrate specificity can be improved).

[0058] In some embodiments, the present disclosure provides a method for the preparation of a polypeptide comprising a polypeptide having an amino acid sequence prior to deletion of the N-terminal domain that is 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 identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76 or 78. , 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more amino acid sequence identity, in which the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 1 is deleted and which has NADH oxidase activity. In some embodiments, the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity to SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124 and has NADH oxidase activity.

[0059] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase that has NADH oxidase activity and lacks the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO:1, wherein the N-terminal domain deletion mutant has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO:1.

[0060] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 3 is deleted, wherein the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO: 3.

[0061] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO:5 is deleted, wherein the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 508 of SEQ ID NO:5.

[0062] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO:7 is deleted, wherein the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO:7.

[0063] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO:9 is deleted, wherein the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO:9.

[0064] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 11 is deleted, wherein the N-terminal domain deletion mutant has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 508 of SEQ ID NO: 11.

[0065] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 13 is deleted, wherein the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO: 13.

[0066] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 15 is deleted, wherein the N-terminal domain deletion mutant has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO: 15.

[0067] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of an NADH oxidase having NADH oxidase activity, in which the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 17 is deleted, wherein the N-terminal domain deletion mutant has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO: 17. In certain embodiments, SEQ ID NO: 172 may be used in place of SEQ ID NO: 17. The same applies to all descriptions of SEQ ID NO: 17 herein. Accordingly, in certain embodiments, the present disclosure provides an N-terminal domain deletion mutant of an NADH oxidase having NADH oxidase activity, in which the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 172 is deleted, wherein the N-terminal domain deletion mutant has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO: 172.

[0068] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 74 is deleted, wherein the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO: 74.

[0069] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 76 is deleted, wherein the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO: 76.

[0070] In another embodiment, the present disclosure provides an N-terminal domain deletion mutant of NADH oxidase having NADH oxidase activity, wherein the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 78 is deleted, wherein the N-terminal domain deletion mutant of NADH oxidase has 90% or more amino acid sequence identity with the amino acid sequence of positions 199 to 509 of SEQ ID NO: 78.

[0071] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an FAD domain and an NADH domain of an NADH oxidase, wherein the amino acid sequences of the homologous regions of the FAD domain and the NADH domain of the NADH oxidase have 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more, amino acid sequence identity with the homologous regions of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 1. Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO: 1.

[0072] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an FAD domain and an NADH domain of an NADH oxidase, wherein the amino acid sequence of the homologous region of the FAD domain and the NADH domain of the NADH oxidase has 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more amino acid sequence identity with the homologous region of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 3, and Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO: 3.

[0073] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an FAD domain and an NADH domain of an NADH oxidase, wherein the amino acid sequences of the homologous regions of the FAD domain and the NADH domain of the NADH oxidase have 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more, amino acid sequence identity with the homologous regions of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 5, and Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO:5.

[0074] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an FAD domain and an NADH domain of an NADH oxidase, wherein the amino acid sequence of the homologous region of the FAD domain and the NADH domain of the NADH oxidase has 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more, amino acid sequence identity with the homologous region of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 7. Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO: 7.

[0075] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an NADH oxidase FAD domain and an NADH domain, wherein the amino acid sequences of the homologous regions of the FAD domain and the NADH domain of the NADH oxidase have 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more, amino acid sequence identity with the homologous regions of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 9. Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase of SEQ ID NO:9.

[0076] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase comprising: ... Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase of SEQ ID NO: 11.

[0077] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an NADH oxidase FAD domain and an NADH domain, wherein the amino acid sequences of the homologous regions of the FAD domain and the NADH domain of the NADH oxidase have 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more amino acid sequence identity with the homologous regions of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 13, and Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO: 13.

[0078] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an NADH oxidase FAD domain and an NADH domain, wherein the amino acid sequence of the homologous region of the FAD domain and the NADH domain of the NADH oxidase has 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more amino acid sequence identity with the homologous region of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 15, and Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO: 15.

[0079] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an NADH oxidase FAD domain and an NADH domain, wherein the amino acid sequence of the homologous region of the FAD domain and the NADH domain of the NADH oxidase has 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more, amino acid sequence identity with the homologous region of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 17, and Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO: 17.In another embodiment, the present disclosure provides a method for the production of an NADH oxidase comprising: ... Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO: 172.

[0080] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an NADH oxidase FAD domain and an NADH domain, wherein the amino acid sequences of the homologous regions of the FAD domain and the NADH domain of the NADH oxidase have 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more, amino acid sequence identity with the homologous regions of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 74, and Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase in SEQ ID NO:74.

[0081] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase comprising: ... Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase of SEQ ID NO:76.

[0082] In another embodiment, the present disclosure provides a method for the production of an NADH oxidase having an N-terminal domain deleted, an NADH oxidase FAD domain and an NADH domain, wherein the amino acid sequence of the homologous region of the FAD domain and the NADH domain of the NADH oxidase has 90% or more, for example 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more amino acid sequence identity with the homologous region of the FAD domain and the NADH domain of the NADH oxidase in SEQ ID NO: 78, and Provided is an NADH oxidase in which the amino acid sequence of the DH domain has 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, 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 amino acid sequence identity to the FAD domain and NADH domain of the NADH oxidase of SEQ ID NO:78.

[0083] Furthermore, the present inventors have surprisingly found that introducing specific amino acid substitutions into NADH oxidase improves the thermostability of the modified NADH oxidase compared to the unmodified NADH oxidase. The present disclosure provides such NADH oxidase mutants.

[0084] In certain embodiments, the NADH oxidase variant of the present disclosure has an amino acid substitution compared to SEQ ID NO: 1 at one or more positions, for example, positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, selected from the group consisting of: a position corresponding to position 503 of SEQ ID NO: 1, a position corresponding to position 203 of SEQ ID NO: 1, a position corresponding to position 246 of SEQ ID NO: 1, a position corresponding to position 283 of SEQ ID NO: 1, a position corresponding to position 284 of SEQ ID NO: 1, a position corresponding to position 329 of SEQ ID NO: 1, a position corresponding to position 378 of SEQ ID NO: 1, a position corresponding to position 467 of SEQ ID NO: 1, a position corresponding to position 383 of SEQ ID NO: 1, and a position corresponding to position 494 of SEQ ID NO: 1, and the thermostability of the variant after the amino acid substitution is improved compared to the variant before the substitution. With respect to the above positions, "an amino acid has been substituted" means that the wild-type amino acid has been substituted with another amino acid, and therefore, the wild-type amino acid is excluded from the substituted amino acids. For example, the position corresponding to position 203 in SEQ ID NO: 1 is phenylalanine (F) in SEQ ID NO: 1, but is leucine (L) in SEQ ID NO: 3. Mutants of the present disclosure include a mutant in which F at position 203 in SEQ ID NO: 1 is replaced with L (F203L). On the other hand, a mutant in which L at position 203 in SEQ ID NO: 3 remains L (L203) does not fall under (is not included in) the mutants of the present disclosure.

[0085] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 503 of SEQ ID NO: 1 may be tyrosine (Y).

[0086] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 203 of SEQ ID NO: 1 may be leucine (L).

[0087] In some embodiments, with respect to an NADH oxidase variant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 246 of SEQ ID NO: 1 may be arginine (R), lysine (K), or threonine (T).

[0088] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 283 of SEQ ID NO: 1 may be glutamic acid (E).

[0089] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 284 of SEQ ID NO: 1 may be asparagine (N).

[0090] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 329 of SEQ ID NO: 1 may be glutamic acid (E).

[0091] In some embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 378 of SEQ ID NO: 1 may be leucine (L), glutamic acid (E), or aspartic acid (D).

[0092] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 467 of SEQ ID NO: 1 may be glutamine (Q).

[0093] In some embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 383 of SEQ ID NO: 1 may be glutamic acid (E) or aspartic acid (D).

[0094] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 494 of SEQ ID NO: 1 may be glutamine (Q).

[0095] Furthermore, the present inventors surprisingly found that by introducing specific amino acid substitutions into NADH oxidase, the substrate specificity of the modified NADH oxidase was improved compared to the unmodified NADH oxidase.

[0096] In certain embodiments, the NADH oxidase mutant of the present disclosure has an amino acid substitution (e.g., at positions 1, 2, 3, 4, 5, or 6) compared to SEQ ID NO: 1 at one or more positions selected from the group consisting of: a position corresponding to position 203 of SEQ ID NO: 1; a position corresponding to position 329 of SEQ ID NO: 1; a position corresponding to position 378 of SEQ ID NO: 1; a position corresponding to position 383 of SEQ ID NO: 1; a position corresponding to position 467 of SEQ ID NO: 1; and a position corresponding to position 494 of SEQ ID NO: 1. The substrate specificity of the mutant after the amino acid substitution is improved compared to the mutant before the substitution. With respect to the above positions, "an amino acid substitution" means that the wild-type amino acid is replaced with another amino acid, and therefore, the wild-type amino acid is excluded from the amino acids after the substitution. For example, the position corresponding to position 203 of SEQ ID NO: 1 is phenylalanine (F) in SEQ ID NO: 1, but is leucine (L) in SEQ ID NO: 3. The mutants of the present disclosure include a mutant (F203L) in which F at position 203 of SEQ ID NO: 1 is replaced with L. On the other hand, a variant in which L at position 203 of SEQ ID NO: 3 remains as L (L203) does not fall under (is not included in) the variants of the present disclosure.

[0097] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 203 of SEQ ID NO: 1 may be leucine (L).

[0098] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 329 of SEQ ID NO: 1 may be glutamic acid (E).

[0099] In some embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 378 of SEQ ID NO: 1 may be aspartic acid (D) or glutamic acid (E).

[0100] In some embodiments, with respect to an NADH oxidase variant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 383 of SEQ ID NO: 1 may be aspartic acid (D), glutamic acid (E), or asparagine (N).

[0101] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 467 of SEQ ID NO: 1 may be glutamine (Q).

[0102] In certain embodiments, with respect to an NADH oxidase mutant of the present disclosure, the substituted amino acid (amino acid after substitution) introduced at the position corresponding to position 494 of SEQ ID NO: 1 can be glutamine (Q) or glutamic acid (E).

[0103] Improved thermostability and improved substrate specificity are separate properties of an enzyme, and generally, mutations that improve thermostability do not necessarily improve substrate specificity. Furthermore, generally, mutations that improve substrate specificity do not necessarily improve thermostability. However, some of the mutations disclosed herein not only improve the thermostability of NADH oxidase, but also improve its substrate specificity. This was unexpected and surprising. In certain embodiments, mutations that improve the thermal stability and substrate specificity of the present disclosure include a mutation substituting the amino acid at position 203 of SEQ ID NO: 1 with leucine (L), a mutation substituting the amino acid at position 329 of SEQ ID NO: 1 with glutamic acid (E), a mutation substituting the amino acid at position 378 of SEQ ID NO: 1 with glutamic acid (E) or aspartic acid (D), a mutation substituting the amino acid at position 383 of SEQ ID NO: 1 with aspartic acid (D), a mutation substituting the amino acid at position 467 of SEQ ID NO: 1 with glutamine (Q), and a mutation substituting the amino acid at position 494 of SEQ ID NO: 1 with glutamine (Q).

[0104] In certain embodiments, the present disclosure provides an N-terminally deleted NADH oxidase mutant 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, 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 amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, and lacking the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 1, and having NADH oxidase activity. The variant may further have an amino acid substitution as described in this disclosure.

[0105] In certain embodiments, the present disclosure provides a method for identifying a nucleic acid sequence that is 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, or 79% or more of the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124.

[0009] The present inventors have found that N-terminally deleted NADH oxidase variants have NADH oxidase activity and share at least 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more, for example, 99% amino acid sequence identity with the NADH oxidase of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17. The present inventors have found that deletion mutants of the NADH oxidases of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, and 17 retain NADH oxidase activity even when the N-terminus of the NADH oxidase is deleted. Furthermore, the present inventors have found that deletion mutants of the NADH oxidases of SEQ ID NOs: 74, 76, and 78, which are derived from different sources from these NADH oxidases, retain NADH oxidase activity even when their N-termini are deleted. Furthermore, the present inventors have found that the introduction of the amino acid substitutions of the present disclosure into the NADH oxidases of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 improves the thermostability and / or substrate specificity. Even when a large mutation, a deletion of 197 amino acids on the N-terminus, was introduced, the effects demonstrated for SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 were also confirmed for SEQ ID NOs: 74, 76, and 78. Therefore, a person skilled in the art would understand that even when a small mutation, an amino acid substitution as disclosed herein, is introduced into the NADH oxidase of SEQ ID NO: 74, 76, or 78, it is highly likely that the same effects will be achieved as with the NADH oxidase of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17.

[0106] In certain embodiments, the present disclosure provides an NADH oxidase 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, 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, e.g., 99% or more, e.g., 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78. In certain embodiments, the NADH oxidase can have an amino acid substitution as described herein (amino acid substitution variant). In some embodiments, the NADH oxidase may lack the N-terminal domain described herein (N-terminal domain deletion mutants). In some embodiments, the amino acid substitution mutants do not include the wild-type sequence itself.

[0107] NADH oxidase is widely distributed in nature and can be obtained by searching for enzymes of microbial, animal, or plant origin, such as actinomycetes, filamentous fungi, yeast, or bacteria. In the present specification, the origin of the NADH oxidase is not particularly limited, and examples thereof include the genus Bacillus, such as Bacillus velezensis, Bacillus subtilis, Bacillus haynesii, Bacillus proteolyticus, Bacillus coagulans, Bacillus amyloliquefaciens, Bacillus cereus, and Bacillus licheniformis; the genus Priestia, such as Priestia aryabhattai; and the genus Acidianus, such as Acidianus ambivalens. ambivalens), the genus Archaeoglobus, for example Archaeoglobus fulgidus, the genus Brevibacterium, for example Brevibacterium sp., the genus Methanocaldococcus, for example Methanocaldococcus jannaschii, the genus Pseudothermotoga, for example Pseudothermotoga hypogea, the genus Pyrococcus, for example Pyrococcus furiosus, Pyrococcus horikoshii, the genus Saccharolobus, for example Saccharolobus Solfataricus (Saccharolobussolfataricus), the genus Streptococcus, for example Streptococcus mutans, the genus Sulfolobus, for example Sulfolobus acidocaldarius, the genus Thermotoga, for example Thermotoga maritima, the genus Geobacillus, for example Geobacillus caldoproteolyticus, Geobacillus caldoxylosilyticus, Geobacillus debilis, Geobacillus galactosidasius, Geobacillus gargensis gargensis, Geobacillus jurassicus, Geobacillus kaustophilus, Geobacillus lituanicus, Geobacillus pallidus, Geobacillus stearothermophilus, Geobacillus stromboliensis, Geobacillus subterraneus, Geobacillus tepidamans, Geobacillus thermocatenulatus, Geobacillus thermodenitrificans thermodenitrificans, Geobacillus thermoglucosidasius, Geobacillus thermoreovoransThe NADH oxidase may be derived from Geobacillus thermoleovorans, Geobacillus toebii, Geobacillus uzensis, Geobacillus vulcani, or Geobacillus zalihae. Unless otherwise specified, the NADH oxidase derived from a specific microorganism includes both wild-type and modified forms thereof.

[0108] (Obtaining a Gene Encoding NADH Oxidase) A gene encoding NADH oxidase (hereinafter simply referred to as "NADH oxidase gene") can be obtained by a commonly used gene cloning method. For example, chromosomal DNA or mRNA can be extracted by a conventional method from microbial cells or various cells capable of producing NADH oxidase. Furthermore, cDNA can be synthesized using mRNA as a template. The chromosomal DNA or cDNA obtained in this manner can be used to prepare a chromosomal DNA or cDNA library.

[0109] Next, a suitable probe DNA is synthesized based on the amino acid sequence of the NADH oxidase, and this is used to select the NADH oxidase gene from a chromosomal DNA or cDNA library. Alternatively, a suitable primer DNA is prepared based on the amino acid sequence, and a suitable polymerase chain reaction (PCR) such as 5' RACE or 3' RACE is used to amplify DNA containing a target gene fragment encoding NADH oxidase. These DNA fragments are then ligated to obtain DNA containing the full-length of the target NADH oxidase gene.

[0110] Examples of the NADH oxidase gene include, but are not limited to, an NADH oxidase gene derived from the genus Bacillus and an NADH oxidase gene derived from the genus Priestia.

[0111] The NADH oxidase gene may be linked to a vector. Examples of vectors include plasmids, bacteriophages, and cosmids, such as pBluescriptII SK+ (Stratagene). Plasmids can be obtained by standard methods. For example, a plasmid containing the NADH oxidase gene can be extracted and purified using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich). The resulting NADH oxidase gene can be manipulated to produce an NADH oxidase mutant gene or to obtain a purified enzyme.

[0112] (Mutation of NADH oxidase gene) Mutation of the NADH oxidase gene can be carried out by any known method depending on the intended mutation form, such as a method of contacting the NADH oxidase gene or a recombinant DNA incorporating the gene with a mutagenic agent, ultraviolet irradiation, genetic engineering techniques, or protein engineering techniques.

[0113] Examples of mutagenic agents used in the above mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil.

[0114] The conditions for this contact and reaction can be adjusted depending on the type of drug used, and are not particularly limited as long as the desired mutation can actually be induced in the NADH oxidase gene. Generally, the desired mutation can be induced by contact and reaction at a drug concentration of preferably 0.5 to 12 M, at a reaction temperature of 20 to 80°C, for 10 minutes or more, preferably 10 to 180 minutes. When ultraviolet irradiation is performed, it can be carried out according to the standard method as described above.

[0115] As a method making full use of protein engineering techniques, a method known as Site-Specific Mutagenesis can generally be used.

[0116] In addition to the above-mentioned gene modification methods, the desired modified NADH oxidase gene can also be directly synthesized by organic synthesis or enzymatic synthesis.

[0117] The nucleotide sequence of the NADH oxidase gene can be confirmed, for example, by using a multi-capillary DNA analysis system, Applied Biosystems 3730x1 DNA Analyzer (manufactured by Thermo Fisher Scientific).

[0118] (Transformation / Transduction) The NADH oxidase gene can be incorporated into a vector such as a bacteriophage, cosmid, or a plasmid used for transforming prokaryotic or eukaryotic cells by standard methods, and then used to transform a host corresponding to each vector by standard methods.

[0119] In certain embodiments, NADH oxidase can be expressed using prokaryotic cells, such as Escherichia microorganisms, e.g., Escherichia coli, Brevibacillus microorganisms, e.g., Brevibacillus choshinensis, Corynebacterium microorganisms, e.g., Corynebacterium glutamicum, or Streptomyces microorganisms, e.g., Streptomyces violaceoruber. Examples of E. coli hosts include, but are not limited to, various E. coli strains, such as K-12, JM109, DH5α, BL21, JM109(DE3), DH5α(DE3), BL21(DE3), TG1, 1100, W3110, and C600. The host is transformed to obtain a host cell (transformant) into which the NADH oxidase gene has been introduced. As a method for introducing a recombinant vector into such host cells, for example, when the host cell is a microorganism belonging to the genus Escherichia coli, a method for introducing recombinant DNA in the presence of calcium ions can be employed, or electroporation can also be used. The NADH oxidase gene can be codon-optimized depending on the expression host.

[0120] In certain embodiments, NADH oxidase can be expressed using eukaryotic cells. An example of a eukaryotic host cell is yeast. Examples of microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Schizosaccharomyces, Saccharomyces, Pichia, and Candida. The transgene may contain a marker gene that enables the selection of transformed cells. Examples of marker genes include genes that complement auxotrophy of the host, such as URA3 and TRP1. Furthermore, the transgene preferably contains a promoter or other regulatory sequence (e.g., an enhancer sequence, a terminator sequence, a polyadenylation sequence, etc.) that can express the gene of interest in the host cell. Specific examples of promoters include the GAL1 promoter and the ADH1 promoter. As a method for transforming yeast, known methods can be used, and transformation can be carried out using any of a variety of techniques including the spheroplast method and the glass bead method.

[0121] Other examples of eukaryotic host cells include fungal cells (including filamentous fungi) such as those of the genus Aspergillus and Trichoderma. The method for producing a fungal cell transformant is not particularly limited, and examples include a method in which a gene encoding NADH oxidase is introduced into a host filamentous fungus in a manner that allows the gene to be expressed, according to a conventional method. Specifically, a DNA construct is prepared in which the gene encoding NADH oxidase is inserted between an expression-inducing promoter and a terminator, and then a host filamentous fungus is transformed with the DNA construct to obtain a transformant that overexpresses the gene encoding NADH oxidase.

[0122] The method for introducing the gene encoding NADH oxidase into the host filamentous fungus in such a manner that the gene is expressed is not particularly limited, and examples thereof include a method of directly inserting the gene into the chromosome of the host organism by utilizing homologous recombination; and a method of introducing the gene into the host filamentous fungus by linking it to a plasmid vector.

[0123] In the method using homologous recombination, a DNA construct is ligated between sequences homologous to the upstream and downstream regions of a recombination site on a chromosome and inserted into the genome of a host filamentous fungus. A transformant can be obtained by self-cloning by overexpressing the construct in the host filamentous fungus under the control of its own high-expression promoter. The high-expression promoter is not particularly limited, but examples include the promoter region of the TEF1 gene (tef1), which is a translation elongation factor, the promoter region of the α-amylase gene (amy), and the promoter region of the alkaline protease gene (alp).

[0124] In the method using a vector, the DNA construct can be inserted into a plasmid vector used for transforming filamentous fungi by a conventional method, and the corresponding host filamentous fungus can be transformed by a conventional method.

[0125] Such a suitable vector-host system is not particularly limited as long as it is a system that allows NADH oxidase to be produced in the 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.

[0126] The DNA construct is preferably introduced into the chromosome of the host filamentous fungus for use; however, as an alternative method, the DNA construct can be incorporated into an autonomously replicating vector (Ozeki et al., Biosci. Biotechnol. Biochem. 59, 1133 (1995)) for use without being introduced into the chromosome.

[0127] The DNA construct may contain a marker gene that allows for the selection of transformed cells. The marker gene is not particularly limited, and examples include genes that complement auxotrophy of the host, such as pyrG, niaD, and adeA; and drug resistance genes for drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably contains a promoter, terminator, or other control sequence (e.g., enhancer, polyadenylation sequence, etc.) that enables overexpression of the gene encoding NADH oxidase in the host cell. The promoter is not particularly limited, and examples include appropriate expression-inducible promoters and constitutive promoters, such as the tef1 promoter, alp promoter, and amy promoter. The terminator is also not particularly limited, and examples include the alp terminator, amy terminator, and tef1 terminator.

[0128] In the DNA construct, an expression control sequence for the gene encoding NADH oxidase is not necessarily required when the DNA fragment containing the gene encoding NADH oxidase to be introduced contains a sequence having an expression control function. Furthermore, when transformation is performed by cotransformation, the DNA construct may not necessarily have a marker gene.

[0129] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, a gene encoding NADH oxidase, the alp gene terminator, and the pyrG marker gene are ligated to an In-Fusion Cloning Site in the multiple cloning site of pUC19.

[0130] The method for transforming a filamentous fungus can be appropriately selected from methods known to those skilled in the art. For example, the protoplast PEG method, in which protoplasts of a host filamentous fungus are prepared and then transformed with polyethylene glycol and calcium chloride (see, for example, Mol. Gen. Genet. 218, 99-104, 1989; JP 2007-222055 A), can be used. A medium for regenerating the transformed filamentous fungus is selected appropriately depending on the host filamentous fungus and the transformation marker gene used. For example, when Aspergillus sojae is used as the host filamentous fungus and the pyrG gene is used as the transformation marker gene, the transformed filamentous fungus can be regenerated in, for example, Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2 M sorbitol.

[0131] Furthermore, for example, to obtain a transformed filamentous fungus, the promoter of the gene encoding NADH oxidase originally present on the chromosome of the host filamentous fungus may be replaced with a high-expression promoter such as tef1 using homologous recombination. In this case, it is also preferable to introduce a transformation marker gene such as pyrG in addition to the high-expression promoter. For example, for this purpose, a transformation cassette consisting of the upstream region of the gene encoding NADH oxidase - transformation marker gene - high-expression promoter - all or part of the gene encoding NADH oxidase can be used, as shown in Example 1 and Figure 1 of Japanese Patent Application Laid-Open No. 2011-239681. In this case, the upstream region of the gene encoding NADH oxidase and all or part of the gene encoding NADH oxidase are used for homologous recombination. The all or part of the gene encoding NADH oxidase can include a region extending from the initiation codon. The length of the region suitable for homologous recombination is preferably 0.5 kb or more.

[0132] The production of a transformed filamentous fungus can be confirmed by culturing the transformed filamentous fungus and then confirming the NADH oxidase activity in the culture medium and / or cell extract obtained after culturing.

[0133] Alternatively, the production of a transformed filamentous fungus may be confirmed by extracting chromosomal DNA from the transformed filamentous fungus, performing PCR using the DNA as a template, and confirming that an amplifiable PCR product is produced if transformation has occurred.

[0134] For example, PCR is carried out using a combination of a forward primer for the nucleotide sequence of the promoter used and a reverse primer for the nucleotide sequence of the transformation marker gene, and it is confirmed that a product of the expected length is produced.

[0135] The host may be a known microorganism, known strain, or an equivalent of the known microorganism or strain described herein. An equivalent refers to a host that exhibits equivalent functions with respect to recombinant expression of a protein. Equivalents include hosts that have been created and modified based on hosts known at the time of filing of this application, which have been developed after the filing of this application, and hosts with properties similar to those known at the time of filing of this application that have been discovered after the filing of this application. Regarding the scientific name or classification of a microorganism, if there is a change in the scientific name, genus name, or classification after the filing of this application, the description in this specification shall take precedence, and the date of filing of this application shall be used as the basis.

[0136] (High-Throughput Screening) NADH oxidase can also be subjected to high-throughput screening to obtain functional NADH oxidase mutants. For example, a library of transformed or transduced strains carrying a mutated NADH oxidase gene can be prepared and subjected to high-throughput screening in microtiter plates or ultra-high-throughput screening using droplet microfluidics. Examples include constructing a combinatorial library of mutant genes encoding variants and then screening a large population of mutant NADH oxidases using phage display (e.g., Chem. Rev. 105 (11): 4056-72, 2005), yeast display (e.g., Comb Chem High Throughput Screen. 2008;11(2): 127-34), bacterial display (e.g., Curr Opin Struct Biol 17: 474-80, 2007), or the like. See also Agresti et al., "Ultrahigh-throughput screening in drop-based microfluidics for directed evolution," Proceedings of the National Academy of Sciences 107 (9): 4004-4009 (Mar. 2010). The description of an ultrahigh-throughput screening method that can be used to screen for NADH oxidase variants is incorporated herein by reference. For example, libraries can be constructed using error-prone PCR. Saturation mutagenesis can also be used to construct libraries by targeting the regions and positions described herein or corresponding regions and positions. The libraries can be transformed into appropriate cells, such as electrocompetent EBY-100 cells, to obtain approximately 10 mutants (10 million mutations). Yeast cells transformed with the libraries can then be subjected to cell sorting. Polydimethoxylsiloxane (PDMS) microfluidic devices fabricated using standard soft lithography techniques can also be used.Monodisperse droplets can be formed using a flow focus device. The formed droplets containing individual mutants can be subjected to an appropriate sorting device. The presence or absence of NADH oxidase activity can be utilized to select cells. For example, a reaction solution whose absorbance changes upon the action of the above-mentioned NADH oxidase may be used. For example, to quantify the amount of NADH consumed by the action of NADH oxidase, absorbance at 340 nm can 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 herein include amino acid substitutions, insertions, deletions, and / or additions.

[0137] For example, one to ten mutations can be introduced into an NADH oxidase, and NADH oxidase activity can be confirmed. Then, starting from an NADH oxidase mutant confirmed to have activity, one to ten additional mutations can be introduced and activity can be confirmed. A series of high-throughput screening (e.g., the above-described method of obtaining and screening approximately 10 to the power of seven mutants) can be repeated for two or more rounds, five or more rounds, ten or more rounds, fifteen or more rounds, for example, twenty or more rounds. By repeating, for example, ten rounds of high-throughput screening, in which one or more, five or more, for example, ten or more mutations are introduced in each round, one can rapidly obtain mutants that have 10 or more, 50 or more, for example, 100 or more mutations introduced from the starting NADH oxidase and still retain activity. Furthermore, by repeating 20 rounds, one can rapidly obtain mutants that have 20 or more, 100 or more, for example, 200 or more mutations introduced from the starting NADH oxidase and still retain activity. Such operations can be performed by repeating a routine process.

[0138] Mutations may be introduced at any one or more positions from the first amino acid to the last amino acid in the full-length amino acid sequence of NADH oxidase, excluding regions important for enzyme function, such as the active center, substrate recognition site, prosthetic group recognition motif, and their vicinity. NADH oxidases have been reported in literature, and those skilled in the art are familiar with regions important for enzyme function, including the active center, substrate recognition site, and prosthetic group recognition motif. In certain embodiments, for example, one or more mutations may be introduced first at positions 1 to 10 of the full-length sequence of NADH oxidase. Next, starting from an NADH oxidase variant confirmed to have activity, one or more mutations may be further introduced at positions 11 to 20, and activity may be confirmed. This process may be repeated n times (n≦51). For example, in the 51st iteration, one or more mutations may be introduced at positions corresponding to positions 501 to 509 of SEQ ID NO: 1. In certain embodiments, for example, in an N-terminal domain-deleted NADH oxidase, one or more mutations may first be introduced into positions 1 to 10 (for example, one or more mutations may be introduced into positions corresponding to positions 198 to 207 of SEQ ID NO: 1). Next, starting from an NADH oxidase variant that has been confirmed to have activity, one or more mutations may be further introduced into positions 208 to 217, and activity may be confirmed. This process may be repeated n times (n≦32). For example, on the 32nd iteration, one or more mutations may be introduced into positions corresponding to positions 508 to 509 of SEQ ID NO: 1. Regions important for enzyme function or regions not intended to be modified may be skipped along the way, as appropriate. This allows any mutation to be introduced at any position in the full-length sequence, except for regions important for the function of the enzyme, and enables the rapid production of active NADH oxidase 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.

[0139] Mutations may be introduced randomly or by rational design. In certain embodiments, mutations introduced by rational design or randomly may be conservative amino acid substitutions. Conservative amino acid substitutions include amino acid substitutions in which the amino acid before and after substitution have similar chemical properties (e.g., Stryer et al., Biochemistry, 5th ed., 2002, pp. 44-49). For example, conservative amino acid substitutions may be selected from the group consisting of: (A) substitution of a basic amino acid with a different basic amino acid; (B) substitution of an acidic amino acid with a different acidic amino acid; (C) substitution of an aromatic amino acid with a different aromatic amino acid; (D) substitution of a nonpolar aliphatic amino acid with a different nonpolar aliphatic amino acid; and (E) substitution of a polar uncharged amino acid with a different polar uncharged amino acid. Basic amino acids may be selected from, for example, arginine, histidine, and lysine. Acidic amino acids may be, for example, aspartic acid or glutamic acid. Aromatic amino acids may be selected from, for example, phenylalanine, tyrosine, and tryptophan. Nonpolar aliphatic amino acids may be selected from, for example, glycine, alanine, proline, valine, leucine, methionine, and isoleucine. Polar uncharged amino acids may be selected from, for example, serine, threonine, cysteine, asparagine, and glutamine.

[0140] In some embodiments, mutations introduced by rational design or randomly introduced include substitutions with functionally similar amino acids. Tables of functionally similar amino acids are widely known in the art. In some embodiments, in substitutions with functionally similar amino acids, the original and substituted amino acids may belong to any of the following amino acid classes: 1) glycine (G), alanine (A); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (N), lysine (K), 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).

[0141] In typical embodiments, conservative amino acid substitutions, or substitutions with functionally similar amino acids, are not present in regions critical to the function of the enzyme, such as the active center of NADH oxidase, the substrate recognition site, the prosthetic group recognition motif, or nearby regions, and therefore do not significantly affect the activity of the enzyme.

[0142] NADH oxidase variants may also include those in which additional amino acids have been inserted compared to the sequence before mutation. In typical embodiments, the amino acid insertion is not located in an area critical to the function of the enzyme, such as the active center, substrate recognition site, prosthetic group recognition motif, or the vicinity thereof, and therefore does not significantly affect the activity of the enzyme. NADH oxidase variants may also include those in which additional amino acids have been added compared to the sequence before mutation. In certain embodiments, the amino acid addition is made to the N-terminus or C-terminus of the NADH oxidase and does not significantly affect the activity of the enzyme. Examples of additions include, but are not limited to, the addition of a short tag sequence of histidine residues (e.g., 2 to 6 histidine residues) to aid in the purification of the NADH oxidase. Examples of additions include, but are not limited to, the addition of a signal peptide to aid in the expression of the NADH oxidase. Examples of signal peptides include known signal sequences or functional equivalents thereof.

[0143] NADH oxidase variants may also contain amino acid deletions compared to the sequence prior to mutation. In some embodiments, deletions may be as short as one or two amino acids. In some embodiments, the amino acid sequence of one NADH oxidase may be compared to that of another NADH oxidase, and if an amino acid is deleted in one sequence, that deletion may be introduced into the other NADH oxidase. Because both NADH oxidases exhibit activity, such deletions are unlikely to significantly affect the activity of the enzyme.

[0144] Mutations can be introduced into NADH oxidase so as not to disrupt secondary structures or structural motifs, such as α-helix structures or β-sheet structures. Regions of secondary structure can be identified, for example, by using a secondary structure prediction algorithm. 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.

[0145] In some limiting embodiments, the substituted amino acid does not back-mutate to an amino acid in a native NADH oxidase sequence (a naturally occurring amino acid). In other embodiments, a substituted amino acid at a position corresponding to, for example, position 503 of SEQ ID NO: 1, can be identical to the amino acid at that position in a native NADH oxidase sequence (a naturally occurring amino acid). In certain embodiments, the NADH oxidases of the present disclosure are free of naturally occurring amino acids.

[0146] (Corresponding Position) In this specification, when a specific position in a reference amino acid sequence corresponds to a specific position in another similar amino acid sequence, this is referred to as a corresponding position. Furthermore, an amino acid at a corresponding position is referred to as a corresponding amino acid. For convenience, this specification will be described using the amino acid sequence of the NADH oxidase derived from the genus Bacillus shown in SEQ ID NO: 1 as a reference. In this case, the "corresponding position" in an amino acid sequence refers to a position in the amino acid sequence of an NADH oxidase derived from another biological species that corresponds to a specific position in the amino acid sequence of the NADH oxidase derived from the genus Bacillus shown in SEQ ID NO: 1.

[0147] A method for identifying "corresponding positions" in amino acid sequences is, for example, to compare amino acid sequences using a known algorithm such as the Lippmann-Parson method, and assign maximum identity to conserved amino acid residues present in the amino acid sequences of each NADH oxidase. By aligning the amino acid sequences of NADH oxidases in this manner, it is possible to determine the positions of homologous amino acid residues in each NADH oxidase sequence, regardless of insertions or deletions in the amino acid sequences. Corresponding positions (homologous positions) are considered to be at the same position in the three-dimensional structure, and can be predicted to have the same or similar effect on the specific function of the target NADH oxidase.

[0148] (Corresponding Position of Mutation) As used herein, the phrase "a position corresponding to position 503 in the amino acid sequence of SEQ ID NO: 1" refers to the position corresponding to position 503 in SEQ ID NO: 1 when the amino acid sequence of a target NADH oxidase is compared with the amino acid sequence of SEQ ID NO: 1. The same applies to other positions in SEQ ID NO: 1, such as positions 203, 246, 283, 284, 329, 467, 378, 383, and 494. For example, the position corresponding to position 503 in the amino acid sequence of SEQ ID NO: 1 is position 503 in SEQ ID NOs: 3, 5, 7, 9, 13, 15, 17, 74, 76, and 78, and position 502 in SEQ ID NO: 11.

[0149] (Corresponding Region) A "corresponding domain" in an amino acid sequence is defined in the same manner as the above-mentioned "corresponding position." For example, the domain corresponding to positions 2 to 198 of SEQ ID NO: 1 corresponds to positions 2 to 198 of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, and 78.

[0150] (Homology, Identity, or Similarity of Amino Acid Sequences) Amino acid sequence homology, identity, or similarity can be calculated using programs such as GENETYX (GENETYX) maximum matching and search homology, DNASIS Pro (Hitachi Solutions) maximum matching and multiple alignment, or CLUSTAL W (https: / / www.genome.jp / tools-bin / clustalw) multiple alignment. To calculate amino acid sequence identity, two or more NADH oxidases can be aligned and the positions of identical amino acids in the two or more NADH oxidases can be examined. Based on this information, identical regions in the amino acid sequences can be determined. Here, for two or more amino acid sequences, the percent identity refers to the percentage calculated when two or more amino acid sequences are aligned using an algorithm such as Blosum62, with the total number of amino acids in the alignable regions as the denominator and the number of positions occupied by identical amino acids as the numerator. Therefore, normally, when two or more amino acid sequences have a region where no identity is observed, for example, when one of the amino acid sequences has an additional sequence at the C-terminus where no identity is observed, the region where no identity is observed cannot be aligned and is therefore not used in calculating the percent identity.

[0151] It is also possible to examine the positions of similar amino acids in two or more NADH oxidases. For example, multiple amino acid sequences can be aligned using CLUSTALW. In this case, the algorithm Blosum62 is used, and amino acids that are determined to be similar when multiple amino acid sequences are aligned may be referred to as similar amino acids. In the variants disclosed herein, amino acid substitutions may be due to substitutions between such similar amino acids. Such alignments allow for the examination of regions of identical amino acid sequences and positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, regions of homology (conserved regions) in the amino acid sequences can be determined.

[0152] Regions of homology can be identified as regions in which identical amino acid residues are conserved in the NADH oxidases shown in FIG. Unless otherwise specified, in this specification, the homologous regions of the FAD domain and the NADH domain of NADH oxidase in SEQ ID NO: 1 are the following positions in SEQ ID NO: 1: 199, 201, 206, 235, 237-244, 247-248, 251-253, 255, 258, 260-263, 265, 267, 270-274, 276, 279, 281, 284-287, 290-292, 294, 298-303, 305-308, 310, 314-320, 323-326, 328-345, 347-348, 350-353, 351-353, 352-354, 353-355, 354-355, 355-356, 357-358, 358-359, 360-361, 361-362, 362-363, 363-364, 364-365, 365-366, 366-367, 367-368, 368-369, 370-371, 371-372, 372-373, 373-374, 374-375, 375-376, 376-377, 378-379, 381-382, 382-383, 383-384, 384-385, 385-386, 386-387 9th place, 360th-368th place, 372nd-374th place, 377th-378th place, 380th-385th place, 387th-388th place, 391st place, 394th-400th place, 402nd ~403rd, 405-406, 408-411, 415-417, 422, 425, 427, 430, 432-433, 435 The homologous region is a region consisting of all of the amino acid sequences at positions 446 to 449, 452 to 455, 459 to 461, 464 to 465, 467, 469, 473, 475 to 481, 487 to 493, 495 to 496, 498 to 500, 502 to 505, and 507 to 508. Unless otherwise specified, the homologous region of the FAD domain and NADH domain of the target NADH oxidase in this specification corresponds to the homologous region of the FAD domain and NADH domain of SEQ ID NO: 1. When comparing homologous regions, their positions correspond to each other. For example, when comparing the homologous region of sequence A with the homologous region of sequence B, the position in sequence A corresponding to position 201 of SEQ ID NO: 1 and the position in sequence B corresponding to position 201 of SEQ ID NO: 1 are compared as corresponding positions. Note that the homology region is defined relative to the sequence before amino acid substitution, and does not preclude the introduction of a mutation into the NADH oxidase into the homology region. For example, position 503 of SEQ ID NO: 1 is included in the homology region of SEQ ID NO: 1, and a mutation may be introduced at this position. The same applies to other mutation positions described herein.Since the homologous regions are sequences common to the sequences shown in Figure 1, the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1 are identical to the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, and 78. Therefore, in this specification, these descriptions are mutually interchangeable.

[0153] In certain embodiments, the NADH oxidase variant of the present disclosure has a full-length amino acid sequence identity of 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, e.g., 99% or more when aligned with an NADH oxidase having the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, and has improved thermal stability compared to the NADH oxidase before modification.

[0154] In certain embodiments, the NADH oxidase 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 503, 203, 246, 283, 284, 329, 467, 378, 383, and 494 of SEQ ID NO: 1, and has improved thermal stability compared to the NADH oxidase before modification. Here, "one or several amino acids" refers to 1 to 15, 1 to 10, 1 to 7, 1 to 5, or 1 to 4, for example, 1 to 3, for example, 1 or 2 amino acids.

[0155] (Production of NADH oxidase) In one embodiment, the present invention provides a method for producing NADH oxidase, comprising culturing a bacterial strain capable of producing NADH oxidase under conditions conducive to expression of the NADH oxidase, and isolating the NADH oxidase from the culture or culture broth. This method can use a host cell transformed with a vector incorporating a gene encoding the NADH oxidase of the present disclosure. Here, conditions conducive to expression of NADH oxidase refer to conditions in which the NADH oxidase gene is transcribed and translated, resulting in the production of a polypeptide encoded by the gene.

[0156] The medium for culturing the above-mentioned strains may contain, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran infusion, to which one or more inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and further contains, as necessary, carbohydrate raw materials, vitamins, and the like.

[0157] Furthermore, the production yield of the target enzyme can be improved by adding to the culture medium substrates on which the NADH oxidase can act or analogous compounds thereof, such as glycated amino acids, glycated peptides, hydrolysates of glycated proteins, or glycated proteins such as glycated hemoglobin and glycated albumin.

[0158] The initial pH of the medium is suitably adjusted to pH 7 to 9. Cultivation is preferably carried out at a culture temperature of 20 to 42°C, preferably around 25 to 37°C, for 4 to 24 hours, more preferably around 25 to 37°C, for 8 to 16 hours by aeration and agitation submerged culture, shaking culture, static culture, or the like.

[0159] After the culture is completed, NADH oxidase can be collected from the culture by conventional enzyme collection methods. For example, the cells can be subjected to ultrasonic disruption, grinding, etc., or the enzyme can be extracted using a lytic enzyme such as lysozyme, or the cells can be lysed by shaking or standing in the presence of toluene, etc., to excrete the enzyme from the cells. The solution can then be filtered, centrifuged, etc. to remove solids, and nucleic acids can be removed, if necessary, with streptomycin sulfate, protamine sulfate, manganese sulfate, etc. After that, the solution is fractionated by adding ammonium sulfate, alcohol, acetone, etc., and the precipitate can be collected to obtain the crude enzyme.

[0160] To obtain a purified enzyme preparation from the crude enzyme, for example, gel filtration using Sephadex, Superdex, Ultrogel, or the like; adsorption-elution using an ion-exchange carrier, a hydrophobic carrier, or hydroxyapatite; electrophoresis using polyacrylamide gel, or the like; sedimentation methods such as sucrose density gradient centrifugation; affinity chromatography; fractionation methods using a molecular sieve membrane or a hollow fiber membrane, or the like, can be appropriately selected or performed in combination to obtain a purified NADH oxidase enzyme preparation.

[0161] (Lyophilized Preparation, Kit, Electrode, Sensor, and NADH Measurement Method) In certain embodiments, the present invention provides a lyophilized preparation, kit, electrode, sensor equipped with the electrode, or method for measuring NADH using NADH oxidase, all of which contain NADH oxidase. The lyophilized preparation, kit, electrode, sensor equipped with the electrode, or method for measuring NADH using NADH oxidase may contain a reagent for measuring reduced compounds, a reagent for measuring hydrogen peroxide, a buffer, a surfactant, salts, preservatives, etc. In addition, solubilizers, stabilizers, reactivity enhancers, reducing agents, bovine serum albumin, sugars (glycerin, lactose, sucrose, etc.), etc. may also be added. The lyophilized preparation, kit, electrode, sensor equipped with the electrode, or method for measuring NADH using NADH oxidase may further contain other known stabilizers, systems for elimination of impurities, etc., as necessary. Techniques used in various conventional freeze-dried preparations, kits, electrodes, sensors equipped with such electrodes, and NADH measurement methods using NADH oxidase can be appropriately modified and used in the kits, electrodes, sensors equipped with such electrodes, or NADH measurement methods using NADH oxidase of the present disclosure.

[0162] Examples of surfactants include nonionic surfactants and ionic surfactants, such as cationic surfactants, anionic surfactants, and amphoteric surfactants.

[0163] Examples of nonionic surfactants include polyoxyethylene alkyl ethers, fatty acid sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides, and alkyl monoglyceryl ethers.

[0164] Examples of cationic surfactants include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkylbenzyldimethylammonium salts, pyridinium salts such as alkylpyridinium salts, phosphonium salts such as alkylphosphonium salts, imidazolium salts such as alkylimidazolium salts, and isoquinonium salts such as alkylisoquinonium salts.

[0165] The reagent for measuring hydrogen peroxide may contain peroxidase and / or a chromogenic substrate. Examples of the chromogenic substrate include, in addition to 4-aminoantipyrine, ADOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), ALOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline), TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine), and DA-64 (N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine).

[0166] Examples of stabilizers include sugars selected from the group consisting of raffinose, melezitose, maltotriose, trehalose, sucrose, lactose, maltose, turanose, cellobiose, ribose, lyxose, xylose, arabinose, glucose, altrose, mannose, galactose, idose, allose, talose, gulose, psicose, fructose, sorbose, tagatose, deoxyribose, fucose, fuculose, rhamnose, ribulose, and xylulose, glycogen, starch (amylose, amylopectin), cellulose, dextrin, glucan, fructan, chitin, chitosan, glucomannan, pectin, alginic acid, hyaluronic acid, and chondroitin. Examples of suitable amino acids include, but are not limited to, polysaccharides selected from the group consisting of glycine sulfate and heparin; sugar alcohols selected from the group consisting of erythritol, threitol, arabinitol, xylitol, ribitol, iditol, galactitol, sorbitol (galactitol), and mannitol; amino acids or salts thereof selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, histidine, lysine, and arginine; and phosphoric acid, carboxylic acid (acetic acid, citric acid, malonic acid, malic acid, etc.) and salts thereof.

[0167] (Method for Measuring NADH) In certain embodiments, the present disclosure provides a method for measuring NADH. NADH measurement can be a qualitative or quantitative method. The quantitative method can include contacting a sample containing NADH with the NADH oxidase of the present disclosure and measuring the reaction products or consumers. The reaction products can include NADH. ＋ , H ２ O ２ The reaction products include NADH, O ２ The term "contact" in this quantitative determination method encompasses any manner in which the enzyme and a sample are physically brought together so that the NADH oxidase can catalyze the oxidation reaction of NADH, and includes, for example, not only mixing free enzyme and NADH in solution, but also adding or dropping a solution sample containing NADH to an enzyme supported on a solid phase carrier. In one embodiment, there is provided a method for detecting NADH, comprising the step of contacting a sample with a sensor containing the NADH oxidase described herein.

[0168] In certain embodiments, the sample used for measurement can be any sample that may contain NADH. In another embodiment, the sample used for measurement can be any sample that can produce NADH by the action of an NAD-dependent dehydrogenase. The sample can be appropriately processed. For example, a sample containing glucose can be appropriately treated with glucose dehydrogenase. This produces D-glucono-δ-lactone and NADH, and the produced NADH can then be measured using the NADH oxidase of the present disclosure. Such samples are also encompassed in the sample used for measurement in the present disclosure.

[0169] By examining the NADH concentration range in which the absorbance of the detected chromogenic substrate decreases proportionally as the amount of NADH added decreases while the amount of enzyme used and reaction time are kept constant, the lowest detectable NADH concentration (detection limit concentration) when using the NADH oxidase can be determined. The amount of enzyme and reaction time can be set so that the detection limit is lower than the NADH concentration in the measurement sample or blood.

[0170] In quantitative measurement, a calibration curve can be prepared in advance by performing regression analysis such as the least squares method on the measured values ​​of absorbance or response current of a control containing NADH of known concentration. The NADH concentration in a sample can be quantified by plotting the measured values ​​of the sample with unknown NADH concentration against the prepared calibration curve.

[0171] The time for allowing NADH oxidase to act on a sample containing NADH can be, for example, 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, but less than 60 minutes, less than 30 minutes, or less than 10 minutes, for example, less than 5 minutes, for example, 0.5 minutes or more but less than 60 minutes, 1 minute or more but less than 30 minutes, 1 minute or more but less than 20 minutes, for example, 1 minute or more but less than 10 minutes, or for example, 1 minute or more but less than 5 minutes. The reaction temperature varies depending on the optimum temperature of the enzyme used, but is, for example, 20 to 45°C, and can be appropriately selected from temperatures used in ordinary enzyme reactions.

[0172] The amount of NADH oxidase used will depend on the amount of substrate contained in the sample solution, but may be added so that the final concentration is 0.1 to 50 U / ml, e.g., 0.2 to 10 U / ml. The pH during reaction can be adjusted using a buffer, taking into consideration the pH at which NADH oxidase can act, e.g., the optimal pH. The reaction pH is, for example, 3 to 11, 5 to 9, 5 to 8, e.g., 6 to 8.

[0173] The measurement of hydrogen peroxide can be carried out simultaneously with the step of generating hydrogen peroxide and can proceed simultaneously with the action of NADH oxidase. Instead of the product, a consumed product can be measured. An example of the consumed product to be measured is dissolved oxygen. The amount of dissolved oxygen in the reaction solution can be measured using a dissolved oxygen meter or the like.

[0174] (Method for measuring NADH oxidase activity) Below, an example of a method for measuring NADH oxidase activity (also referred to as NADH oxidation activity) using NADH as a substrate is described, but the measurement method is not limited to this. NADH may be commercially available. Unless otherwise specified, in this specification, the enzyme titer is defined as the amount of enzyme that oxidizes 1 μmol of NADH per minute when measured at 37° C. and pH 7.5 using NADH as a substrate, with 1 U being the amount of enzyme that oxidizes 1 μmol of NADH per minute.

[0175] A: Reagents for activity measurement (Reagent 1) 250 mM potassium phosphate buffer, pH 7.5 (Reagent 2) 1 mM flavin adenine dinucleotide (FAD) solution (Reagent 3) 2 mM β-nicotinamide adenine dinucleotide (reduced form, β-NADH) solution (Sample solution) Sample containing NADH oxidase B: Activity measurement method Mix 150 μl of Reagent 1, 25 μl of Reagent 2, V μl of sample solution, and (500-V) μl of deionized water and incubate at 37°C for 2 minutes. Then, add 75 μl of Reagent 3 and mix well. Then, measure the absorbance (A) of light at a wavelength of 340 nm using a spectrophotometer U-3900 (Hitachi High-Tech Science) with the cell holder kept at 37°C. ３４０ ) was measured and the A per minute ３４０ The absolute value of the decrease (ΔA Ｓ As a control experiment, 75 μl of deionized water was added instead of 75 μl of Reagent 3, and the absorbance (A) of light with a wavelength of 340 nm was calculated. ３４０ ) was measured and the A per minute ３４０ The absolute value of the decrease (ΔA ０ ) was calculated.

[0176] The NADH oxidase activity (U / ml) can be calculated based on the following formula: where "6.22" is the millimolar absorption coefficient (mM －１ cm －１ ) [Formula] U / ml = (ΔA Ｓ -ΔA ０ )×750×df / (6.22×V) =120.6×(ΔA Ｓ -ΔA ０ ) × df / V

[0177] In certain embodiments, the present disclosure provides a polynucleotide encoding an NADH oxidase variant. In certain embodiments, the present disclosure provides a vector comprising such a polynucleotide. In certain embodiments, the present disclosure provides a host cell transformed with such a vector, i.e., a host cell comprising such a vector. In certain embodiments, the present disclosure provides a method for producing an NADH oxidase variant, comprising culturing such a host cell to produce the NADH oxidase variant, and obtaining the produced NADH oxidase variant. In certain embodiments, the present disclosure provides a method for contacting an NADH oxidase variant, or an electrode, sensor, or kit comprising the same, with a sample containing NADH, a sample capable of containing NADH, or a sample capable of producing NADH, to oxidize NADH contained in the sample. In certain embodiments, the method is capable of detecting NADH. In certain embodiments, the method is capable of measuring NADH.

[0178] The present enzyme has a wide stable pH range and excellent thermal stability. Therefore, when used, for example, as a reagent or food processing aid, the present enzyme is less susceptible to changes in the environment, such as pH or temperature, and is therefore more stable than known NADH oxidases. The term "stable pH" refers to the pH range in which the relative activity (%) of an NADH oxidase solution prepared at each pH is 80% or higher after heating it in a water bath maintained at 40°C for 60 minutes. For example, the stable pH of NOX-b shown in Figure 6 is between pH 6.0 and 8.0. Furthermore, the optimal pH range of the present enzyme is more neutral than known NADH oxidases, opening up new applications in pH ranges where known NADH oxidases could not be expected to be sufficiently effective, potentially expanding the variety of industrial applications. The optimal pH refers to the pH range in which the relative activity (%) of an NADH oxidase solution is 80% or higher. For example, the optimum pH of NOX-b shown in FIG. 3 is pH 5.0 to 7.5.

[0179] In some embodiments, the NADH oxidases of the present disclosure exclude naturally occurring NADH oxidases. In some embodiments, the NADH oxidases of the present disclosure have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, e.g., at least 99% amino acid sequence identity to SEQ ID NO: 1 and have NADH oxidase activity, excluding polypeptides consisting of the amino acid sequence of SEQ ID NO: 5. In some embodiments, the NADH oxidases of the present disclosure have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, e.g., at least 99% amino acid sequence identity to SEQ ID NO: 11 and have NADH oxidase activity, excluding those NADH oxidases with wild-type back mutations (excluding naturally occurring sequences). In some embodiments, the NADH oxidase of the present disclosure has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, e.g., at least 99% amino acid sequence identity to SEQ ID NO: 13 and has NADH oxidase activity, excluding wild-type back-mutations from the NADH oxidase (excluding naturally occurring sequences). In some embodiments, the NADH oxidase of the present disclosure has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, e.g., at least 99% amino acid sequence identity to SEQ ID NO: 15 and has NADH oxidase activity, excluding wild-type back-mutations from the NADH oxidase (excluding naturally occurring sequences). In some embodiments, the NADH oxidase of the present disclosure has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, e.g., at least 99% amino acid sequence identity to SEQ ID NO: 74 and has NADH oxidase activity, excluding wild-type back-mutations from the NADH oxidase (excluding naturally occurring sequences). In some embodiments, the NADH oxidase of the present disclosure has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, e.g., at least 99% amino acid sequence identity to SEQ ID NO: 76 and has NADH oxidase activity, excluding wild-type back-mutations from the NADH oxidase (excluding naturally occurring sequences).In some embodiments, the NADH oxidase of the present disclosure has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, e.g., 99% or more amino acid sequence identity with SEQ ID NO: 76 and has NADH oxidase activity, excluding those NADH oxidases that have reversion mutations to wild-type (naturally occurring sequences). In some embodiments, the NADH oxidase of the present disclosure excludes the polypeptides consisting of the amino acid sequence of SEQ ID NO: 1, 11, 13, 15, 74, 76, and 78. Other known sequences (e.g., sequences registered in databases, sequences described in public literature, etc.) are also excluded.

[0180] The NADH oxidases of the present disclosure are further illustrated by the following examples, which are provided for illustrative purposes only and are not intended to limit the present disclosure in any way.

[0181] 1. Construction of Plasmid for Expression of NADH Oxidase (NOX) A plasmid (pET22b-NOX-a) for expression of NADH oxidase (NOX-a) derived from Bacillus sp. having the amino acid sequence of SEQ ID NO: 1 was constructed using NEBuilder HiFi DNA Assembly (New England Biolabs).

[0182] Twelve bases (SEQ ID NO: 37) were added upstream of atg on the 5' end of the NOX-a gene (SEQ ID NO: 2), and 12 bases (SEQ ID NO: 38) were added downstream of taa on the 3' end, and synthesis was outsourced to Integrated DNA Technologies.

[0183] The plasmid fragment was prepared by PCR using the pET-22b(+) plasmid as a template and primers of SEQ ID NO: 39 and SEQ ID NO: 40. 1.0 μl of DpnI (New England BioLabs) was added to the PCR solution, which was then treated at 37° C. for 1 hour, and the amplified fragment was purified using a GFX PCR DNA and Gel Band Purification Kit (Cytiva).

[0184] The composition shown in Table 1 was reacted at 50°C for 15 minutes to obtain a plasmid for expressing NOX-a (pET22b-NOX-a). The resulting plasmid was transformed into E. coli JM109. The resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed by DNA sequence analysis to be the desired sequence.

[0185]

[0186] Similarly, for the NOX-b gene (SEQ ID NO: 4), NOX-c gene (SEQ ID NO: 6), NOX-d gene (SEQ ID NO: 8), NOX-e gene (SEQ ID NO: 10), NOX-f gene (SEQ ID NO: 12), NOX-g gene (SEQ ID NO: 14), NOX-h gene (SEQ ID NO: 16), and NOX-i gene (SEQ ID NO: 18), 12 bases (SEQ ID NO: 37) were added upstream of atg on the 5'-end side, and 12 bases (SEQ ID NO: 38) were added downstream of taa on the 3'-end side, and synthesis was outsourced to Integrated DNA Technologies. Each expression plasmid (pET22b-NOX-b, pET22b-NOX-c, pET22b-NOX-d, pET22b-NOX-e, pET22b-NOX-f, pET22b-NOX-g, pET22b-NOX-h, and pET22b-NOX-i) was obtained using the same procedures as used to construct pET22b-NOX-a, and DNA sequence analysis confirmed that it had the desired sequence.

[0187] An expression plasmid (pKK223-3-NOX-j) for NADH oxidase (NOX-j) derived from Geobacillus thermoleovorans having the amino acid sequence of SEQ ID NO: 74 was prepared using In-Fusion Snap Assembly Master Mix (manufactured by Takara Bio).

[0188] Twelve bases (SEQ ID NO: 126) were added upstream of atg on the 5' end of the NOX-j gene (SEQ ID NO: 75), and 12 bases (SEQ ID NO: 127) were added downstream of taa on the 3' end, and synthesis was outsourced to Integrated DNA Technologies.

[0189] The plasmid fragment was prepared by PCR using the pKK223-3 plasmid as a template and primers of SEQ ID NO: 128 and SEQ ID NO: 129. 1.0 μl of DpnI (New England BioLabs) was added to the post-PCR solution, which was then treated at 37° C. for 1 hour, and the amplified fragment was purified using a GFX PCR DNA and Gel Band Purification Kit (Cytiva).

[0190] The composition shown in Table 2 was reacted at 50°C for 15 minutes to obtain a plasmid for expressing NOX-a (pKK223-3-NOX-j). The resulting plasmid was transformed into E. coli JM109. The resulting transformant was cultured, and the base sequence of the extracted plasmid was confirmed by DNA sequence analysis to be the desired sequence.

[0191]

[0192] Similarly, for the gene (SEQ ID NO:77) encoding NOX-k (SEQ ID NO:76) and the gene (SEQ ID NO:79) encoding NOX-l (SEQ ID NO:78), 12 bases (SEQ ID NO:126) were added upstream of the 5'-terminal atg and 12 bases (SEQ ID NO:127) were added downstream of the 3'-terminal taa, and synthesis was outsourced to Integrated DNA Technologies, Inc. The respective expression plasmids (pKK223-3-NOX-k and pKK223-3-NOX-l) were obtained using the same procedures as used to construct pKK223-3-NOX-j, and DNA sequence analysis confirmed that they had the intended sequences.

[0193] 2. Construction of NOX-producing strains E. coli BL21(DE3) was transformed with pET22b-NOX-a, pET22b-NOX-b, pET22b-NOX-c, pET22b-NOX-d, pET22b-NOX-e, pET22b-NOX-f, pET22b-NOX-g, pET22b-NOX-h, or pET22b-NOX-i to obtain various NOX-producing strains.

[0194] E. coli JM109 strain transformed with pKK223-3-NOX-j, pKK223-3-NOX-k, or pKK223-3-NOX-l was used as a strain producing various NOXs.

[0195] 3. Recombinant Production of NOX The NOX-producing strain was inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 μg / ml) placed in a test tube and cultured overnight at 37°C and 130 rpm. 0.5 ml of the seed culture was inoculated into 250 ml of LB-amp medium (ampicillin concentration 50 μg / ml) placed in a Sakaguchi flask and cultured at 30°C and 130 rpm for 3 hours. IPTG was then added to a final concentration of 0.1 mM, and the culture was continued at 25°C and 130 rpm for 20 hours.

[0196] The culture medium from six Sakaguchi flasks was centrifuged at 6,500 × g for 10 minutes to obtain a pellet, which was then resuspended in 50 ml of 10 mM potassium phosphate buffer (PPB) pH 7.5. The bacterial cell suspension was then sonicated and centrifuged at 15,000 rpm for 15 minutes to obtain a supernatant, which was used as a crude NOX enzyme solution.

[0197] When the NOX crude enzyme solution was not purified, the NOX-producing strain was cultured on a smaller scale. The NOX-producing strain was inoculated into 2.5 ml of LB-amp medium (ampicillin concentration: 50 μg / ml) placed in a test tube and cultured overnight at 37°C and 130 rpm. 0.5 ml of the seed culture was inoculated into 30 ml of LB-amp-IPTG medium (ampicillin concentration: 100 μg / ml, IPTG concentration: 0.1 mM) placed in an Erlenmeyer flask and cultured at 25°C and 160 rpm for 20 hours.

[0198] The culture medium was centrifuged at 6,500 × g for 10 minutes, and the resulting pellet was resuspended in 3 ml of 10 mM potassium phosphate buffer (PPB) pH 7.5. The bacterial cell suspension was then sonicated and centrifuged at 15,000 rpm for 15 minutes. The supernatant was collected and used as a crude NOX enzyme solution.

[0199] 4. Purification of NOX The crude enzyme solution of NOX was applied to Q Sepharose® Fast Flow (manufactured by Cytiva, column volume (CV) 144.4 ml) equilibrated with 10 mM PPB pH 7.5 to bind to the anion exchange resin.

[0200] The resin was then washed with 1,444 ml (10 CV) of 10 mM PPB (pH 7.5) containing 100 mM NaCl, and the NaCl concentration in 10 mM PPB (pH 7.5) was increased linearly at a rate of 10 mM / CV while the resin was pumped to elute the NOX bound to the resin.

[0201] Further purification was performed using Q Sepharose (registered trademark) Fast Flow (manufactured by Cytiva, resin volume 23.1 ml) equilibrated with 10 mM PPB (pH 7.5) at different resin volumes. 10 mM PPB (pH 7.5) was used for resin equilibration and elution.

[0202] The resin was then washed with 231 ml (10 CV) of 10 mM PPB (pH 7.5) containing 100 mM NaCl, and the resin-bound NOX was eluted by linearly increasing the NaCl concentration in 10 mM PPB (pH 7.5) at a rate of 3.3 mM / CV. Elution peaks for NOX-b, NOX-c, and NOX-f appeared at NaCl concentrations of 290 mM, 220 mM, and 400 mM, respectively.

[0203] The eluted fractions were concentrated using an Amicon Ultra Ultracel-10K column. The purity of each eluted fraction was evaluated by SDS-PAGE, and fractions free of contaminating proteins were collected and used as a purified NOX preparation. As an example, Figure 2 shows the results of SDS-PAGE analysis of the purity of purified NOX-b. The molecular weight of NOX-b was approximately 54 kDa, which was at the same level as the molecular weight (approximately 55 kDa) predicted from its amino acid sequence.

[0204] 5. Measurement of NADH oxidation activity Flavin adenine dinucleotide (FAD, manufactured by Tokyo Chemical Industry Co., Ltd.) was used as a reagent for measuring NADH oxidation activity. The NOX solution was diluted with 10 mM PPB (pH 7.5) containing 0.15% bovine serum albumin (BSA, manufactured by Sigma-Aldrich).

[0205] 675 μl of the reagent in Table 3 was incubated at 37°C for 2 minutes, and then 75 μl of 2 mM β-nicotinamide adenine dinucleotide (reduced form, β-NADH, manufactured by Oriental Yeast Co., Ltd.) solution was added and mixed. The absorbance (A) of light at a wavelength of 340 nm was measured using a spectrophotometer U-3900 (manufactured by Hitachi High-Tech Science) with the cell holder kept at 37°C. ３４０ ) was measured and the A per minute ３４０ The absolute value of the decrease (ΔA Ｓ Measurements were also carried out by adding 75 μl of ion-exchanged water instead of the substrate solution (β-NADH solution), and the A per minute was calculated. ５５５ The absolute value of the decrease (ΔA ０ ) was calculated.

[0206]

[0207] NADH oxidation activity (U / ml) was calculated based on the following formula: 1 U is defined as the amount of enzyme required to oxidize 1 μmol of NADH in 1 minute at 37°C and pH 7.5. The "6.22" in the formula is the millimolar absorption coefficient of NADH for light with a wavelength of 340 nm (mM －１ cm －１ ) where df represents the dilution rate of the enzyme solution. [Formula] U / ml = (ΔA Ｓ -ΔA ０ )×750×df / (6.22×V) =120.6×(ΔA Ｓ -ΔA ０ ) × df / V

[0208] 6. Measurement of NADPH oxidation activity Measurement of NADPH oxidation activity was carried out in the same manner as in "5. Measurement of NADH oxidation activity," except that 75 μl of 2 mM β-nicotinamide adenine dinucleotide phosphate (reduced form, β-NADPH, manufactured by Oriental Yeast Co., Ltd.) solution was used as the substrate instead of 75 μl of 2 mM β-nicotinamide adenine dinucleotide (reduced form, β-NADH) solution.

[0209] The NADPH oxidation activity (U / ml) was calculated based on the following formula: 1 U is defined as the amount of enzyme required to oxidize 1 μmol of NADPH in 1 minute at 37°C and pH 7.5. The "6.22" in the formula is the millimolar absorption coefficient (mM －１ cm －１ ) where df represents the dilution rate of the enzyme solution. [Formula] U / ml = (ΔA Ｓ -ΔA ０ )×750×df / (6.22×V) =120.6×(ΔA Ｓ -ΔA ０ ) × df / V

[0210] 7. Evaluation of NADPH / NADH Activity Ratio The NADPH / NADH activity ratio was calculated from the NADPH oxidation activity ratio when the NADH oxidation activity of each NOX was set to 1.

[0211] The NADPH / NADH activity ratios of the purified enzyme solutions of NOX-b, NOX-c, and NOX-f are as shown in the table below, and the substrate specificity of each enzyme was characterized by lower reactivity to NADPH compared to reactivity to NADH.

[0212]

[0213] 8. Evaluation of NOX Thermal Stability The crude NOX enzyme solution or purified NOX solution was diluted with 10 mM PPB pH 7.5 to a final NOX concentration of 0.17 U / ml. Subsequently, 240 μl of the 0.17 U / ml NOX solution was mixed with 160 μl of 250 mM PPB pH 7.5 and heated for 15 minutes in a water bath maintained at 50°C or 75°C. After heating, the NOX solution was quickly cooled on ice, and activity was measured using 375 μl of the NOX solution. The residual activity of the sample after heating was calculated by setting the activity of the sample cooled on ice without heating as 1.

[0214]

[0215] After heating at 50°C for 15 minutes, the residual activity of each NOX varied from 4% to 81%.

[0216] 9. Evaluation of Optimal pH for NOX Catalytic Reaction When measuring the optimal pH for NOX catalytic reaction, the PPB pH 7.5 contained in the activity measurement reagent in Table 3 was replaced with citrate buffer (pH 5.0 or 5.5), MES buffer (pH 5.0, 5.5, 6.0, 6.5, or 7.0), PPB (pH 6.5, 7.0, 7.5, or 8.0), Tris buffer (pH 7.5, 8.0, 8.5, or 9.0), or CHES buffer (pH 9.0, 9.5, or 10.0), and the activity was measured.

[0217] 3, 4, and 5 show the pH dependence of the NADH oxidation activity of NOX-b, NOX-c, and NOX-f, respectively. The vertical axis represents relative activity (%), and the horizontal axis represents pH. The relative activity is the relative NADH oxidation activity when the NADH oxidation activity under buffer conditions at which the activity was highest was taken as 100%. The optimal pH for NOX-b, NOX-c, and NOX-f, i.e., the pH at which the relative activity was 80% or higher, was pH 5.0 to 7.5, pH 7.0 to 8.0, and pH 5.0 to 6.0, respectively.

[0218] 10. Evaluation of Stable pH of NOX To measure the stable pH of the NOX catalytic reaction, the NOX crude enzyme solution was diluted with 10 mM PPB pH 7.5 to a final NOX concentration of 2.4 U / ml, and then the pH of the enzyme solution was adjusted to the composition shown in Table 6. The "Buffer" in Table 6 was citrate buffer (pH 3.0, 3.5, 4.0, 4.5, 5.0, or 5.5), MES buffer (pH 5.0, 5.5, 6.0, or 6.5), PPB (pH 6.0, 6.5, 7.0, 7.5, or 8.0), Tris buffer (pH 7.5, 8.0, 8.5, or 9.0), CHES buffer (pH 9.0, 9.5, or 10.0), or CAPS buffer (pH 10.0, 10.5, or 11.0).

[0219]

[0220] The NOX solutions prepared at each pH were heated for 60 minutes in a water bath maintained at 40°C. After heating, the NOX solutions were quickly cooled on ice, and 375 μl of the NOX solutions were used to measure NOX activity according to the composition shown in Table 3. The relative activity was calculated from the relative oxidase activity when the highest oxidase activity was measured under buffer conditions, which was set at 100%.

[0221] 6, 7, and 8 are graphs showing the stable pH of NOX-b, NOX-c, and NOX-f, respectively. The vertical axis represents the relative activity of NOX (%), and the horizontal axis represents pH. The stable pH of NOX-b, NOX-c, and NOX-f, i.e., the pH at which the relative activity is 80% or higher, was pH 6.0 to 8.0, pH 5.0 to 10.0, and pH 7.5 to 9.5, respectively.

[0222] As shown in Figure 2, the molecular weight of purified NOX-b was approximately 54 kDa, which was comparable to the molecular weight (approximately 55 kDa) predicted from its amino acid sequence. Subsequently, while investigating the mutants, the inventors analyzed cell extracts containing various NOXs, which had been prepared and stored refrigerated, by SDS-PAGE (Figure 9). The molecular weights of NOX-a, NOX-b, NOX-c, NOX-d, NOX-e, NOX-f, NOX-g, NOX-h, and NOX-i were approximately 51 kDa, 45 kDa, 51 kDa, 53 kDa, 52 kDa, 52 kDa, 51 kDa, 51 kDa, and 53 kDa, respectively, which, except for NOX-b, were comparable to the molecular weight (approximately 55 kDa) predicted from their amino acid sequences. However, we found by chance that the molecular weight of only NOX-b on SDS-PAGE was approximately 10 kDa smaller than the predicted molecular weight (approximately 55 kDa) based on the amino acid sequence. Furthermore, the NOX-b enzyme preparation shown in Figure 9 retained its activity. This result indicates that NOX-b retains its enzymatic activity as NOX even when a partial region of its polypeptide sequence is deleted. This was completely unexpected and a great surprise. To the inventors' knowledge, there have been no reports to date of the ability to retain enzymatic activity even when approximately 10 kDa is deleted from the NOX sequence. Following this result, we conducted extensive research and created the N-terminal deletion mutants of each NOX shown below.

[0223] When cell extracts containing NOX-j, NOX-k, and NOX-1 were analyzed by SDS-PAGE, the molecular weights of NOX-j, NOX-k, and NOX-1 were found to be approximately 52 kDa, 52 kDa, and 53 kDa, respectively.

[0224] 11. Construction of a Plasmid for Expression of a NOX Deletion Mutant. Site-specific deletion was performed using pET22b-NOX-a as a template to obtain a plasmid carrying a gene encoding the NOX-a deletion mutant (NOX-aΔ197, SEQ ID NO: 19). The PCR reaction mixture was prepared by mixing 10 μl of KOD one PCR Master Mix (Toyobo), 3 μl of 2 μM Fw primer, 3 μl of 2 μM Rv primer, 0.5 μl of 40 μg / ml template DNA, and 3.5 μl of ion-exchanged water. The PCR reaction conditions were a cycle of 98°C for 10 seconds, 53°C for 5 seconds, and 68°C for 60 seconds, repeated 34 times.

[0225] 1 μl of DpnI was added to the post-PCR solution and treated at 37 ° C for 1 hour to degrade the template DNA. 2 μl of the resulting DpnI-treated solution was mixed with 5 μl of Ligation High Ver. 2 (Toyobo), 1 μl of 5 U / μl T4 polynucleotide kinase, and 7 μl of ion-exchanged water, and the mixture was reacted at 16 ° C for 1 hour. The reaction solution was then used to transform E. coli JM109 strain. The base sequence of the plasmid extracted from the resulting transformant was confirmed to be the desired sequence by DNA sequence analysis.

[0226] By carrying out the same procedure as above, NOX-b deletion mutant (NOX-bΔ197, SEQ ID NO: 21), NOX-c deletion mutant (NOX-cΔ197, SEQ ID NO: 23), NOX-d deletion mutant (NOX-dΔ197, SEQ ID NO: 25), NOX-e deletion mutant (NOX-eΔ197, SEQ ID NO: 27), NOX-f deletion mutant (NOX-fΔ197, SEQ ID NO: 29), NOX-g deletion mutant (NOX-gΔ197, SEQ ID NO: 31), NOX-h deletion mutant (NOX-hΔ 197, SEQ ID NO: 33), NOX-i deletion mutant (NOX-iΔ197, SEQ ID NO: 35), various NOX-j deletion mutants, specifically, NOX-jΔ197-4 (SEQ ID NO: 80), NOX-jΔ197-5 (SEQ ID NO: 82), NOX-jΔ197-7 (SEQ ID NO: 84), NOX-jΔ197-8 (SEQ ID NO: 86), NOX-jΔ197-9 (SEQ ID NO: 88), NOX-jΔ197-10 (SEQ ID NO: 90), NOX-jΔ197-11 (SEQ ID NO: 92), NOX-k deletion mutants, specifically NOX-kΔ197-4 (SEQ ID NO: 94), NOX-kΔ197-5 (SEQ ID NO: 96), NOX-kΔ197-6 (SEQ ID NO: 98), NOX-kΔ197-8 (SEQ ID NO: 100), NOX-kΔ197-9 (SEQ ID NO: 102), NOX-kΔ197-10 (SEQ ID NO: 104), NOX-kΔ197-11 (SEQ ID NO: 106), various NOX-l deletion mutants, specifically NOX-lΔ197-j4 (SEQ ID NO: 108), Plasmids carrying genes encoding NOX-lΔ197-j9 (SEQ ID NO: 110), NOX-lΔ197-j11 (SEQ ID NO: 112), NOX-lΔ197-k4 (SEQ ID NO: 114), NOX-lΔ197-k5 (SEQ ID NO: 116), NOX-lΔ197-k8 (SEQ ID NO: 118), NOX-lΔ197-k9 (SEQ ID NO: 120), NOX-lΔ197-k10 (SEQ ID NO: 122), and NOX-lΔ197-k11 (SEQ ID NO: 124) were obtained. The Fw primer, Rv primer, and template DNA used for PCR are shown in Tables 7 and 8.

[0227]

[0228] Production strains for each NOX deletion mutant were prepared according to the same method as in "2. Preparation of NOX-producing strains." Production strains for the NOX-a, b, c, d, e, f, g, h, and i deletion mutants were prepared by transforming E. coli BL21(DE3). Production strains for the NOX-j, k, and l deletion mutants were prepared by transforming E. coli JM109.

[0229] A crude enzyme solution of each NOX deletion mutant was prepared according to the method described in "3. Recombinant production of NOX" and used for various evaluations.

[0230] Analysis of cell extracts containing various NOXs by SDS-PAGE revealed that the molecular weights of NOX-a, NOX-b, NOX-c, NOX-d, NOX-e, NOX-f, NOX-g, NOX-h, and NOX-i were approximately 51 kDa, 45 kDa, 51 kDa, 53 kDa, 52 kDa, 52 kDa, 51 kDa, 51 kDa, and 53 kDa, respectively, which, except for NOX-b, were at the same level as the molecular weight (approximately 55 kDa) predicted from their amino acid sequences.

[0231] 12. Evaluation of heat resistance of each NOX deletion mutant The heat resistance of each NOX deletion mutant was evaluated using the same method as in "8. Evaluation of NOX thermal stability." The remaining activity (%) of the crude solution of each NOX deletion mutant after heating at 50°C for 15 minutes is shown in the table below.

[0232]

[0233] The residual activity of NOX-a, d, e, g, h, and i increased by 1% to 48% by deleting positions 2-198, and the deletions improved thermostability. In other words, the residual activity increased by 3% to 358% compared to the wild type.

[0234] The residual activity of NOX-j, -k, -l, and their deletion mutants was evaluated for heat resistance of each NOX deletion mutant using the same method as in "8. Evaluation of NOX thermal stability." The residual activity (%) of the crudely purified solution of each NOX deletion mutant after heating at 75°C for 15 minutes is shown in the table below.

[0235]

[0236] The residual activity of NOX-j increased by 3% to 16% by deleting the N-terminal 197 amino acids, and the deletions improved its thermostability. In other words, the residual activity increased by 4% to 24% compared to the wild-type.

[0237] The residual activity of NOX-k was increased by 21% to 36% by deleting the N-terminal 197 amino acids, and the deletion improved thermostability, i.e., the residual activity was increased by 48% to 82% compared to the wild-type.

[0238] The residual activity of NOX-1 was increased by 11% to 24% by deleting 197 amino acids from the N-terminus and then substituting the N-terminus, and the thermostability was improved by the deletions. In other words, the residual activity was improved by 22% to 47% compared to the wild-type.

[0239] Each NOX deletion mutant was purified according to the method described in "4. Purification of NOX" and used for various evaluations. Elution peaks of NOX-bΔ197, NOX-cΔ197, and NOX-fΔ197 appeared at NaCl concentrations of 290 mM, 200 mM, and 270 mM, respectively.

[0240] 13. Evaluation of Optimal pH for Catalytic Reaction of Each NOX Deletion Isolate The optimal pH for the catalytic reaction of each NOX deletion Isolate was evaluated using the same method as in "9. Evaluation of Optimal pH for Catalytic Reaction of NOX." Figures 10, 11, and 12 show the pH dependence of the NADH oxidation activity of NOX-bΔ197, NOX-cΔ197, and NOX-fΔ197, respectively. The optimal pH for NOX-bΔ197, NOX-cΔ197, and NOX-fΔ197, i.e., the pH at which the relative activity was 80% or higher, was pH 5.0-7.0, pH 5.0-8.0, and pH 5.0-6.0, respectively.

[0241] 14. Evaluation of the Stable pH of Each NOX Deletion Isolate The stable pH of each NOX deletion Isolate was evaluated using the same method as in "10. Evaluation of the Stable pH of NOX." Figures 13, 14, and 15 show the stable pH of NOX-bΔ197, NOX-cΔ197, and NOX-fΔ197, respectively. The stable pH of NOX-bΔ197, NOX-cΔ197, and NOX-fΔ197, i.e., the pH at which the relative activity was 80% or higher, was pH 6.0 to 8.0, pH 5.5 to 10.0, and pH 6.5 to 9.5, respectively.

[0242] 15. Construction of Modified NOX-cΔ197 Site-directed mutagenesis was performed using the NOX-cΔ197 expression plasmid (pET22b-NOX-cΔ197) as a template to obtain a plasmid carrying the gene encoding modified NOX-cΔ197. The PCR reaction mixture was prepared by mixing 10 μl of KOD one PCR Master Mix (Toyobo), 3 μl of 2 μM Fw primer, 3 μl of 2 μM Rv primer, 0.5 μl of 40 μg / ml template DNA (pET22b-NOX-cΔ197), and 3.5 μl of ion-exchanged water. The names of the mutants and the combinations of Fw and Rv primers used are shown in Table 11. The PCR reaction conditions were 15 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 68°C for 35 seconds. An expression plasmid for the multiple mutant NOX-cΔ197 was constructed by repeated single mutagenesis. For example, an expression plasmid for the double mutant NOX-cΔ197 / F378E / F503Y was constructed by PCR using pET22b-NOX-cΔ197 / F378E as a template and primers represented by SEQ ID NOs: 49 and 50.

[0243]

[0244] The strains producing each modified NOX-cΔ197 were prepared according to the same method as in "2. Preparation of NOX-producing strains."

[0245] Crude enzyme solutions of each modified NOX-cΔ197 were prepared according to the method described in "3. Recombinant production of NOX" and used for various evaluations.

[0246] 16. Evaluation of thermostability of modified NOX-cΔ197 PCR of the mutants prepared based on Table 11, recombinant production of NOX, activity measurement, and evaluation of thermostability were all performed in the same manner as for NOX described above. The table below shows the residual activity of modified NOX-Δ197 after heating at 50°C for 15 minutes. When experiments were performed multiple times, the average value is shown.

[0247]

[0248] The residual activity of the modified NOX-cΔ197 shown in Table 12 above was increased by 5% to 18% compared to NOX-cΔ197 without any additional modifications. In other words, the modified NOX-cΔ197 had improved thermal stability compared to NOX-cΔ197 without any additional modifications.

[0249] 17. Evaluation of substrate specificity (NADPH / NADH (%)) of modified NOX-cΔ197. PCR of the mutants prepared based on Table 11, recombinant production of NOX, activity measurement, and calculation of the NADPH / NADH activity ratio were all performed in the same manner as for NOX above. When experiments were performed multiple times, the average values ​​are shown.

[0250]

[0251] The NADPH / NADH (%) ratio, i.e., substrate specificity, of the modified NOX-cΔ197 shown in Table 13 above was improved by 0.7% to 4.5% compared to NOX-cΔ197 without any additional modifications. These results indicate a decrease in the degree of reactivity to NADPH relative to reactivity to NADH, and all modified NOX-cΔ197 had improved substrate specificity compared to NOX-cΔ197 without any additional modifications. The present inventors found that deletion mutants of the NADH oxidases of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, and 17 retained NADH oxidase activity even when the N-terminus was deleted. Furthermore, the present inventors found that deletion mutants of the NADH oxidases of SEQ ID NOS: 74, 76, and 78, which are derived from different sources from these NADH oxidases, retained NADH oxidase activity even when the N-terminus was deleted. Furthermore, the present inventors have found that the introduction of the amino acid substitutions of the present disclosure improves the thermostability and / or substrate specificity of the NADH oxidase of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17. Even when a large mutation, such as the deletion of 197 amino acids from the N-terminus, is introduced, the effects demonstrated for SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 are also confirmed for SEQ ID NOs: 74, 76, and 78. Therefore, those skilled in the art will understand that the introduction of small mutations, such as the amino acid substitutions of the present disclosure, into the NADH oxidase of SEQ ID NO: 74, 76, and 78 is likely to produce the same effects as the NADH oxidase of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17.

[0252] 18. Investigation of NOX Freeze-Drying Conditions Freeze-drying of purified NOX solution was carried out using the composition shown in Table 14. In the control test without adding any stabilizer, freeze-drying was carried out without adding any stabilizer.

[0253]

[0254] (Method for measuring NOX activity and stability test) The stability test of the NOX-b lyophilized preparation was performed by comparing the residual activity after 2 weeks of storage at 37° C. The residual activity of the enzyme solution after 2 weeks of storage at 37° C. was expressed as a relative amount (%) when the change in absorbance before storage was set at 100%.

[0255] (Effect of the Coexistence of the Sugar Trehalose) 20 mM Tris-HCl buffer (pH 7.5) containing trehalose at concentrations of 13, 26, and 53 mM was prepared as a stabilizer and freeze-dried. A storage test was performed on this freeze-dried powder at 37°C for 2 weeks, and the enzyme activity before and after the storage test was compared to evaluate the effect of stabilizing sugar alcohols. The results are shown in Table 15. The residual activity was 100% when trehalose was added at 13 mM or more. This indicates that the storage stability of the NOX freeze-dried preparation was significantly improved by adding trehalose at a concentration of 13 mM or more.

[0256]

[0257] (Effect of Coexistence of Sodium L-Glutamate) 10 mM Tris-HCl buffer (pH 7.5) containing 27, 53, and 107 M sodium L-glutamate was prepared and lyophilized. A storage test was performed on this lyophilized powder at 37°C for 2 weeks, and the enzyme activity before and after storage was compared to evaluate the effect of stabilizing amino acids. The results, as shown in Table 16, demonstrated that 100% of the enzyme activity was maintained compared to the enzyme activity before storage. In other words, the storage stability of the NOX lyophilized formulation was significantly improved by adding sodium L-glutamate and lyophilizing it.

[0258]

[0259] The NADH oxidase of the present disclosure can be used in NADH measurement and NADH sensors.

[0260] Numerous documents, including patent applications and manufacturer's manuals, are cited herein. The disclosures of these documents, while not considered relevant to the patentability of this invention, are hereby incorporated by reference in their entirety. More particularly, all referenced documents are hereby incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

[0261]

Claims

1. The following properties: (i) Action: Oxidizes NADH to NAD ＋ (ii) thermostability: having 65% or more of the residual activity after heat treatment at 75°C for 15 minutes; (iii) having a flavin compound as a prosthetic group; and (iv) molecular weight: having a molecular weight of about 52 kDa when the lysate is measured by SDS-polyacrylamide electrophoresis.

2. An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 74, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO: 74 (excluding those having 100% amino acid sequence identity with SEQ ID NO: 74, those having 100% amino acid sequence identity with SEQ ID NO: 76, and those having 100% amino acid sequence identity with SEQ ID NO: 78).

3. The following properties: (i) Action: Oxidizes NADH to NAD ＋ (ii) thermostability: having 40% or more residual activity after heat treatment at 75°C for 15 minutes; (iii) having a flavin compound as a prosthetic group; and (iv) molecular weight: having a molecular weight of about 52 kDa when the lysate is measured by SDS-polyacrylamide electrophoresis.

4. An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 76, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO: 76 (excluding those having 100% amino acid sequence identity with SEQ ID NO: 74, those having 100% amino acid sequence identity with SEQ ID NO: 76, and those having 100% amino acid sequence identity with SEQ ID NO: 78).

5. The following properties: (i) Action: Oxidizes NADH to NAD ＋ (ii) thermostability: having 50% or more of the residual activity after heat treatment at 75°C for 15 minutes; (iii) having a flavin compound as a prosthetic group; and (iv) molecular weight: having a molecular weight of about 53 kDa when the lysate is measured by SDS-polyacrylamide electrophoresis.

6. An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 78, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO: 78 (excluding those having 100% amino acid sequence identity with SEQ ID NO: 74, those having 100% amino acid sequence identity with SEQ ID NO: 76, and those having 100% amino acid sequence identity with SEQ ID NO: 78).

7. The following properties (i) to (vii): (i) Action: Oxidizes NADH to NAD ＋ (ii) substrate specificity: lower reactivity to NADPH compared to reactivity to NADH; (iii) thermostability: retaining 60% or more of the activity after heat treatment at 50°C for 15 minutes; (iv) having a flavin compound as a prosthetic group; (v) optimum pH: 5.0 to 7.5; (vi) stable pH: 6.0 to 8.0; and (vii) molecular weight: the molecular weight is about 54 kDa as measured by SDS-polyacrylamide electrophoresis.

8. An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 3, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO:

3.

9. The following properties (i) to (vii): (i) Action: Oxidizes NADH to NAD ＋ (ii) substrate specificity: lower reactivity to NADPH compared to reactivity to NADH; (iii) thermostability: retaining 80% or more of the activity after heat treatment at 50°C for 15 minutes; (iv) having a flavin compound as a prosthetic group; (v) optimum pH: 7.0 to 8.0; (vi) stable pH: 5.0 to 10.0; and (vii) molecular weight: the molecular weight is about 51 kDa as measured by SDS-polyacrylamide electrophoresis.

10. An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 5, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO:

5.

11. The following properties (i) to (vii): (i) Function: Oxidizes NADH to NAD ＋ (ii) substrate specificity: lower reactivity to NADPH compared to reactivity to NADH; (iii) thermostability: retaining 30% or more of the activity after heat treatment at 50°C for 15 minutes; (iv) having a flavin compound as a prosthetic group; (v) optimum pH: 5.0 to 6.0; (vi) stable pH: 7.5 to 9.5; and (vii) molecular weight: the molecular weight is about 52 kDa as measured by SDS-polyacrylamide electrophoresis.

12. An NADH oxidase having an amino acid sequence identity of 90% or more with SEQ ID NO: 11, or an NADH oxidase in which the amino acid sequences of the homologous regions of the FAD domain and NADH domain of the NADH oxidase have an amino acid sequence identity of 90% or more with the homologous regions of the FAD domain and NADH domain of SEQ ID NO: 1, and the full-length amino acid sequence of the NADH oxidase has an amino acid sequence identity of 70% or more with SEQ ID NO:

11.

13. An N-terminal domain deletion mutant of NADH oxidase, in which all or part of the N-terminal domain is deleted and which retains NADH oxidase activity.

14. An N-terminal domain deletion mutant of NADH oxidase, which has a deletion of a sequence consisting of 197 consecutive amino acids on the N-terminal side and retains NADH oxidase activity.

15. The sequence consisting of 197 consecutive amino acids on the N-terminal side is: positions 4 to 200 of SEQ ID NO:74, positions 5 to 201 of SEQ ID NO:74, positions 6 to 202 of SEQ ID NO:74, positions 7 to 203 of SEQ ID NO:74, positions 8 to 204 of SEQ ID NO:74, positions 9 to 205 of SEQ ID NO:74, positions 10 to 206 of SEQ ID NO:74, and positions 11 to 207 of SEQ ID NO:74; positions 4 to 200 of SEQ ID NO:76, positions 5 to 201 of SEQ ID NO:76, positions 6 to 202 of SEQ ID NO:76, positions 7 to 203 of SEQ ID NO:76, positions 8 to 204 of SEQ ID NO:76, positions 9 to 205 of SEQ ID NO:76, positions 10 to 206 of SEQ ID NO:76, and positions 11 to 207 of SEQ ID NO:76; 15. The N-terminal domain deletion mutant of NADH oxidase according to claim 13 or 14, wherein the mutant has a sequence corresponding to a position selected from the group consisting of positions 4 to 200 of SEQ ID NO:78, positions 5 to 201 of SEQ ID NO:78, positions 6 to 202 of SEQ ID NO:78, positions 7 to 203 of SEQ ID NO:78, positions 8 to 204 of SEQ ID NO:78, positions 9 to 205 of SEQ ID NO:78, positions 10 to 206 of SEQ ID NO:78, and positions 11 to 207 of SEQ ID NO:

78.

16. An N-terminal domain deletion mutant of NADH oxidase according to claim 13 or 14, which lacks the N-terminal domain corresponding to positions 2 to 198 of SEQ ID NO: 1 and has NADH oxidase activity.

17. The N-terminal domain deletion mutant of NADH oxidase according to any one of claims 13 to 16, wherein the NADH oxidase is derived from the genus Bacillus, Priestia, Achydianus, Archaeoglobus, Brevibacterium, Methanocaldococcus, Pseudothermotoga, Pyrococcus, Saccharolobus, Streptococcus, Sulfolobus, Thermotoga, or Geobacillus.

18. The N-terminal domain-deleted mutant of NADH oxidase according to any one of claims 13 to 17, wherein the amino acid sequence before deletion of the N-terminal domain has 90% or more amino acid sequence identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78; or the amino acid sequence after deletion of the N-terminal domain has 90% or more amino acid sequence identity with SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124.

19. An N-terminal domain deletion mutant of NADH oxidase, in which the N-terminal domain of NADH oxidase is deleted and which has the FAD domain and NADH domain of NADH oxidase, wherein the amino acid sequences of the FAD domain and NADH domain of NADH oxidase have 90% or more amino acid sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, or have 90% or more amino acid sequence identity to SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124.

20. An NADH oxidase variant having amino acid substitutions at positions corresponding to positions 503, 203, 246, 283, 284, 329, 378, 383, 467, and / or 494 of SEQ ID NO:

1.

21. The amino acid substituted at position 503 of SEQ ID NO: 1 is tyrosine (Y); The amino acid substituted at position 203 of SEQ ID NO: 1 is leucine (L); The amino acid substituted at position 246 of SEQ ID NO: 1 is arginine (R), lysine (K), or threonine (T); The amino acid substituted at position 283 of SEQ ID NO: 1 is glutamic acid (E), and / or The amino acid substituted at position 284 of SEQ ID NO: 1 is asparagine (N); The amino acid substituted at position 329 of SEQ ID NO: 1 is glutamic acid (E); The amino acid substituted at position 378 of SEQ ID NO: 1 is leucine (L), glutamic acid (E), or aspartic acid (D); The amino acid substituted at position 383 of SEQ ID NO: 1 is aspartic acid (D), asparagine (N), or glutamic acid (E).

21. The NADH oxidase mutant of claim 20, wherein the amino acid after substitution at the position corresponding to position 467 of SEQ ID NO: 1 is glutamine (Q), and / or the amino acid after substitution at the position corresponding to position 494 of SEQ ID NO: 1 is glutamine (Q) or glutamic acid (E).

22. The NADH oxidase before amino acid substitution is an NADH oxidase having 90% or more amino acid sequence identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, or an NADH oxidase that lacks the N-terminal domain of NADH oxidase and has the FAD domain and NADH domain of NADH oxidase, wherein the amino acid sequences of the FAD domain and the NADH domain of NADH oxidase have 90% or more amino acid sequence identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 74, 76, or 78, or have 90% or more amino acid sequence identity with SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124; 22. The NADH oxidase mutant according to claim 20 or 21, which is an N-terminal domain deletion mutant of NADH oxidase.

23. A freeze-dried preparation comprising NADH oxidase and a stabilizer, wherein the stabilizer is selected from the group consisting of trehalose, raffinose, melezitose, maltotriose, sucrose, lactose, maltose, turanose, cellobiose, ribose, lyxose, xylose, arabinose, glucose, altrose, mannose, galactose, idose, allose, talose, gulose, psicose, fructose, sorbose, tagatose, deoxyribose, fucose, fuculose, rhamnose, ribulose, and xylulose, glycogen, starch, cellulose, dextrin, glucan, fructan, chitin, chitosan, glucomannan, and penicillin. a polysaccharide selected from the group consisting of cutin, alginic acid, hyaluronic acid, chondroitin sulfate, and heparin; a sugar alcohol selected from the group consisting of erythritol, threitol, arabinitol, xylitol, ribitol, iditol, galactitol, sorbitol, and mannitol; an amino acid or a salt thereof selected from the group consisting of glutamic acid, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, asparagine, glutamine, aspartic acid, histidine, lysine, and arginine; or a phosphoric acid, a carboxylic acid, or a salt thereof.

24. The freeze-dried preparation according to claim 23, wherein the NADH oxidase is the NADH oxidase according to any one of claims 1 to 22.

25. A kit for measuring NADH, comprising the NADH oxidase according to any one of claims 1 to 22.

26. An electrode comprising the NADH oxidase of any one of claims 1 to 22.

27. A sensor comprising the electrode of claim 26.

28. A method for measuring NADH in a sample, comprising oxidizing NADH in the sample using the NADH oxidase according to any one of claims 1 to 22, and measuring the hydrogen peroxide produced or the oxygen consumed.

29. A method for measuring NADH in a sample, comprising the step of contacting the electrode of claim 26 with the sample.

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