NADH oxidase mutant with improved enzyme activity

Abstract

The invention provides an NADH oxidase mutant with improved enzyme activity. Compared with the amino acid sequence as shown in SEQ ID NO: 1, the NADH oxidase has the advantages that the amino acid sequence is more stable; the amino acid sequence has substitution mutation at any one position, any two positions, any three positions, any four positions, any five positions, any more than six positions, any more than seven positions or all eight positions of the 120th position, the 152nd position, the 157th position, the 158th position, the 176th position, the 181th position, the 183th position and the 239th position.

Description

Technical Field

[0001] This invention belongs to the field of catalysis, and more specifically, this invention relates to a mutant of NADH oxidase with enhanced enzyme activity. Background Technology

[0002] Industrial biocatalysis has emerged as a promising new direction in the field of biotechnology, with enzymes playing a crucial role as important catalysts. Current data indicates that only a small percentage of discovered enzymes are used in industry, with oxidoreductases accounting for the largest proportion and possessing significant application value in industrial catalysis. It is known that most oxidoreductase catalysis processes require coenzymes (NAD(P)+ / NAD(P)H) to participate in electron transfer and promote substrate conversion. However, coenzymes are expensive, unstable in solution, and have low reusability. The large-scale addition of coenzymes significantly increases the cost of industrial catalysis, severely limiting the further development of oxidoreductases. Therefore, it is necessary to establish efficient, environmentally friendly, and low-cost coenzyme regeneration systems to solve the problems of high cost and low utilization rate of coenzymes in industrial applications.

[0003] NADH oxidase, also known as NADH oxidase complex, NADH:coenzyme Q reductase, or complex I (EC 1.6.5.3), is an enzyme located on the inner mitochondrial membrane that catalyzes the transfer of electrons from NADH to coenzyme Q. This enzyme is the "entry enzyme" for oxidative phosphorylation in mitochondria. It can directly oxidize NADH to NAD+ in the presence of oxygen, as shown in the following reaction process:

[0004]

[0005] NADH oxidases are widely found in various organisms, including microorganisms, plants, and animals. However, the NADH oxidases with promising industrial applications mainly originate from microorganisms. With the increasing industrial application of oxidoreductases, the catalytic efficiency of NADH dehydrogenases has become a research hotspot. NOX, due to its advantages of not requiring the addition of oxygen as a reactant, its simple product structure, and its lack of impact on the separation and purification of the main product, has shown great application potential in the regeneration of the oxidized coenzyme NAD+.

[0006] However, wild-type NADH oxidase has low enzyme activity in enzyme-catalyzed reactions, which limits its application. Summary of the Invention

[0007] The purpose of this invention is to provide a mutant NADH oxidase with enhanced enzyme activity. The NADH oxidase of this invention, compared with the amino acid sequence shown in SEQ ID NO:1, has substitution mutations at any one, two, three, four, five, six or more, seven or more, or all eight positions among positions 120, 152, 157, 158, 176, 181, 183, and 239.

[0008] In a first aspect of the invention, an isolated NADH oxidase is provided, said NADH oxidase:

[0009] (a) Compared with the amino acid sequence shown in SEQ ID NO:1, its amino acid sequence has substitution mutations at any one, two, three, four, five, six or more, seven or more, or all eight positions among positions 120, 152, 157, 158, 176, 181, 183, and 239; or

[0010] (b) A protein derived from (a) having N-deoxyribotransferase II function, formed by substituting, deleting, or adding one or more amino acid residues of the amino acid sequence of (a), wherein the protein corresponds to the amino acid residues at positions 120, 152, 157, 158, 176, 181, 183, and 239 of SEQ ID NO:1 as described in (a); or

[0011] (c) A protein derived from (a) having more than 80% sequence identity with the amino acid sequence of (a) and having N-deoxyribotransferase II function, wherein the amino acid residues corresponding to positions 120, 152, 157, 158, 176, 181, 183 and 239 of SEQ ID NO:1 as described in (a).

[0012] In one or more embodiments, the mutation at position 120 is a mutation of isoleucine to a nonpolar amino acid selected from valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or glycine.

[0013] In one or more embodiments, the mutation at position 152 is a mutation of valine to an uncharged polar amino acid selected from serine, threonine, glutamine, asparagine, tyrosine, or cysteine.

[0014] In one or more embodiments, the mutation at position 157 is a tyrosine mutation to a nonpolar amino acid selected from glycine, valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or isoleucine.

[0015] In one or more embodiments, the mutation at position 158 is a mutation of isoleucine to an uncharged polar amino acid selected from serine, threonine, cysteine, tyrosine, asparagine, or glutamine.

[0016] In one or more embodiments, the mutation at position 176 is a mutation of isoleucine to an uncharged polar amino acid selected from serine, threonine, cysteine, tyrosine, asparagine, or glutamine.

[0017] In one or more embodiments, the mutation at position 181 is an arginine mutation to an uncharged polar amino acid selected from serine, threonine, cysteine, tyrosine, asparagine, or glutamine.

[0018] In one or more embodiments, the mutation at position 183 is a mutation of methionine to a polar, positively charged amino acid selected from lysine, arginine, or histidine.

[0019] In one or more embodiments, the mutation at position 239 is a cysteine ​​mutation to a nonpolar amino acid selected from glycine, valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or isoleucine.

[0020] In one or more embodiments, the mutation at position 120 is a mutation of isoleucine to valine, leucine, alanine, or glycine; the mutation at position 152 is a mutation of valine to serine or threonine; the mutation at position 157 is a mutation of tyrosine to glycine, alanine, valine, leucine, or isoleucine; the mutation at position 158 is a mutation of isoleucine to glutamine or asparagine; the mutation at position 176 is a mutation of isoleucine to cysteine, asparagine, or glutamine; the mutation at position 181 is a mutation of arginine to threonine, serine, or cysteine; the mutation at position 183 is a mutation of methionine to lysine or arginine; and the mutation at position 239 is a mutation of cysteine ​​to glycine, alanine, valine, leucine, or isoleucine.

[0021] In one or more embodiments, the mutation at position 120 is a mutation of isoleucine to leucine or valine; the mutation at position 152 is a mutation of valine to serine; the mutation at position 157 is a mutation of tyrosine to glycine or alanine; the mutation at position 158 is a mutation of isoleucine to glutamine; the mutation at position 176 is a mutation of isoleucine to cysteine; the mutation at position 181 is a mutation of arginine to threonine; the mutation at position 183 is a mutation of methionine to arginine; and the mutation at position 239 is a mutation of cysteine ​​to glycine or alanine.

[0022] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the NADH oxidase has a substitution mutation at least at position 120 and / or at position 152, and has substitution mutations at any one, any two, any three, any four, or all of positions 157, 158, 176, 181, 183, and 239.

[0023] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the NADH oxidase has a substitution mutation at at least one or all of positions 120 and 152, a substitution mutation at one or all of positions 157, 158, and 176, and the corresponding substitution mutation at one or all of positions 181, 183, and 239.

[0024] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the NADH oxidase has substitution mutations at least at positions 120, 152, and 158, and the corresponding substitution mutations at any one, any two, any three, any four, or all of positions 157, 176, 181, 183, and 239.

[0025] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the NADH oxidase has substitution mutations at least at positions 120, 152, 158 and 176, and the corresponding substitution mutations at any one, any two, any three or all of positions 157, 181, 183 and 239.

[0026] In one or more embodiments, the mutation of the NADH oxidase compared to the amino acid sequence shown in SEQ ID NO:1 is selected from any combination of the following:

[0027] (1) Mutations at positions 120 and 157;

[0028] (2) Mutations at positions 152, 176, and 183;

[0029] (3) Mutations at positions 120, 157, 181 and 239;

[0030] (4) Mutations at positions 120, 152, 158, 176, and 181;

[0031] (5) Mutations at positions 120, 152, 158, 176, and 183;

[0032] (6) Mutations at positions 120, 152, 158, 176, 181, and 183;

[0033] (7) The 120th, 152nd, 158th and 176th positions;

[0034] (8) Mutations at positions 120, 152, 157, 158, 176, 181, 183 and 239.

[0035] In one or more embodiments, the mutation at bit 120 is I120V; the mutation at bit 152 is V152S; the mutation at bit 157 is Y157G; the mutation at bit 158 ​​is I158Q; the mutation at bit 176 is I176C; the mutation at bit 181 is R181T; the mutation at bit 183 is M183R; and the mutation at bit 239 is C239G.

[0036] In one or more embodiments, compared with SEQ ID NO:1, the mutation of the NADH oxidase is selected from any of the following: (1) I120V; (2) V152S; (3) Y157G; (4) I158Q; (5) I176C; (6) R181T; (7) M183R; (8) C239G; (9) I120V and Y157G; (10) V152S, I176C and M183R; (11) I120V, Y157G, R181T and C239G; (12) I120V, V 152S, I158Q, I176C and R181T; (13) I120V, V152S, I158Q, I176C and M183R; (14) I120V, V152S, I158Q, I176C, R181T and M183R; (15) I120V, V152S, I158Q and I176C; (16) I120V, V152S, Y157G, I158Q, I176C, R181T, M183R and C239G.

[0037] In one or more embodiments, the amino acid sequence of the NADH oxidase is shown in any one of SEQ ID NO:2 to 16.

[0038] In a second aspect of the invention, a polynucleotide molecule or its complementary sequence thereof encoding the NADH oxidase described in any embodiment of the invention is provided; or a nucleic acid construct containing the polynucleotide molecule or its complementary sequence thereof.

[0039] In one or more embodiments, the nucleic acid construct is an expression cassette or vector.

[0040] In one or more embodiments, the nucleic acid construct is an expression vector or a recombinant vector.

[0041] In a third aspect of the invention, a host cell is provided, said host cell:

[0042] (1) Expressing the NADH oxidase as described in any embodiment of the present invention; and / or

[0043] (2) Contains the polynucleotide molecule or its complementary sequence as described in any embodiment of the present invention, or a nucleic acid construct.

[0044] In a fourth aspect of the invention, NADH oxidase expressed using a host cell as described in any embodiment of the invention is provided.

[0045] In a fifth aspect of the invention, an enzyme preparation is provided, characterized in that the enzyme preparation contains the NADH oxidase described in any embodiment of the invention.

[0046] In one or more embodiments, the enzyme preparation further comprises a buffer solution; preferably, the buffer solution is a PBS buffer with a pH of 6.5 to 8.0, more preferably 6.5 to 7.5.

[0047] In one or more embodiments, the enzyme preparation is a powder, such as a lyophilized powder.

[0048] In one or more embodiments, the enzyme preparation is the supernatant obtained after host cell lysis as described in any embodiment of the present invention, or its concentrate or lyophilized product.

[0049] In a sixth aspect of the invention, the NADH oxidase described in any embodiment of the invention is provided for use in catalyzing the generation of NAD+ from NADH; or for use in the preparation of formulations that catalyze the generation of NAD+ from NADH.

[0050] In a seventh aspect of the invention, a method for catalytic synthesis of NAD+ is provided, the method comprising: using NADH and oxygen as substrates, and catalytically synthesizing NAD+ using an NADH oxidase mutant as described in any embodiment of the invention.

[0051] In one or more embodiments, the method has one or more of the following features:

[0052] The oxygen is dissolved oxygen; more preferably, the concentration of dissolved oxygen can be 1 to 10 mg / L, more preferably 3 to 3.5 mg / L;

[0053] The concentration of the NADH oxidase is 10–1000 mg / L, more preferably 200–400 mg / L;

[0054] The concentration of NADH is 1–10 mM, more preferably 2–4 mM;

[0055] The reaction temperature is 20–35°C, more preferably 22–30°C;

[0056] The reaction system contains PBS buffer;

[0057] The pH of the reaction system is 6.5–8.0, more preferably 6.5–7.5.

[0058] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0059] Figure 1 Example 1: Detection of NADH oxidase mutant nucleic acid by gel electrophoresis.

[0060] Figure 2Example 1: Comparison of NADH oxidase mutants (SEQ ID NO: 2-9) with wild-type NADH oxidase. The horizontal axis represents the NADH oxidase mutants, and the vertical axis represents the relative enzyme activity of the mutants compared to wild-type NADH oxidase.

[0061] Figure 3 Example 1: Comparison of NADH oxidase mutants (SEQ ID NO: 10-16) with wild-type NADH oxidase activity. The horizontal axis represents the NADH oxidase mutant, and the vertical axis represents the relative enzyme activity of the mutant compared to wild-type NADH oxidase. Detailed Implementation

[0062] To address the technical problem of low enzyme activity of NADH oxidase in enzymatic reactions in existing technologies, this invention provides several NADH oxidase mutants. These NADH oxidase mutants exhibit increased enzyme activity compared to the wild type, which can be used to improve the reaction efficiency of NAD+ regeneration systems while reducing costs.

[0063] NADH oxidase and its mutants

[0064] The NADH oxidases described herein particularly include NADH oxidases containing any of the amino acid sequences shown in SEQ ID NO: 1 to 16, and also include mutants that have 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with any of the amino acid sequences shown in SEQ ID NO: 1 and retain the biological activity of the NADH oxidase shown in SEQ ID NO: 1. This mutant is preferably derived from *Lactobacillus brevis*.

[0065] In this document, unless otherwise stated, the terms "NADH oxidase mutant," "mutant NADH oxidase," "mutant NOX," and "NOX mutant" are used interchangeably and generally refer to mutants that have undergone mutations relative to wild-type NADH oxidase (the amino acid sequence of wild-type NADH oxidase is shown in SEQ ID NO:1, NCBI accession number WP_035465043.1). Preferably, the mutation site is selected from positions 120, 152, 157, 158, 176, 181, 183, and 239 of SEQ ID NO:1. Specifically, this N-deoxyribotransferase mutant, relative to SEQ ID NO:1, is a mutant with a mutation relative to wild-type NADH oxidase. NO:1 may have a substitution mutation at any one of the following positions: 120, 152, 157, 158, 176, 181, 183, and 239; or at any two, three, four, five, six, seven, or all eight positions. In some embodiments, this NADH oxidase mutant is also derived from *Lactobacillus brevis*.

[0066] As used herein, "isolated NADH oxidase" refers to an NADH oxidase mutant that is substantially free of other naturally occurring proteins, lipids, carbohydrates, or other substances associated with it. Those skilled in the art can purify NADH oxidase mutants using standard protein purification techniques. Substantially pure protein produces a single master band on a non-reducing polyacrylamide gel.

[0067] In this document, the mutation at position 120 is a mutation of isoleucine (I) to a nonpolar amino acid, such as valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or glycine. Preferably, the mutation at position 120 is a mutation of isoleucine (I) to valine, leucine, alanine, or glycine. More preferably, the mutation at position 120 is a mutation of isoleucine (I) to leucine or valine. More preferably, the mutation at position 120 is a mutation of isoleucine (I) to valine (V).

[0068] In this document, the mutation at position 152 is a mutation of valine (V) to an uncharged polar amino acid, such as serine, threonine, glutamine, asparagine, tyrosine, or cysteine. Preferably, the mutation at position 152 is a mutation of valine (V) to serine or threonine. More preferably, the mutation at position 152 is a mutation of valine (V) to serine (S).

[0069] In this document, the mutation at position 157 is a mutation of tyrosine (Y) to a nonpolar amino acid, such as glycine, valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or isoleucine. Preferably, the mutation at position 157 is a mutation of tyrosine (Y) to glycine, alanine, valine, leucine, or isoleucine. More preferably, the mutation at position 157 is a mutation of tyrosine (Y) to glycine or alanine. More preferably, the mutation at position 157 is a mutation of tyrosine (Y) to glycine (G).

[0070] In this document, the mutation at position 158 is a mutation of isoleucine (I) to an uncharged polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine, or glutamine. Preferably, the mutation at position 158 is a mutation of isoleucine (I) to glutamine or asparagine. More preferably, the mutation at position 158 is a mutation of isoleucine (I) to glutamine (Q).

[0071] In this document, the mutation at position 176 is a mutation of isoleucine (I) to an uncharged polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine, or glutamine. Preferably, the mutation at position 176 is a mutation of isoleucine (I) to cysteine, asparagine, or glutamine. More preferably, the mutation at position 176 is a mutation of isoleucine (I) to cysteine ​​(C).

[0072] In this document, the mutation at position 181 is a mutation of arginine (R) to an uncharged polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine, or glutamine. Preferably, the mutation at position 181 is a mutation of arginine (R) to threonine, serine, or cysteine. More preferably, the mutation at position 181 is a mutation of arginine (R) to threonine (T).

[0073] In this document, the mutation at position 183 is a mutation of methionine (M) to a polar, positively charged amino acid, such as lysine, arginine, or histidine. Preferably, the mutation at position 183 is a mutation of methionine (M) to lysine or arginine. More preferably, the mutation at position 183 is a mutation of methionine (M) to arginine (R).

[0074] In this document, the mutation at position 239 is a mutation of cysteine ​​(C) to a nonpolar amino acid, such as glycine, valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or isoleucine. Preferably, the mutation at position 239 is a mutation of cysteine ​​(C) to glycine, alanine, valine, leucine, or isoleucine. More preferably, the mutation at position 239 is a mutation of cysteine ​​(C) to glycine (G).

[0075] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO:1, the NADH oxidase mutant described herein undergoes the mutations described below at one or more of the following amino acid sites:

[0076] (1) The isoleucine (I) at position 120 is mutated to leucine (L) or valine (V);

[0077] (2) The valine (V) at position 152 is mutated to either serine (S) or threonine (T);

[0078] (3) The tyrosine (Y) at position 157 is mutated to glycine (G) or alanine (A);

[0079] (4) The isoleucine (I) at position 158 is mutated to glutamine (Q) or asparagine (N);

[0080] (5) The isoleucine at position 176 (I) is mutated to cysteine ​​(C), asparagine (N), or glutamine (Q);

[0081] (6) The arginine (R) at position 181 is mutated to threonine (T), serine (S) or cysteine ​​(C);

[0082] (7) The methionine (M) at position 183 is mutated to lysine (K), arginine (R), or histidine (H);

[0083] (8) The cysteine ​​(C) at position 239 is mutated to glycine (G) or alanine (A).

[0084] In one or more preferred embodiments, the mutations of the NADH oxidase mutant described herein compared to the amino acid sequence shown in SEQ ID NO:1 are selected from: I120V, V152S, Y157G, I158Q, I176C, R181T, M183R and C239G.

[0085] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the NADH oxidase has a substitution mutation at least at position 120 and / or at position 152, and has substitution mutations at any one, any two, any three, any four, or all of positions 157, 158, 176, 181, 183, and 239.

[0086] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the NADH oxidase has a substitution mutation at at least one or all of positions 120 and 152, a substitution mutation at one or all of positions 157, 158, and 176, and the corresponding substitution mutation at one or all of positions 181, 183, and 239.

[0087] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the NADH oxidase has substitution mutations at least at positions 120, 152, and 158, and the corresponding substitution mutations at any one, any two, any three, any four, or all of positions 157, 176, 181, 183, and 239.

[0088] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the NADH oxidase has substitution mutations at least at positions 120, 152, 158 and 176, and the corresponding substitution mutations at any one, any two, any three or all of positions 157, 181, 183 and 239.

[0089] In one or more embodiments, the mutation of the NADH oxidase compared to the amino acid sequence shown in SEQ ID NO:1 is selected from any combination of the following:

[0090] (1) Mutations at positions 120 and 157;

[0091] (2) Mutations at positions 152, 176, and 183;

[0092] (3) Mutations at positions 120, 157, 181 and 239;

[0093] (4) Mutations at positions 120, 152, 158, 176, and 181;

[0094] (5) Mutations at positions 120, 152, 158, 176, and 183;

[0095] (6) Mutations at positions 120, 152, 158, 176, 181, and 183;

[0096] (7) The 120th, 152nd, 158th and 176th positions;

[0097] (8) Mutations at positions 120, 152, 157, 158, 176, 181, 183 and 239.

[0098] In one or more embodiments, the mutation of the NADH oxidase compared to the amino acid sequence shown in SEQ ID NO:1 is selected from any combination of the following:

[0099] (1) Mutations at positions 120 and 157; where position 120 is mutated to I120L or I120V, and position 157 is mutated to Y157G or Y157A;

[0100] (2) Mutations at positions 152, 176 and 183; where position 152 is mutated to V152S or V152T, position 176 is mutated to I176C, I176N or I176Q, and position 183 is mutated to M183K, M183R or M183H.

[0101] (3) Mutations at positions 120, 157, 181 and 239; where position 120 is mutated to I120L or I120V, position 157 is mutated to Y157G or Y157A, position 181 is mutated to R181T, R181S or R181C, and position 239 is mutated to C239G or C239A;

[0102] (4) Mutations at positions 120, 152, 158, 176 and 181; where position 120 is mutated to I120L or I120V, position 152 is mutated to V152S or V152T, position 158 is mutated to I158Q or I158N, position 176 is mutated to I176C, I176N or I176Q, and position 181 is mutated to R181T, R181S or R181C;

[0103] (5) Mutations at positions 120, 152, 158, 176, and 183; where position 120 is mutated to I120L or I120V, position 152 is mutated to V152S or V152T, position 158 is mutated to I158Q or I158N, position 176 is mutated to I176C, I176N, or I176Q, and position 183 is mutated to M183K, M183R, or M183H;

[0104] (6) Mutations at positions 120, 152, 158, 176, 181, and 183; wherein position 120 is mutated to I120L or I120V, position 152 is mutated to V152S or V152T, position 158 is mutated to I158Q or I158N, position 176 is mutated to I176C, I176N, or I176Q, position 181 is mutated to R181T, R181S, or R181C, and position 183 is mutated to M183K, M183R, or M183H;

[0105] (7) Positions 120, 152, 158 and 176; wherein position 120 is mutated to I120L or I120V, position 152 is mutated to V152S or V152T, position 158 is mutated to I158Q or I158N, and position 176 is mutated to I176C, I176N or I176Q;

[0106] (8) Mutations at positions 120, 152, 157, 158, 176, 181, 183 and 239, wherein position 120 is mutated to I120L or I120V, position 152 is mutated to V152S or V152T, position 157 is mutated to Y157G or Y157A, position 158 is mutated to I158Q or I158N, position 176 is mutated to I176C, I176N or I176Q, position 181 is mutated to R181T, R181S or R181C, position 183 is mutated to M183K, M183R or M183H, and position 239 is mutated to C239G or C239A.

[0107] In one or more preferred embodiments, the mutation of the NADH oxidase compared to the amino acid sequence shown in SEQ ID NO:1 is selected from any combination of the following:

[0108] (1) I120V and Y157G; (2) V152S, I176C and M183R; (3) I120V, Y157G, R181T and C239G; (4) I120V, V152S, I158Q, I176C and R181T; (5) I120V, V152S, I158Q, I176C and M183R; (6) I120V, V152S, I158Q, I176C, R181T and M183R; (7) I120V, V152S, I158Q and I176C; (8) I120V, V152S, Y157G, I158Q, I176C, R181T, M183R and C239G.

[0109] In one or more preferred embodiments, the amino acid sequence of the NADH oxidase mutant is as shown in any one of SEQ ID NO:2 to 16, compared to the amino acid sequence shown in SEQ ID NO:1.

[0110] This invention also includes fragments, derivatives, and analogs of the NADH oxidase mutant described herein. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the native NADH oxidase mutant of this invention. The protein fragments, derivatives, or analogs of this invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing an additional amino acid sequence to this protein sequence (such as a leader sequence or secretory sequence or a sequence used to purify this protein or a proteogenic sequence, or a fusion protein). Preferably, the fragments, derivatives, and analogs retain at least one mutation described herein, corresponding to an amino acid substitution at at least one of the mutations at positions 120, 152, 157, 158, 176, 181, 183, and 239 in SEQ ID NO:1.

[0111] In this invention, the term "NADH oxidase mutant" also includes (but is not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-20, more preferably 1-10, and even more preferably 1-8, 1-5, 1-3, or 1-2), and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding or deleting one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. Preferably, the mutant retains at least one mutation described in this application, corresponding to at least one amino acid substitution from the mutations at positions 120, 152, 157, 158, 176, 181, 183, and 239 in SEQ ID NO:1. In some implementations, the NADH oxidase mutant is also derived from *Levilactobacillus brevis*.

[0112] In this invention, the term "NADH oxidase mutant" also includes (but is not limited to): derived proteins that retain their protein activity and have at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, such as at least 98% or 99%, sequence identity with the NADH oxidase mutants described herein. Similarly, these mutants retain at least one mutation described herein, namely, a mutation corresponding to at least one amino acid substitution at positions 120, 152, 157, 158, 176, 181, 183, and 239 in SEQ ID NO:1. In some embodiments, such NADH oxidase mutants are also derived from *Levilactobacillus brevis*. Identification between two sequences can be calculated using methods well known in the art, such as BLASTP.

[0113] In the NADH oxidase mutants, fragments, derivatives and analogs of the present invention, the mutation sites and their mutated amino acids described herein are conserved.

[0114] The proteins of this invention can be recombinant proteins, natural proteins, or synthetic proteins, with recombinant proteins being preferred. The proteins of this invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology.

[0115] Polynucleotide molecules

[0116] The present invention also provides a multinucleotide molecule encoding the NADH oxidase mutant of the present invention or its conserved variant protein and its complementary sequence.

[0117] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0118] The polynucleotide molecule encoding the mature protein of the mutant includes: a coding sequence that encodes only the mature protein; a coding sequence for the mature protein and various additional coding sequences; a coding sequence for the mature protein (and optional additional coding sequences) and a non-coding sequence.

[0119] A “polynucleotide molecule encoding a protein” can be a polynucleotide that includes the protein, or it can include polynucleotides that also include additional coding and / or non-coding sequences.

[0120] The polynucleotide molecules of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0121] Nucleic acid constructs

[0122] This document also provides a nucleic acid construct containing the polynucleotide molecule described herein. The nucleic acid construct may also contain one or more regulatory sequences, such as promoters, terminators, and / or enhancers, operatively linked to the polynucleotide molecule described herein.

[0123] In this text, "operable linkage" or a similar description refers to the arrangement of elements, wherein the components are arranged in a specific shape to perform their required functions. Thus, a given promoter operably linked to a coding sequence enables the efficient expression of that coding sequence in the presence of the correct transcription factors, etc. The promoter does not need to be adjacent to the coding sequence, as long as it serves to guide the expression of that sequence. Therefore, sequences that do not participate in translation but are transcribed can exist between the promoter sequence and the coding sequence, just like transcribed introns; and the promoter sequence can still be considered "operable linked" to the coding sequence.

[0124] The polynucleotide encoding the present invention can be manipulated in various ways to ensure the expression of the NADH oxidase mutant or its conserved variant protein. The manipulation of the polynucleotide molecule prior to its insertion into the expression vector may be desired or necessary depending on the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0125] The regulatory sequence can be a suitable promoter, a nucleotide sequence recognized by the host cell that expresses the polynucleotide molecule described herein. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0126] In Escherichia coli, suitable promoters include, but are not limited to, lac (lactose promoter), trp (tryptophan promoter), and tac (a hybrid promoter of lactose and tryptophan).

[0127] The regulatory sequence also includes a suitable transcription terminator, which is recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the polynucleotide molecule described herein. Any terminator that is functional in a selected host cell can be used in this invention. In some embodiments, the transcription terminator sequence is a functional terminator in *E. coli*.

[0128] In some implementations, the nucleic acid construct is an expression cassette or expression box containing a promoter and a transcription terminator operatively linked to the polynucleotide molecule described herein.

[0129] carrier

[0130] The nucleic acid constructs described in this article can also be vectors, including but not limited to expression vectors and cloning vectors.

[0131] In expression vectors, various nucleic acid molecules and regulatory sequences can be linked together to produce recombinant expression vectors comprising one or more restriction sites that allow insertion of the polynucleotide molecules or nucleic acid constructs described herein at such sites. During the fabrication of the expression vector, the polynucleotide molecules described herein are located within the vector to enable their operative linking to appropriate regulatory sequences.

[0132] The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0133] Recombinant expression vectors can be any vector (such as plasmids or viruses) that can readily undergo recombinant DNA methods and result in the expression of nucleotide sequences of interest. The choice of vector generally depends on its compatibility with the host cell in which it is introduced. The vector can be a linear or closed circular plasmid.

[0134] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and whose replication does not depend on chromosome replication, such as a plasmid, extrachromosomal element, mini-chromosome, or artificial chromosome. The vector can contain any means necessary to ensure self-replication. Alternatively, the vector can be a vector that, when introduced into a host cell, integrates into the genome and replicates along with the chromosome into which it has already been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, containing the total DNA to be introduced into the host cell's genome, can be used.

[0135] The vectors described herein may contain one or more selectable markers that allow for easy selection of cells for transformation, transfection, transduction, etc. These selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophic to auxotrophic traits, etc.

[0136] The vectors described herein may contain elements that allow the vector to integrate into the host cell genome or to replicate autonomously in the cell independently of the genome.

[0137] One or more copies of the polynucleotide of the present invention can be inserted into a host cell to increase the yield of the gene product. The increase in polynucleotide copy number can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selector gene and the polynucleotide. Cells containing an amplified selector gene and thereby containing additional copies of the polynucleotide can be screened by culturing the cells in the presence of a suitable selector.

[0138] The expression vectors described in this article are preferably vectors that can be used for expression in Escherichia coli, such as pET-30a.

[0139] Cloning vectors containing the polynucleotide sequences described herein can be used to replicate a sufficient number of target plasmids. Therefore, the cloning vectors described herein contain strong self-replication elements, such as replication initiation sites. Typically, the cloning vectors of this invention do not contain expression elements.

[0140] The expression vectors described herein can be constructed using methods well-known in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Vectors containing the aforementioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0141] host cells

[0142] The host cell of this invention can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include: *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, and *Agrobacterium*; eukaryotic cells such as yeast and plant cells. In a specific embodiment of this invention, *Escherichia coli* is used as the host cell.

[0143] Recombinant DNA technology can be used to express or produce recombinant NADH oxidase mutants using the polynucleotide molecules of the present invention. This expression or production typically includes the following steps: (1) transforming or transducing suitable host cells with the polynucleotide encoding the NADH oxidase mutant of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) culturing the host cells in a suitable culture medium; and (3) isolating and purifying the protein from the culture medium or cells.

[0144] In a specific embodiment of the present invention, the NADH oxidase mutant was expressed and purified in Escherichia coli.

[0145] enzyme preparations

[0146] In some embodiments, the present invention provides an enzyme preparation containing the NADH oxidase mutant described in any embodiment of the present invention. In some embodiments, the enzyme preparation is a buffer solution containing the NADH oxidase mutant.

[0147] In some embodiments, the enzyme preparation is a powder, such as a lyophilized powder.

[0148] In some embodiments, the enzyme preparation is the supernatant obtained after lysis of host cells expressing the NADH oxidase mutant, such as *E. coli*. Lysis can be performed using methods well known in the art. In some embodiments, the enzyme preparation can be a concentrate of the supernatant, and the solvent in the supernatant can be removed using conventional methods. In some embodiments, the enzyme preparation can be a lyophilized version of the supernatant.

[0149] application

[0150] The NADH oxidase mutant described in this invention can be used (e.g., in the presence of oxygen) to catalyze the production of NAD+ from NADH; or to prepare reagents (including catalytic reagents, such as enzyme preparations) or culture media that catalyze the production of NAD+ from NADH.

[0151] In a preferred embodiment, the oxygen is present in the form of dissolved oxygen, wherein the concentration of dissolved oxygen can be 1–10 mg / L, preferably 3–3.5 mg / L. In a preferred embodiment, the concentration of NADH can be 1–10 mM, for example 2–4 mM. The suitable temperature for the NADH oxidase mutant is 20–35°C, for example 22–30°C, preferably 25°C. The suitable pH for the NADH oxidase mutant is 6.5–8.0, for example 6.5–7.5.

[0152] The NADH oxidase mutant of the present invention or the host cell expressing the NADH oxidase mutant can be formulated in an industrially acceptable vector to obtain a composition suitable for catalytic reaction or suitable for storage.

[0153] The NADH oxidase mutant of the present invention, or host cells containing it, can also be included in the kit for industrial use or commercialization. Furthermore, the kit may also contain auxiliary reagents such as reaction buffers. Generally, the kit also includes instructions for use.

[0154] It should be understood that the NADH oxidase mutant described in this invention can be used to catalyze the production of NAD+ from NADH in the presence of oxygen without the need for additional substrate. The catalytic product is water, with no other byproducts produced, making it environmentally friendly and possessing great potential for industrial application.

[0155] NADH oxidase catalyzes the production of NAD from NADH. +

[0156]

[0157] In this invention, NADH is used as a substrate, and in the presence of oxygen, the NADH oxidase mutant described in this invention catalyzes the oxidation of H on NADH to form NAD. + .

[0158] In this reaction, the oxygen may be in the form of dissolved oxygen, wherein the concentration of dissolved oxygen may be 1–10 mg / L, for example 3–3.5 mg / L. In the reaction system described above, the concentration of the NADH oxidase mutant may be 10–1000 mg / L, for example 200–400 mg / L. In the reaction system, the concentration of NADH may be 1–10 mM, for example 2–4 mM. The reaction temperature may be 20–35°C, for example 22–30°C. A PBS buffer may be used to provide the pH required for the reaction. An exemplary PBS buffer may have a pH of 6.5–8.0, for example 6.5–7.5.

[0159] In some embodiments, the amino acid sequence of the NADH oxidase may be as shown in any of SEQ ID NO: 2-16. The NADH oxidase may be the NADH oxidase described in any embodiment herein. It should be understood that more than one NADH oxidase and / or its mutants may be used in the same reaction. In this invention, the NADH oxidase mutant has higher enzyme activity compared to the wild-type NADH oxidase. Enzyme activity can be determined by various methods. For example, since NADH oxidase can oxidize NADH to NAD+, the activity can be determined by detecting the rate of decrease of NADH at 340 nm. Alternatively, the oxidation of NADH can be coupled with the reduction of 2,6-dichlorophenol indigo blue (DCPIP), where the blue DCPIP is reduced to colorless DCPIP, and the NADH oxidase activity can be calculated by measuring the reduction rate of the blue DCPIP at 600 nm. These methods for determining the enzyme activity of NADH oxidase are all included in or can be used in this invention, including but not limited to: standard curve method and colorimetric method.

[0160] Having obtained information about the NADH oxidase mutant described in this invention, those skilled in the art will understand how to use this mutant to catalyze the synthesis of NAD+ using NADH and oxygen as substrates. Various intracellular or extracellular catalytic synthesis methods are included in this invention or can be used in this invention.

[0161] Therefore, the present invention also provides a method for catalytic synthesis of NAD+, the method comprising: using NADH and oxygen as substrates, and catalytically synthesizing NAD+ using the NADH oxidase mutant described in this invention. In this method, a buffer solution (e.g., PBS buffer) can be used as the reaction solvent to allow the reaction to proceed under specific pH conditions. In this reaction, the oxygen can exist in the form of dissolved oxygen, wherein the concentration of dissolved oxygen can be 1–10 mg / L, for example 3–3.5 mg / L. In the reaction system described above, the concentration of the NADH oxidase mutant can be 10–1000 mg / L, for example 100–500 mg / L or 200–400 mg / L. In the reaction system, the concentration of NADH can be 1–10 mM, for example 1–5 mM or 2–4 mM. The reaction temperature can be 20–35°C, for example 22–30°C, preferably 25–30°C. The exemplary pH of the PBS buffer can be 6.5–8.0, for example 6.5–7.5.

[0162] The beneficial effects of this invention include:

[0163] (1) The NADH oxidase mutant obtained in this invention has higher enzyme activity than wild-type NADH oxidase, and can efficiently catalyze the generation of NAD+ in the presence of NADH and oxygen. The catalytic efficiency of the NADH oxidase mutant of this invention can reach up to 3.2 times that of the wild type, which greatly increases the catalytic efficiency and significantly improves the enzyme's ability to catalyze the generation of NAD+.

[0164] (2) The NADH oxidase mutant obtained in this invention can be used to catalyze the production of NAD+ from NADH in the presence of oxygen without the need for additional substrate. The catalytic product is water and no other byproducts are produced. It is environmentally friendly and has great potential for industrial application.

[0165] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form preferred technical solutions.

[0166] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. The materials and reagents used in the embodiments are conventional materials and reagents in the art, unless otherwise stated, such as materials and reagents that are commercially available.

[0167] Example 1: Construction and expression of recombinant bacteria containing a gene encoding a NADH oxidase mutant

[0168] Using the sequence of SEQ ID NO:1 as a template, directed evolution was performed using conventional PCR, iterative saturation mutagenesis, and combinatorial mutagenesis. The mutants were then transformed into *E. coli* BL21(DE3) competent cells and evenly spread on LB agar plates containing 50 μg / ml kanamycin (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, pH 7.2), and incubated statically at 37°C for 18 h. The mutants from the transformed plates were then transferred to 96-well plates using sterile toothpicks and incubated overnight at 37°C and 250 rpm in a shaker. 50 μL of bacterial culture was transferred from the wells of the primary plate to the corresponding wells of the secondary plate. After incubation at 37°C and 250 rpm for 2–3 h, IPTG was added to a final concentration of 0.2 mM, and the mixture was incubated at 30°C for 20 h to obtain the corresponding mutants. 500 μL of K701 lysozyme (2 mg / ml) and nuclease (1 mg / ml) were added to each well, and the mixture was placed in a shaker at 30°C for 2 h. After centrifugation at 4000 rpm for 20 min, the supernatant enzyme solution was collected and added to the prepared reaction system. The reaction was incubated for 5 min, with a 30 s interval between each reaction. 4 μL of the reaction solution was collected and subjected to high-throughput screening using a microplate reader to identify mutants with significantly increased activity for gene sequencing. The protein sequences of the obtained NADH oxidase mutants are shown in SEQ ID NO: 2–16.

[0169] SEQ ID NO:1, Wild-type NADH oxidase

[0170] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSG SWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIGAELAEAYSTTGHDVTLIDAMDRVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIK TDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVST GLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0171] SEQ ID NO:2, NADH oxidase mutant (I120V)

[0172] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPK V PGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIGAELAEAYSTTGHDVTLIDAMDRVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0173] SEQ ID NO:3, NADH oxidase mutant (V152S)

[0174] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRIT SIGAGYIGAELAEAYSTTGHDVTLIDAMDRVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0175] SEQ ID NO:4, NADH oxidase mutant (Y157G) [[ID=B]]

[0176] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAG G IGAELAEAYSTTGHDVTLIDAMDRVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0177] SEQ ID NO:5, NADH oxidase mutant (I158Q)

[0178] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGY Q GAELAEAYSTTGHDVTLIDAMDRVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0179] SEQ ID NO:6, NADH oxidase mutant (I176C)

[0180] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIGAELAEAYSTTGHDVTL CDAMDRVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0181] SEQ ID NO:7, NADH oxidase mutant (R181T)

[0182] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIGAELAEAYSTTGHDVTLIDAMD T VMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0183] SEQ ID NO:8, NADH oxidase mutant (M183R)

[0184] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIGAELAEAYSTTGHDVTLIDAMDRV R PKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0185] SEQ ID NO:9, NADH oxidase mutant (I120V / Y157G)

[0186] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPK V PGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAG GIGAELAEAYSTTGHDVTLIDAMDRVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0187] SEQ ID NO:10, NADH oxidase mutant (V152S / I176C / M183R)

[0188] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRIT S IGAGYIGAELAEAYSTTGHDVTL C DAMDRV R PKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0189] SEQ ID NO:11, NADH oxidase mutant (I120V / Y157G / R181T / C239G)

[0190] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPK V PGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAG G IGAELAEAYSTTGHDVTLIDAMD T VMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAIL G IGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0191] SEQ ID NO:12, NADH oxidase mutant (I120V / V152S / I158Q / I176C / R181T)

[0192] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPK V PGIDSDRVKLCKNWAHAQALIEDAKEAKRIT S IGAGY Q GAELAEAYSTTGHDVTL C DAMD TVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0193] SEQ ID NO:13, NADH oxidase mutant (I120V / V152S / I158Q / I176C / M183R)

[0194] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPK V PGIDSDRVKLCKNWAHAQALIEDAKEAKRIT S IGAGY Q GAELAEAYSTTGHDVTL C DAMDRV R PKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0195] SEQ ID NO:14, NADH oxidase mutant

[0196] (I120V / V152S / I158Q / I176C / R181T / M183R)

[0197] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPK V PGIDSDRVKLCKNWAHAQALIEDAKEAKRIT S IGAGY Q GAELAEAYSTTGHDVTL C DAMD T V R PKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0198] SEQ ID NO:15, NADH oxidase mutant (I120V / V152S / I158Q / I176C)

[0199] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPK V PGIDSDRVKLCKNWAHAQALIEDAKEAKRIT S IGAGY Q GAELAEAYSTTGHDVTL CDAMDRVMPKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0200] SEQ ID NO:16, NADH oxidase mutant

[0201] (I120V / V152S / Y157G / I158Q / I176C / R181T / M183R / C239G)

[0202] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTSHAQTTESYDKLVMTSGSWPIVPK V PGIDSDRVKLCKNWAHAQALIEDAKEAKRIT S IGAG GQ GAELAEAYSTTGHDVTL C DAMD T V R PKYFDADFTDVIEQDYRDHGVQLALSETVESFTDSATGLTIKTDKNSYETDLAIL G IGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGVNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0203] Example 2: Enzyme activity assay of NADH oxidase mutant

[0204] The enzyme activity of NADH oxidase was determined using the following method:

[0205] The reaction system consisted of PBS buffer, NADH, and NADH oxidase solutions of varying concentrations. The reaction conditions were 25°C and pH 7.0. The absorbance of NAD at 340 nm was measured using a microplate reader.

[0206] Calculation formula: U=ΔA*N*V0 / K*V1

[0207] In the formula, ΔA represents the change in absorbance, N represents the dilution factor, K represents the slope of the standard curve, V0 represents the reaction system, and V1 represents the enzyme addition volume.

[0208] Enzyme activity is defined as the amount of enzyme required to consume or generate 1 μmol of NADH per minute, which is one enzyme activity unit (U).

[0209] Preparation of standard curve:

[0210]

[0211]

[0212] Total volume of sample added (200 μL)

[0213] 1 2 3 4 5 6 A 180 170 160 150 140 130 B 20 20 20 20 20 20 C 0 10 20 30 40 50

[0214] The enzyme activities of NADH oxidase mutant and wild-type NADH oxidase were measured separately, and the relative enzyme activities of NADH oxidase mutant were calculated:

[0215] Relative enzyme activity = enzyme activity of NADH oxidase mutant ÷ enzyme activity of wild-type NADH oxidase.

[0216] The results are as follows Figure 2 , Figure 3 As shown in Table 1.

[0217] Table 1. Relative enzyme activities of NADH oxidase mutants

[0218]

[0219]

[0220] From Table 1 and Figure 3 It can be seen that the NADH oxidase mutant SEQ ID NO:16 showed the best activity improvement, with an activity 3.2 times higher than that of wild-type NADH oxidase.

[0221] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications, equivalent substitutions, and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. An isolated NADH oxidase, characterized in that, the NADH oxidase: (a) has a substitution mutation at any one of positions, any two of positions, any three of positions, any four of positions, any five of positions, any six or more of positions, any seven or more of positions, or all eight of positions 120, 152, 157, 158, 176, 181, 183, and 239 of the amino acid sequence set forth in SEQ ID NO: 1; or (b) is derived from the amino acid sequence of (a) by substitution, deletion, or addition of one or more amino acid residues and has an N- d e o x y r i b o s y l t r a n s f e r a s e II function, wherein the protein corresponds to the amino acid residues at positions 120, 152, 157, 158, 176, 181, 183, and 239 of SEQ ID NO: 1 as set forth in (a); or (c) has 80% or more sequence identity to the amino acid sequence of (a) and has an N-d e o x y r i b o s y l t r a n s f e r a s e II function, and the protein corresponds to the amino acid residues at positions 120, 152, 157, 158, 176, 181, 183, and 239 of SEQ ID NO: 1 as set forth in (a).

2. The NADH oxidase of claim 1, wherein: the mutation at position 120 is a substitution of isoleucine with a non-polar amino acid selected from valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or glycine; the mutation at position 152 is a substitution of valine with an uncharged polar amino acid selected from serine, threonine, glutamine, asparagine, tyrosine, or cysteine; the mutation at position 157 is a substitution of tyrosine with a non-polar amino acid selected from glycine, valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or isoleucine; the mutation at position 158 is a substitution of isoleucine with an uncharged polar amino acid selected from serine, threonine, cysteine, tyrosine, asparagine, or glutamine; the mutation at position 176 is a substitution of isoleucine with an uncharged polar amino acid selected from serine, threonine, cysteine, tyrosine, asparagine, or glutamine; the mutation at position 181 is a substitution of arginine with an uncharged polar amino acid selected from serine, threonine, cysteine, tyrosine, asparagine, or glutamine; the mutation at position 183 is a substitution of methionine with a positively charged polar amino acid selected from lysine, arginine, or histidine; the mutation at position 239 is a substitution of cysteine with a non-polar amino acid selected from glycine, valine, leucine, proline, alanine, phenylalanine, tryptophan, methionine, or isoleucine; preferably, the mutation at position 120 is a substitution of isoleucine with valine, leucine, alanine, or glycine; the mutation at position 152 is a substitution of valine with serine or threonine; the mutation at position 157 is tyrosine to glycine, alanine, valine, leucine or isoleucine; the mutation at position 158 is isoleucine to glutamine or asparagine; the mutation at position 176 is isoleucine to cysteine, asparagine or glutamine; the mutation at position 181 is arginine to threonine, serine or cysteine; the mutation at position 183 is methionine to lysine or arginine; the mutation at position 239 is cysteine to glycine, alanine, valine, leucine or isoleucine; more preferably, the mutation at position 120 is isoleucine to leucine or valine; the mutation at position 152 is valine to serine; the mutation at position 157 is tyrosine to glycine or alanine; the mutation at position 158 is isoleucine to glutamine; the mutation at position 176 is isoleucine to cysteine; the mutation at position 181 is arginine to threonine; the mutation at position 183 is methionine to arginine; the mutation at position 239 is cysteine to glycine or alanine.

3. The NADH oxidase of claim 1 or 2, wherein, the NADH oxidase has at least a substitution mutation at position 120 and / or a substitution mutation at position 152, and has a substitution mutation at any one, any two, any three, any four or all of positions 157, 158, 176, 181, 183 and 239, as compared with the amino acid sequence shown in SEQ ID NO: 1; and / or, the NADH oxidase has at least a substitution mutation at any one or all of positions 120, 152, a substitution mutation at any one, any two or all of positions 157, 158, 176, and a corresponding substitution mutation at any one, any two or all of positions 181, 183 and 239, as compared with the amino acid sequence shown in SEQ ID NO: 1; and / or, the NADH oxidase has at least a substitution mutation at positions 120, 152 and 158, and has a corresponding substitution mutation at any one, any two, any three, any four or all of positions 157, 176, 181, 183 and 239, as compared with the amino acid sequence shown in SEQ ID NO: 1; and / or, the NADH oxidase has at least a substitution mutation at positions 120, 152, 158 and 176, and has a corresponding substitution mutation at any one, any two, any three or all of positions 157, 181, 183 and 239, as compared with the amino acid sequence shown in SEQ ID NO:

1. Preferably, the mutation of the NADH oxidase is selected from any combination of the following: (1) mutation at position 120 and 157; (2) mutation at position 152, 176 and 183; (3) mutation at position 120, 157, 181 and 239; (4) mutation at position 120, 152, 158, 176 and 181; (5) mutation at position 120, 152, 158, 176 and 183; (6) mutation at position 120, 152, 158, 176, 181 and 183; (7) mutation at position 120, 152, 158 and 176; (8) mutation at position 120, 152, 157, 158, 176, 181, 183 and 239.

4. The NADH oxidase of any one of claims 1-3, wherein: the mutation at position 120 is I120V; the mutation at position 152 is V152S; the mutation at position 157 is Y157G; the mutation at position 158 is I158Q; the mutation at position 176 is I176C; the mutation at position 181 is R181T; the mutation at position 183 is M183R; the mutation at position 239 is C239G.

5. The NADH oxidase as set forth in claim 1, wherein the mutation of the NADH oxidase is selected from any of the following: (1) I120V; (2) V152S; (3) Y157G; (4) I158Q; (5) I176C; (6) R181T; (7) M183R; (8) C239G; (9) I120V and Y157G; (10) V152S, I176C and M183R; (11) I120V, Y157G, R181T and C239G; (12) I120V, V152S, I158Q, I176C and R181T; (13) I120V, V152S, I158Q, I176C and M183R; (14) I120V, V152S, I158Q, I176C, R181T and M183R; (15) I120V, V152S, I158Q and I176C; (16) I120V, V152S, Y157G, I158Q, I176C, R181T, M183R and C239G. Preferably, the amino acid sequence of the NADH oxidase is as set forth in any one of SEQ ID NO: 2-16.

6. A polynucleotide molecule encoding the NADH oxidase of any one of claims 1-5, or a complement thereof, or a nucleic acid construct comprising said polynucleotide molecule or a complement thereof; Preferably, the nucleic acid construct is an expression cassette or a vector; more preferably, the nucleic acid construct is an expression vector or a recombinant vector.

7. A host cell, characterized in that, the host cell: (1) expressing the NADH oxidase of any one of claims 1 to 5; and / or (2) containing the polynucleotide molecule of claim 6 or its complementary sequence, or a nucleic acid construct.

8. NADH oxidase expressed by the host cell of claim 7.

9. An enzyme preparation, characterized in that, The enzyme preparation contains the NADH oxidase of any one of claims 1 to 5 and 8; Preferably, the enzyme preparation further contains a buffer; preferably, the buffer is PBS buffer, and the pH is 6.5 to 8.0, preferably 6.5 to 7.5; or Preferably, the enzyme preparation is in the form of a powder, such as a lyophilized powder; or Preferably, the enzyme preparation is a supernatant obtained after lysis of the host cell of claim 7, or a concentrate or lyophilizate thereof.

10. Use of the NADH oxidase of any one of claims 1 to 5 and 8 for catalyzing the generation of NAD+ from NADH, or in the preparation of an agent for catalyzing the generation of NAD+ from NADH.

11. A method of catalytically synthesizing NAD+, the method comprising: NAD+ is synthesized using the NADH oxidase mutant of any one of claims 1 to 5 and 8 as a catalyst, with NADH and oxygen as substrates; Preferably, the method has one or more of the following features: The oxygen is dissolved oxygen; more preferably, the concentration of the dissolved oxygen is 1 to 10 mg / L, more preferably 3 to 3.5 mg / L; The concentration of the NADH oxidase is 10 to 1000 mg / L, more preferably 200 to 400 mg / L; The concentration of the NADH is 1 to 10 mM, more preferably 2 to 4 mM; The reaction temperature is 20 to 35 °C, more preferably 22 to 30 °C; The reaction system contains PBS buffer; The pH of the reaction system is 6.5 to 8.0, more preferably 6.5 to 7.5.

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