Protein having 3-hydroxybutyrate dehydrogenase activity

Abstract

The present invention relates to a protein having 3-hydroxybutyratedehydrogenase activity which has 80% or more sequence identity with an amino acid sequence represented by SEQ ID NO: 1, 10, 11, 12, 13, or 14, in which the amino acid residue corresponding to position 162 of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid residue other than a leucine residue, or the amino acid residue corresponding to position 33, 47, 52, 54, or 96 of the amino acid sequence represented by SEQ ID NO: 10, 11, 12, 13, or 14 is an amino acid residue other than a valine residue.

Description

Protein Having 3-Hydroxybutyrate Dehydrogenase Activity

[0001] The present invention relates to a protein having 3-hydroxybutyrate dehydrogenase activity.

[0002] When carbohydrates (glucose) in the body are depleted and energy obtained from carbohydrate metabolism is insufficient, lipid metabolism is promoted and ketone bodies are produced. The produced ketone bodies are used as an energy source in place of carbohydrates. Ketone bodies present in the body include acetone, acetoacetic acid, and 3-hydroxybutyrate.

[0003] When glucose cannot be used as an energy source due to diabetes, starvation, fasting, or other conditions, ketone bodies need to be used as an energy source. This promotes the production of ketone bodies in the body, and accumulation of ketone bodies in the body leads to a condition called ketosis. Further accumulation of ketone bodies in the body causes the blood to become acidic, leading to a condition called ketoacidosis. Ketoacidosis causes dehydration or impaired consciousness, and can be fatal in the worst cases. Particularly for diabetic patients, there is a risk of developing ketoacidosis as described above, and there is a need for continuous monitoring of the concentration of ketone bodies (e.g., 3-hydroxybutyrate).

[0004] As a technique for measuring ketone bodies in blood, for example, Patent Document 1 discloses a method for measuring ketone bodies using a specific 3-hydroxybutyrate dehydrogenase.

[0005] Japanese Unexamined Patent Application Publication No. 2003-339385

[0006] As disclosed in Patent Document 1, 3-hydroxybutyrate dehydrogenase is sometimes used for measurement of ketone bodies. However, there remains room for improvement in properties such as thermostability and substrate specificity of the enzyme.

[0007] Accordingly, an object of the present invention is to provide a novel protein having 3-hydroxybutyrate dehydrogenase activity that exhibits excellent thermostability and substrate specificity.

[0008] The present invention includes, for example, the following inventions: [1-A] A protein comprising an amino acid sequence selected from (1) to (6) below, and having 3-hydroxybutyrate dehydrogenase activity. (1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid residue corresponding to the 162nd position in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than a leucine residue. (2) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid residue corresponding to the 33rd position in the amino acid sequence shown in SEQ ID NO: 10 is an amino acid residue other than a valine residue. (3) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 11, wherein the amino acid residue corresponding to the 47th position in the amino acid sequence shown in SEQ ID NO: 11 is an amino acid residue other than a valine residue. (4) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, wherein the amino acid residue corresponding to the 52nd position in the amino acid sequence shown in SEQ ID NO: 12 is an amino acid residue other than a valine residue. (5) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid residue corresponding to the 54th position in the amino acid sequence shown in SEQ ID NO: 13 is an amino acid residue other than a valine residue. (6) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 14, wherein the amino acid residue corresponding to the 96th position in the amino acid sequence shown in Sequence ID No. 14 is an amino acid residue other than a valine residue. [1] A protein having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, wherein the amino acid residue other than the leucine residue is an amino acid residue other than a leucine residue, and having 3-hydroxybutyrate dehydrogenase activity. [2] The protein according to [1-A] or [1], wherein the amino acid residue other than the leucine residue is an amino acid residue with a larger side chain volume compared to the leucine residue.[3] The protein according to [2], wherein the amino acid residue with a larger side chain volume compared to the leucine residue is an amino acid residue selected from the group consisting of phenylalanine residue, tryptophan residue, tyrosine residue, methionine residue, arginine residue, and lysine residue. [4] The protein according to any one of [1-A] and [1] to [3], wherein the amino acid residue other than the leucine residue is a hydrophobic amino acid residue. [5] The protein according to [4], wherein the hydrophobic amino acid residue is an amino acid residue with a greater degree of hydrophobicity compared to the leucine residue. [6] The protein according to any one of [1-A] and [1] to [5], wherein the amino acid residue other than the leucine residue is a phenylalanine residue. [6-A] The protein according to [1-A], wherein the amino acid residue other than the valine residue is an amino acid residue with a larger side chain volume compared to the valine residue. [6-B] The protein according to [6-A], wherein the amino acid residue with a larger side chain volume compared to the valine residue is an amino acid residue selected from the group consisting of leucine residue, isoleucine residue, phenylalanine residue, tryptophan residue, tyrosine residue, methionine residue, glutamine residue, glutamic acid residue, histidine residue, arginine residue, and lysine residue. [6-C] The protein according to any one of [1-A] and [6-A] to [6-B], wherein the amino acid residue other than the valine residue is a hydrophobic amino acid residue. [6-D] The protein according to [6-C], wherein the hydrophobic amino acid residue is an amino acid residue with a greater degree of hydrophobicity compared to the valine residue. [6-E] The protein according to any one of [1-A] and [6-A] to [6-D], wherein the amino acid residue other than the valine residue is an isoleucine residue. [7] A nucleic acid comprising a base sequence encoding the protein according to any one of [1-A], [1] to [6], and [6-A] to [6-E]. [8] An expression vector comprising the nucleic acid described in [7]. [9] A host cell comprising the nucleic acid described in [7].

[10] A composition for measuring D-3-hydroxybutyrate comprising the protein described in any of [1-A], [1] to [6], and [6-A] to [6-E].

[11] A kit for measuring D-3-hydroxybutyrate, comprising a composition containing the protein described in any of [1-A], [1] to [6], and [6-A] to [6-E].

[12] A sensor for measuring D-3-hydroxybutyrate, comprising an electrode on which the protein described in any of [1-A], [1] to [6], and [6-A] to [6-E] is immobilized.

[13] A sample containing D-3-hydroxybutyrate and β-NAD. ＋ A method for measuring D-3-hydroxybutyric acid, comprising the steps of: contacting a protein described in any of [1-A], [1] to [6], and [6-A] to [6-E] to produce acetoacetic acid and β-NADH; and calculating the amount of D-3-hydroxybutyric acid based on the signal caused by the β-NADH.

[0009] According to the present invention, it is possible to provide a novel protein having 3-hydroxybutyrate dehydrogenase activity with good heat resistance and substrate specificity.

[0010] This graph shows the results of evaluating the relative activity of 3-hydroxybutyrate dehydrogenase in which the 162nd leucine residue was replaced with a phenylalanine residue, when reacted with various substrates.

[0011] The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

[0012] [Protein] The protein according to this embodiment is a protein that comprises an amino acid sequence selected from (1) to (6) below and has 3-hydroxybutyrate dehydrogenase activity. (1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, where the amino acid residue corresponding to the 162nd position in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than a leucine residue. (2) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10, where the amino acid residue corresponding to the 33rd position in the amino acid sequence shown in SEQ ID NO: 10 is an amino acid residue other than a valine residue. (3) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 11, where the amino acid residue corresponding to the 47th position in the amino acid sequence shown in SEQ ID NO: 11 is an amino acid residue other than a valine residue. (4) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, where the amino acid residue corresponding to the 52nd position in the amino acid sequence shown in SEQ ID NO: 12 is an amino acid residue other than a valine residue. (5) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, where the amino acid residue corresponding to the 54th position in the amino acid sequence shown in SEQ ID NO: 13 is an amino acid residue other than a valine residue. (6) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, wherein the amino acid residue corresponding to the 96th amino acid sequence shown in SEQ ID NO: 14 is an amino acid residue other than a valine residue.

[0013] The protein according to this embodiment may have 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue corresponding to the 162nd position in the amino acid sequence shown in Sequence ID No. 1 is an amino acid residue other than a leucine residue, and may be a protein having 3-hydroxybutyrate dehydrogenase activity.

[0014] The amino acid sequence shown in Sequence ID No. 1 is the same as the amino acid sequence of 3-hydroxybutyrate dehydrogenase from Pseudomonas sp. J452 (Sequence ID No. 2), but with the 162nd leucine residue replaced by a phenylalanine residue.

[0015] The protein according to this embodiment may be a protein comprising an amino acid sequence selected from (2) to (6) below and having 3-hydroxybutyrate dehydrogenase activity. (1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10, where the amino acid residue corresponding to the 33rd position in the amino acid sequence shown in SEQ ID NO: 10 is an amino acid residue other than a valine residue. (2) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 11, where the amino acid residue corresponding to the 47th position in the amino acid sequence shown in SEQ ID NO: 11 is an amino acid residue other than a valine residue. (3) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, where the amino acid residue corresponding to the 52nd position in the amino acid sequence shown in SEQ ID NO: 12 is an amino acid residue other than a valine residue. (4) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, where the amino acid residue corresponding to the 54th position in the amino acid sequence shown in SEQ ID NO: 13 is an amino acid residue other than a valine residue. (5) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, where the amino acid residue corresponding to the 96th position in the amino acid sequence shown in SEQ ID NO: 14 is an amino acid residue other than a valine residue.

[0016] The protein according to this embodiment may have 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10, and the amino acid residue corresponding to the 33rd position in the amino acid sequence shown in SEQ ID NO: 10 is an amino acid residue other than a valine residue, and may be a protein having 3-hydroxybutyrate dehydrogenase activity.

[0017] The amino acid sequence shown in Sequence ID No. 10 is the same as the amino acid sequence of 3-hydroxybutyrate dehydrogenase from Pseudomonas sp. J452 (Sequence ID No. 2), but with the 33rd valine residue replaced by an isoleucine residue.

[0018] The protein according to this embodiment may have 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 11, and the amino acid residue corresponding to the 47th position in the amino acid sequence shown in SEQ ID NO: 11 is an amino acid residue other than a valine residue, and may be a protein having 3-hydroxybutyrate dehydrogenase activity.

[0019] The amino acid sequence shown in Sequence ID No. 11 is the same as the amino acid sequence of 3-hydroxybutyrate dehydrogenase from Pseudomonas sp. J452 (Sequence ID No. 2), but with the 47th valine residue replaced by an isoleucine residue.

[0020] The protein according to this embodiment may have 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, and the amino acid residue corresponding to the 52nd position in the amino acid sequence shown in SEQ ID NO: 12 is an amino acid residue other than a valine residue, and may be a protein having 3-hydroxybutyrate dehydrogenase activity.

[0021] The amino acid sequence shown in Sequence ID No. 12 is the same as the amino acid sequence of 3-hydroxybutyrate dehydrogenase from Pseudomonas sp. J452 (Sequence ID No. 2), but with the 52nd valine residue substituted with an isoleucine residue.

[0022] The protein according to this embodiment may have 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and the amino acid residue corresponding to the 54th position in the amino acid sequence shown in SEQ ID NO: 13 is an amino acid residue other than a valine residue, and may be a protein having 3-hydroxybutyrate dehydrogenase activity.

[0023] The amino acid sequence shown in Sequence ID No. 13 is the same as the amino acid sequence of 3-hydroxybutyrate dehydrogenase from Pseudomonas sp. J452 (Sequence ID No. 2), but with the 54th valine residue replaced by an isoleucine residue.

[0024] The protein according to this embodiment may have 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, and the amino acid residue corresponding to the 96th position in the amino acid sequence shown in SEQ ID NO: 14 is an amino acid residue other than a valine residue, and may be a protein having 3-hydroxybutyrate dehydrogenase activity.

[0025] The amino acid sequence shown in Sequence ID No. 14 is the same as the amino acid sequence of 3-hydroxybutyrate dehydrogenase from Pseudomonas sp. J452 (Sequence ID No. 2), but with the 96th valine residue replaced by an isoleucine residue.

[0026] 3-hydroxybutyrate dehydrogenase (hereinafter also referred to as "HBDH") belongs to the short-chain dehydrogenase / reductase (SDR) family and is a β-NAD ＋ It is an enzyme that reversibly catalyzes the oxidation reaction of D-3-hydroxybutyrate to acetoacetic acid using as a coenzyme, and forms a homotetramer structure. In this specification, "3-hydroxybutyrate dehydrogenase activity" refers to the activity that catalyzes the following reaction (1): D-3-hydroxybutyrate + β-NAD ＋ ←→Acetoacetate + β-NADH + H ＋ …(1)

[0027] The protein according to this embodiment only needs to have 80% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 1, 10, 11, 12, 13, or 14. For example, it may be 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or it may be 100%.

[0028] In this specification, sequence identity refers to the percentage (%) of matching bases or residues when the base sequences or amino acid sequences being compared are multiple-aligned. Multiple alignment refers to aligning base sequences or amino acid sequences by inserting appropriate gaps so that corresponding base sequences or amino acid sequence portions are aligned, in order to make the base sequences or amino acid sequences comparable to each other. For multiple alignment, a known multiple alignment program can be used. For example, Crystal W, Crystal X, and the BLAST program can be suitably used.

[0029] The protein according to this embodiment may include an amino acid sequence in which amino acid residues 1-20, 1-15, 1-10, 1-5, and 1-3 are deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NOs: 1, 10, 11, 12, 13, or 14, provided that it has 3-hydroxybutyrate dehydrogenase activity. The substituted, inserted, or added amino acid residues may be native amino acid residues or non-native amino acid residues.

[0030] A protein according to one embodiment contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid residue corresponding to the 162nd position in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than a leucine residue, thus exhibiting good heat resistance. Furthermore, in this protein, at least one amino acid residue selected from the group consisting of the 33rd, 47th, 52nd, 54th, and 96th positions in the amino acid sequence shown in SEQ ID NO: 1 may be an amino acid residue other than a valine residue.

[0031] The protein according to one embodiment contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10, and the amino acid residue corresponding to the 33rd position in SEQ ID NO: 10 is an amino acid residue other than a valine residue, thus having good heat resistance. Furthermore, the protein may also have an amino acid residue other than a leucine residue corresponding to the 162nd position in the amino acid sequence shown in SEQ ID NO: 10, and / or at least one amino acid residue selected from the group consisting of the 47th, 52nd, 54th, and 96th positions in the amino acid sequence shown in SEQ ID NO: 10 may be an amino acid residue other than a valine residue.

[0032] A protein according to one embodiment contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 11, and the amino acid residue corresponding to the 47th position in SEQ ID NO: 11 is an amino acid residue other than a valine residue, thus exhibiting good heat resistance. Furthermore, the protein may also have an amino acid residue other than a leucine residue corresponding to the 162nd position in the amino acid sequence shown in SEQ ID NO: 11, and / or at least one amino acid residue selected from the group consisting of the 33rd, 52nd, 54th, and 96th positions in the amino acid sequence shown in SEQ ID NO: 11 may be an amino acid residue other than a valine residue.

[0033] The protein according to one embodiment contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, and the amino acid residue corresponding to the 52nd position in SEQ ID NO: 12 is an amino acid residue other than a valine residue, thus having good heat resistance. Furthermore, the protein may also have an amino acid residue other than a leucine residue corresponding to the 162nd position in the amino acid sequence shown in SEQ ID NO: 12, and / or at least one amino acid residue selected from the group consisting of the 33rd, 47th, 54th, and 96th positions in the amino acid sequence shown in SEQ ID NO: 12 may be an amino acid residue other than a valine residue.

[0034] The protein according to one embodiment contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and the amino acid residue corresponding to the 54th position in SEQ ID NO: 13 is an amino acid residue other than a valine residue, thus having good heat resistance. Furthermore, the protein may also have an amino acid residue other than a leucine residue corresponding to the 162nd position in the amino acid sequence shown in SEQ ID NO: 13, and / or at least one amino acid residue selected from the group consisting of the 33rd, 47th, 52nd, and 96th positions in the amino acid sequence shown in SEQ ID NO: 13 may be an amino acid residue other than a valine residue.

[0035] The protein according to one embodiment contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, and the amino acid residue corresponding to the 96th position in SEQ ID NO: 14 is an amino acid residue other than a valine residue, thus providing good heat resistance. Furthermore, the protein may also have an amino acid residue other than a leucine residue corresponding to the 162nd position in the amino acid sequence shown in SEQ ID NO: 14, and / or at least one amino acid residue selected from the group consisting of the 33rd, 47th, 52nd, and 54th positions in the amino acid sequence shown in SEQ ID NO: 14 may be an amino acid residue other than a valine residue.

[0036] In this specification, "amino acid residue" means a portion of a peptide or protein molecule that corresponds to a unit of amino acids constituting the peptide or protein. If an optically active form of an "amino acid residue" is present and not specifically designated, it includes L-isomers, D-isomers, and DL-isomers. Furthermore, in this specification, "amino acid residue corresponding to the 162nd amino acid residue in SEQ ID NO: 1" means the amino acid residue in the amino acid sequence of the target protein that corresponds to the position of the 162nd phenylalanine residue in SEQ ID NO: 1 when the amino acid sequence of the target protein is aligned with the amino acid sequence shown in SEQ ID NO: 1. In addition, in this specification, "amino acid residue other than a leucine residue" means an amino acid residue other than an L-leucine residue. Amino acid residues other than leucine residues may be native amino acid residues or unnatural amino acid residues.

[0037] Other native amino acid residues besides leucine residues may be amino acid residues selected from the group consisting of, for example, L-alanine residues, L-asparagine residues, L-aspartic acid residues, L-glutamine residues, L-glutamic acid residues, glycine residues, L-histidine residues, L-isoleucine residues, L-lysine residues, L-arginine residues, L-methionine residues, L-phenylalanine residues, L-proline residues, L-serine residues, L-threonine residues, L-tryptophan residues, L-tyrosine residues, L-valine residues, and L-cysteine ​​residues.

[0038] In this specification, "amino acid residue corresponding to the 33rd amino acid residue in SEQ ID NO: 10" means the amino acid residue in the amino acid sequence of the target protein that, when the amino acid sequence of the target protein is aligned with the amino acid sequence shown in SEQ ID NO: 10, is located at the position corresponding to the 33rd isoleucine residue in SEQ ID NO: 10.

[0039] In this specification, "amino acid residue corresponding to the 47th amino acid residue in SEQ ID NO: 11" means the amino acid residue in the amino acid sequence of the target protein that, when the amino acid sequence of the target protein is aligned with the amino acid sequence shown in SEQ ID NO: 11, is located at the position corresponding to the 47th isoleucine residue in SEQ ID NO: 11.

[0040] In this specification, "amino acid residue corresponding to the 52nd amino acid residue in SEQ ID NO: 12" means the amino acid residue in the amino acid sequence of the target protein that, when the amino acid sequence of the target protein is aligned with the amino acid sequence shown in SEQ ID NO: 12, is located at the position corresponding to the 52nd isoleucine residue in SEQ ID NO: 12.

[0041] In this specification, "amino acid residue corresponding to the 54th amino acid residue in SEQ ID NO: 13" means the amino acid residue in the amino acid sequence of the target protein that, when the amino acid sequence of the target protein is aligned with the amino acid sequence shown in SEQ ID NO: 13, is located at the position corresponding to the 54th isoleucine residue in SEQ ID NO: 13.

[0042] In this specification, "amino acid residue corresponding to the 96th amino acid residue in SEQ ID NO: 14" means the amino acid residue in the amino acid sequence of the target protein that, when the amino acid sequence of the target protein is aligned with the amino acid sequence shown in SEQ ID NO: 14, is located at the position corresponding to the 96th isoleucine residue in SEQ ID NO: 14.

[0043] In this specification, "amino acid residues other than valine residues" means amino acid residues other than L-valine residues. Amino acid residues other than valine residues may be native amino acid residues or non-native amino acid residues.

[0044] Other native amino acid residues besides valine residues may be amino acid residues selected from the group consisting of, for example, L-alanine residues, L-asparagine residues, L-aspartic acid residues, L-glutamine residues, L-glutamic acid residues, glycine residues, L-histidine residues, L-isoleucine residues, L-lysine residues, L-arginine residues, L-methionine residues, L-phenylalanine residues, L-proline residues, L-serine residues, L-threonine residues, L-tryptophan residues, L-tyrosine residues, L-leucine residues, and L-cysteine ​​residues.

[0045] Non-natural amino acid residues other than leucine or valine residues may be amino acid residues selected from the group consisting of, for example, β-amino acid residues, γ-amino acid residues, D-amino acid residues, and α-amino acid residues whose side chains differ from those of natural amino acid residues.

[0046] Herein, in this specification, "side chain of amino acid residue" refers to the group and / or atom bonded to the carbon atom to which the amino group and carboxyl group are bonded (α-carbon) in the case of an α-amino acid residue. For example, the methyl group of Ala is a side chain of an amino acid residue. In the case of a β-amino acid residue, the group and / or atom bonded to the α-carbon and / or β-carbon become the side chain of the amino acid residue, and in the case of a γ-amino acid residue, the group and / or atom bonded to the α-carbon, β-carbon, and / or γ-carbon can become the side chain of the amino acid residue. Furthermore, in this specification, "back chain of amino acid residue" refers to the chain portion composed of the amino group, α-carbon, and carboxyl group in the case of an α-amino acid residue, the chain portion composed of the amino group, β-carbon, α-carbon, and carboxyl group in the case of a β-amino acid residue, and the chain portion composed of the amino group, γ-carbon, β-carbon, α-carbon, and carboxyl group in the case of a γ-amino acid residue.

[0047] Amino acid residues other than leucine residues are not particularly limited, but are preferably amino acid residues having a larger side chain volume than leucine residues. As used herein, the term "side chain volume" means the volume of the space occupied by the side chain of an amino acid residue, can be calculated from the van der Waals radii of the atoms constituting the amino acid, and is described in a known non-patent document (Peptides 29: 1798-1805 (2008)).

[0048] The side chain volume of a leucine residue is 378.76 Å ３ , therefore, "an amino acid residue having a larger side chain volume than a leucine residue" is "378.76 Å ３ can also be rephrased as an amino acid residue having an excess side chain volume.

[0049] The side chain volume of an amino acid residue having a larger side chain volume than a leucine residue is, for example, 400 Å ３ or more, 500 Å ３ or more, 600 Å ３ or more, or 700 Å ３ or more, or 700 Å ３ or less, 800 Å ３ or less, or 900 Å ３ The amino acid residue may have the following side chain volume. The side chain volume of an amino acid residue having a larger side chain volume than a leucine residue is, for example, 400 Å ３ or more and 900 Å ３ or less, 500 Å ３ or more and 900 Å ３ or less, or 500 Å ３ or more and 800 Å ３ The amino acid residue may have the following side chain volume.

[0050] Specifically, the amino acid residue having a larger side chain volume than the leucine residue may be an amino acid residue selected from the group consisting of, for example, phenylalanine residues, tryptophan residues, tyrosine residues, methionine residues, arginine residues, and lysine residues.

[0051] The amino acid residues other than the leucine residue may be hydrophilic or hydrophobic, but hydrophobic amino acid residues are preferred. A "hydrophobic amino acid" is an amino acid that does not have a polar side chain and whose side chain is not charged at pH 7.4 (physiological pH). For example, among naturally occurring amino acids, hydrophobic amino acids are alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and proline.

[0052] If an amino acid residue other than the leucine residue is a hydrophobic amino acid residue, it is preferable that the hydrophobic amino acid residue has a greater degree of hydrophobicity than the leucine residue. In this specification, the degree of hydrophobicity is calculated according to the method described in a known non-patent document (J. Mol. Biol. 179:125-142 (1984)). According to the above non-patent document, the degree of hydrophobicity of the leucine residue is 1.060, so "an amino acid residue with a greater degree of hydrophobicity than the leucine residue" can also be rephrased as "an amino acid residue with a degree of hydrophobicity greater than 1.060".

[0053] The amino acid residues other than the leucine residue mentioned above may be phenylalanine residues.

[0054] The amino acid residues other than the valine residue are not particularly limited, but it is preferable that they are amino acid residues with a larger side chain volume compared to the valine residue. According to known non-patent literature (Peptides 29:1798-1805 (2008)), the side chain volume of the valine residue is 256.74 Å. ３ Therefore, "amino acid residues with a larger side chain volume compared to leucine residues" are "256.74 Å". ３ This can also be rephrased as "amino acid residues with a side chain volume exceeding [a certain value]".

[0055] For amino acid residues with a larger side chain volume compared to valine residues, the side chain volume is, for example, 300 Å. ３ Above 400 Å ３ Above or above, or 500 Å ３ Above or above, or 500 Å ３ Below, 600 Å ３ Below, 700Å３ Below, 800 Å ３ The following, or 900 Å ３ The amino acid residue may have the following side chain volumes. For example, the side chain volume of an amino acid residue with a larger side chain volume compared to a valine residue is 300 Å. ３ The above is 900 Å. ３ Below, 300Å ３ Above 800 Å ３ Below, 300Å ３ More than 700Å ３ Below, 300Å ３ The above is 600 Å. ３ The following, or 300 Å ３ Above 500 Å ３ The amino acid residue may have the following side chain volumes.

[0056] The amino acid residues with a larger side chain volume compared to the valine residues mentioned above may specifically be amino acid residues selected from the group consisting of, for example, leucine residues, isoleucine residues, phenylalanine residues, tryptophan residues, tyrosine residues, methionine residues, glutamine residues, glutamic acid residues, histidine residues, arginine residues, and lysine residues.

[0057] The amino acid residues other than the valine residue may be hydrophilic or hydrophobic, but it is preferable that they be hydrophobic. If the amino acid residues other than the valine residue are hydrophobic, it is preferable that the hydrophobic amino acid residues have a greater degree of hydrophobicity than the valine residue. According to a known non-patent document (J. Mol. Biol. 179:125-142 (1984)), the degree of hydrophobicity of the valine residue is 1.080, so "an amino acid residue with a greater degree of hydrophobicity than the valine residue" can also be rephrased as "an amino acid residue with a degree of hydrophobicity greater than 1.080".

[0058] The amino acid residues other than the valine residue mentioned above may be isoleucine residues.

[0059] The protein according to this embodiment is not particularly limited as long as it contains an amino acid sequence selected from (1) to (6) above and has 3-hydroxybutyrate dehydrogenase activity, for example, it may be a bacterial 3-hydroxybutyrate dehydrogenase, or it may be a modified version of a bacterial 3-hydroxybutyrate dehydrogenase. The protein according to this embodiment is not particularly limited as long as it has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid residue corresponding to the 162nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than a leucine residue, for example, it may be a bacterial 3-hydroxybutyrate dehydrogenase, or it may be a modified version of a bacterial 3-hydroxybutyrate dehydrogenase.

[0060] Examples of the above-mentioned bacteria include bacteria belonging to the genera Pseudomonas, Geobacillus, Rhodobacter, Alcaligenes, and Paraburgholderia, with Pseudomonas being preferred.

[0061] The protein according to this embodiment has good heat resistance. Therefore, the protein according to this embodiment has excellent storage properties. The heat resistance according to this embodiment can be evaluated by measuring the residual activity rate after incubation in a pH 6.5 solution at 50°C or 55°C for 15 minutes, as follows.

[0062] The residual activity of the protein according to this embodiment can be evaluated, for example, by the method described in the examples below. Specifically, a sample solution containing the protein according to this embodiment is diluted with 10 mM PPB (pH 6.5) so that the final concentration of the protein is 0.8 U / mL. Subsequently, the sample solution and 250 mM PPB (pH 6.5) are mixed in a volume ratio of 3:2 and heated in a water bath maintained at 50°C or 55°C for 15 minutes. Immediately after heating, the sample solution is cooled on ice and the 3-hydroxybutyrate dehydrogenase activity is measured according to the method described below. The residual activity (%) of the sample after heating is calculated, with the 3-hydroxybutyrate dehydrogenase activity of the sample solution cooled on ice without heating set as 100%.

[0063] The 3-hydroxybutyrate dehydrogenase activity of the protein according to this embodiment can be evaluated as follows. The reaction described in (1) above is performed using the protein according to this embodiment, and the amount of β-NADH produced is measured by measuring the absorbance of light at a wavelength of 340 nm (A340) for 120 seconds, and the increase in A340 per minute (ΔA) is evaluated. Ｓ ) is calculated. Measurements are also performed with 12.5 μL of deionized water added instead of the substrate (D-3-hydroxybutyrate) solution, and the increase in A340 per minute (ΔA) is calculated. ０ Calculate the measured ΔA. Ｓ and ΔA ０ The value can be used to calculate the following equation. Note that "6.22" in the equation is the millimolar extinction coefficient (mM) of β-NADH for light with a wavelength of 340 nm. －１ cm －１ The formula shows the dilution ratio of the measurement sample containing HBDH. 1 U is defined as the amount of enzyme that produces 1 μmol of β-NADH per minute at 37°C and pH 8.5. (Formula) HBDH activity (U / mL) = (ΔA Ｓ -ΔA ０ )×df×150 / (6.22×5)=(ΔA Ｓ -ΔA ０ ) × df × 4.82

[0064] The protein according to this embodiment may have a residual activity rate of more than 20%, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more after incubation in a pH 6.5 solution at 55°C for 15 minutes. The protein according to this embodiment may have a residual activity rate of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more after incubation in a pH 6.5 solution at 55°C for 15 minutes.

[0065] The protein according to this embodiment is D-3-hydroxybutyric acid and β-NAD ＋ When the activity of the protein according to this embodiment is reacted with D-3-hydroxybutyric acid, it is also possible that when L-3-hydroxybutyric acid is used instead of D-3-hydroxybutyric acid in the reaction, the activity of the protein according to this embodiment may be 1.0% or less, 0.95% or less, 0.9% or less, 0.85% or less, 0.8% or less, 0.75% or less, 0.7% or less, 0.65% or less, 0.6% or less, 0.55% or less, 0.5% or less, 0.45% or less, 0.4% or less, 0.35% or less, 0.3% or less, 0.25% or less, 0.2% or less, 0.15% or less, or 0.1% or less.

[0066] The protein according to this embodiment is D-3-hydroxybutyric acid and β-NAD ＋ When the activity of the protein according to this embodiment is reacted with D-3-hydroxybutyric acid, it is possible that the activity of the protein according to this embodiment will be 0.25% or less, 0.2% or less, 0.15% or less, 0.1% or less, 0.05% or less, or 0.01% or less when L-threonine is used instead of D-3-hydroxybutyric acid in the reaction.

[0067] The protein according to this embodiment is D-3-hydroxybutyric acid and β-NAD ＋ When the activity of the protein according to this embodiment is reacted with β-NAD, the activity is set to 100%. ＋ Instead, β-NADP ＋When reacted using [the specified agent], the activity of the protein according to this embodiment may be 1.0% or less, 0.95% or less, 0.9% or less, 0.85% or less, 0.8% or less, 0.75% or less, 0.7% or less, 0.65% or less, 0.6% or less, 0.55% or less, 0.5% or less, 0.45% or less, 0.4% or less, 0.35% or less, 0.3% or less, 0.25% or less, 0.2% or less, 0.15% or less, or 0.1% or less.

[0068] The protein according to this embodiment can be obtained, for example, by introducing a nucleic acid containing the base sequence encoding the protein into a host cell and expressing it in the host cell.

[0069] [Nucleic Acid] The nucleic acid according to this embodiment includes a base sequence that codes for the protein according to this embodiment. The nucleic acid according to this embodiment may be DNA or RNA.

[0070] A specific example of the nucleic acid according to this embodiment is, for example, a nucleic acid containing a base sequence having 90% or more sequence identity with the base sequence shown in Sequence ID No. 3. The above sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or it may be 100%. Furthermore, the nucleic acid according to this embodiment may be a nucleic acid consisting of the base sequence shown in Sequence ID No. 3.

[0071] The nucleic acid according to this embodiment is not particularly limited as long as it contains a base sequence encoding the protein according to this embodiment. For example, the base sequence may have codons optimized according to the type of host cell to be transformed.

[0072] As described above, the nucleic acids according to this embodiment can be obtained, for example, by designing primers to introduce the desired mutation using the cDNA of a wild-type protein as a template, and then obtaining them by PCR or by artificial gene synthesis.

[0073] [Expression Vector] The expression vector according to this embodiment contains the nucleic acid according to this embodiment. The expression vector according to this embodiment is not particularly limited as long as it contains the nucleic acid according to this embodiment and can express the protein according to this embodiment. For example, it may be a viral vector, a plasmid vector (sometimes simply called a plasmid), a cosmid vector, an artificial chromosome vector, etc., or a fragment thereof. It may be an episomal plasmid vector, a constitutive expression vector, an inducible expression vector, or a transient expression vector. Furthermore, after being introduced into cells, part or all of the introduced plasmid vector may be inserted into the chromosome. It can be appropriately selected depending on the type of cell to be introduced and the purpose.

[0074] The expression vector according to this embodiment may be capable of autonomous replication within the host cell, or it may be incorporated into the host chromosome and replicated along with chromosome replication.

[0075] The expression vector according to this embodiment has, in addition to the nucleic acid according to this embodiment, one or more regulatory sequences operably linked to the base sequence of the nucleic acid. The regulatory sequences are sequences that control protein expression in the host (e.g., promoters, enhancers, ribosome-binding sequences, transcription termination sequences, etc.) and can be appropriately selected depending on the type of host.

[0076] The expression vector according to this embodiment may include the base sequence of a selection marker gene for selecting a transformed strain. Examples of selection marker genes include genes that confer antibiotic resistance and genes that complement nutritional requirements.

[0077] The expression vector according to this embodiment is not particularly limited as long as it contains the base sequence of the nucleic acid according to this embodiment and is capable of expressing the protein of the present invention.

[0078] [Host Cells] The host cells according to this embodiment include the nucleic acid according to this embodiment. The host cells according to this embodiment are not particularly limited as long as they are host cells into which the nucleic acid according to this embodiment has been introduced and which are capable of expressing the protein according to the present invention.

[0079] The host cell according to this embodiment is not particularly limited and may be a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells include bacterial cells. Examples of eukaryotic cells include yeast cells, filamentous fungal cells, insect cells, animal cells, and plant cells.

[0080] The host cells according to this embodiment can be obtained by transforming host cells by introducing the nucleic acid according to this embodiment. The nucleic acid may be introduced into the host cells as is, or it may be introduced into the host cells as an expression vector containing the nucleic acid according to this embodiment. The method for introducing the nucleic acid according to this embodiment can be a method known in the art, depending on the type of nucleic acid or expression vector, the type of host, etc. The nucleic acid according to this embodiment may be introduced by being incorporated into the genome of the host cells according to this embodiment, or it may be introduced without being incorporated into the genome of the host cells according to this embodiment.

[0081] The method for incorporating the nucleic acid according to this embodiment into the genome of a host cell according to this embodiment is not particularly limited and can be carried out by methods commonly used in the art. For example, it can be carried out by methods such as random recombination, homologous recombination, or site-directed recombination.

[0082] The method for introducing the nucleic acid according to this embodiment into the genome of a host cell according to this embodiment without incorporating it is not particularly limited and can be carried out by methods commonly used in the art. If the host cell is a prokaryotic cell, for example, competent cells can be prepared and the process can be carried out by a transformation method using heat shock. If the host cell is a eukaryotic cell, for example, the process can be carried out by a lipofection method using a transfection reagent, a calcium phosphate method, an electroporation method, etc.

[0083] [Composition for measuring D-3-hydroxybutyric acid] The composition for measuring D-3-hydroxybutyric acid according to this embodiment (hereinafter also referred to as "the composition according to this embodiment") contains the protein according to this embodiment.

[0084] As described above, 3-hydroxybutyrate dehydrogenase is sometimes used to measure D-3-hydroxybutyrate. The protein according to this embodiment has 3-hydroxybutyrate dehydrogenase activity and good heat resistance and substrate specificity, so a composition containing the protein according to this embodiment can be suitably used to measure D-3-hydroxybutyrate. The composition according to this embodiment can also be used to constitute, for example, a kit for measuring D-3-hydroxybutyrate according to this embodiment, as described later. It can also be used in the method for measuring D-3-hydroxybutyrate according to this embodiment, as described later.

[0085] In the composition according to this embodiment, the protein content according to this embodiment may be, for example, 1% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, 96% by mass or more, 97% by mass or more, 98% by mass or more, 99% by mass or more, or 100% by mass, based on the total mass of the composition, and may also be 99% by mass or less, 98% by mass or less, 97% by mass or less, 96% by mass or less, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, or 5% by mass or less.

[0086] The composition according to this embodiment may further contain, in addition to the protein according to this embodiment, additives such as excipients, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, and emulsifiers.

[0087] [Kit for Measuring D-3-Hydroxybutyric Acid] The kit for measuring D-3-hydroxybutyric acid according to this embodiment (hereinafter also referred to as "the kit according to this embodiment") comprises a composition containing the protein according to this embodiment. The kit according to this embodiment can be used, for example, in the method for measuring D-3-hydroxybutyric acid according to this embodiment described later.

[0088] The kit according to this embodiment contains β-NAD ＋ The composition may further contain the following. The kit according to this embodiment may also contain the composition containing the protein according to this embodiment, and β-NAD ＋ In addition to the composition containing the above, the composition may also contain a buffer, a surfactant, salts, a protease inhibitor, and a reagent for measuring D-3-hydroxybutyrate (for example, a reagent capable of detecting β-NADH), a sensor for measuring D-3-hydroxybutyrate (for example, a sensor for measuring D-3-hydroxybutyrate according to the present embodiment described later).

[0089] [Sensor for measuring D-3-hydroxybutyrate] The sensor for measuring D-3-hydroxybutyrate according to this embodiment (hereinafter also referred to as "the sensor according to this embodiment") includes an electrode on which the protein according to this embodiment is immobilized. The electrode is β-NAD ＋ It would be fine if that were even more fixed.

[0090] The sensor according to this embodiment may have a working electrode and a counter electrode, or a working electrode, a counter electrode, and a reference electrode. A carbon electrode, a gold electrode, a platinum electrode, etc., can be used as the working electrode, and the protein according to this embodiment can be immobilized on the working electrode. The electrode may contain the protein according to this embodiment and / or β-NAD. ＋Methods for immobilizing the protein include, for example, using a crosslinking reagent, encapsulating it in a polymer matrix, coating it with a dialysis membrane, and using a photocrosslinkable polymer, a conductive polymer, or a redox polymer. Alternatively, the protein may be immobilized in a polymer together with a mediator or adsorbed onto an electrode. These methods may be combined. When immobilizing the protein according to this embodiment onto an electrode, this may be done by immobilizing the composition according to this embodiment.

[0091] More specifically, for example, using glutaraldehyde to produce the protein and / or β-NAD according to this embodiment. ＋ After immobilizing the material on a carbon electrode, the glutaraldehyde is blocked by treating it with a reagent containing an amine group. The sensor according to this embodiment can be used for various electrochemical measurements. Examples of electrochemical measurement methods include amperometry, such as chronoamperometry and potential-step chronoamperometry; voltammetry, such as cyclic voltammetry, differential pulse voltammetry, potentiometry, and coulometry.

[0092] For example, when a sample containing D-3-hydroxybutyric acid is brought into contact with an electrode, the D-3-hydroxybutyric acid in the sample reacts with the protein according to this embodiment, which is immobilized on the electrode, generating a weak electric current via β-NADH. The strength of the current depends on the concentration of D-3-hydroxybutyric acid in the sample brought into contact with the electrode. Therefore, the concentration of D-3-hydroxybutyric acid can be measured by measuring the current.

[0093] [Method for measuring D-3-hydroxybutyric acid] The method for measuring D-3-hydroxybutyric acid according to this embodiment involves a sample containing D-3-hydroxybutyric acid and β-NAD ＋ The method includes a step of contacting the protein according to this embodiment with the protein to produce acetoacetic acid and β-NADH (production step), and a step of calculating the amount of D-3-hydroxybutyric acid based on the signal caused by the β-NADH (calculation step).

[0094] In the production process, a sample containing D-3-hydroxybutyric acid and β-NAD ＋ By contacting the protein according to this embodiment, D-3-hydroxybutyric acid and β-NAD contained in the sample are released. ＋ The protein according to this embodiment is then reacted with the protein to produce acetoacetic acid and β-NADH (reaction (1) above).

[0095] A sample containing D-3-hydroxybutyric acid (hereinafter also simply referred to as "sample") is a solution in which D-3-hydroxybutyric acid is dissolved. The sample is not particularly limited as long as it contains D-3-hydroxybutyric acid, but may be derived from a specimen obtained from a living organism, for example. The specimen may be at least one selected from the group consisting of blood (whole blood, plasma, or serum), interstitial fluid, urine, and sweat.

[0096] The production process involves the sample and β-NAD ＋ The step may involve mixing the sample with the protein according to this embodiment and incubating it. ＋ The order in which the protein according to this embodiment is mixed is arbitrary.

[0097] The production process involves the sample and β-NAD ＋ The sample, the protein immobilized on the sensor according to this embodiment, and β-NAD may be brought into contact with the sample, the protein immobilized on the sensor according to this embodiment, and β-NAD ＋ They may be brought into contact with each other.

[0098] In the reaction system of the production process, the concentration of the protein according to this embodiment is not particularly limited, but may be 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, or 0.1% by mass or more, relative to the total amount of the reaction system, or it may be 10% by mass or less, 1% by mass or less, or 0.1% by mass or less.

[0099] In the reaction system of the production process, β-NAD ＋The concentration is not particularly limited, but for example, it may be 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, or 0.1% by mass or more, relative to the total amount of the reaction system, or it may be 1% by mass or less, 0.1% by mass or less, or 0.01% by mass or less.

[0100] The contact time in the production process is not particularly limited as long as acetoacetic acid and β-NADH are produced. For example, it may be 30 seconds or more, 1 minute or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, or 1 hour or more, and may also be 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less.

[0101] The temperature in the production process is not particularly limited as long as acetoacetic acid and β-NADH are produced. For example, it may be 25°C or higher, 30°C or higher, or 35°C or higher, and may also be 55°C or lower, 50°C or lower, 45°C or lower, or 40°C or lower.

[0102] The pH in the production process is not particularly limited as long as acetoacetic acid and β-NADH are produced. For example, it may be 5.0 or higher, 5.5 or higher, 6.0 or higher, 6.5 or higher, or 7.0 or higher, or 9.0 or lower, 8.5 or lower, 8.0 or lower, or 7.5 or lower.

[0103] The production process involves the sample and β-NAD ＋ In addition to the proteins according to this embodiment, the process may also be carried out in the presence of buffers, water, pH adjusters, surfactants, metal ions, protease inhibitors, etc.

[0104] In the calculation step, the amount of D-3-hydroxybutyrate in the sample is calculated based on the signal caused by β-NADH produced in the contact step. In this specification, "signal caused by β-NADH" means a measurable physical or chemical change that occurs due to the presence of β-NADH.

[0105] Signals caused by β-NADH include, for example, changes in absorbance (e.g., changes in absorbance at 340 nm), electric current, luminescence, and colorimetric analysis. The calculation process can be carried out by a known method as appropriate, taking into consideration the reaction equation in (1) above and other necessary values.

[0106] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0107] [Test Example 1: Heat Resistance Test of 3-Hydroxybutyrate Dehydrogenase] A heat resistance test was conducted on HBDH derived from Pseudomonas sp. J452 (HBDH consisting of the amino acid sequence shown in SEQ ID NO: 2 (hereinafter also referred to as "HBDH_2").

[0108] (Construction of a plasmid for 1-1:3-hydroxybutyrate dehydrogenase (HBDH) expression) Twelve bases (SEQ ID NO: 4) were added upstream of the 5' start codon (atg) and twelve bases (SEQ ID NO: 5) downstream of the 3' stop codon (taa) of the base sequence encoding HBDH_2 in SEQ ID NO: 1, and the plasmid was synthesized by Integrated DNA Technologies.

[0109] Plasmid fragments were prepared by PCR using primers SEQ ID NO: 6 and SEQ ID NO: 7, with the reaction mixture shown in Table 1, followed by 35 cycles of 98°C for 10 seconds, 53°C for 5 seconds, and 68°C for 20 seconds. After PCR, 1.0 μL of DpnI (New England BioLabs) was added to the solution and treated at 37°C for 1 hour to degrade the template plasmid, after which the PCR product was purified.

[0110]

[0111] The above PCR products were subjected to an in-fusion reaction at 50°C for 15 minutes with the composition shown in Table 2 to obtain an HBDH_2 expression plasmid (pETBA-HBDH_2). Subsequently, Escherichiacol JM109 strain was transformed with the obtained plasmid. The resulting transformants were cultured, and the base sequence of the extracted plasmid was confirmed to be the desired sequence by DNA sequencing analysis.

[0112]

[0113] (1-2: Recombinant production of HBDH_2) E. coli BL21 (DE3) strain was transformed with pETBA-HBDH_2 containing the nucleotide sequence encoding HBDH_2 to produce an HBDH_2-producing strain. The HBDH_2-producing strain was inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 μg / mL) in a test tube and cultured overnight at 37°C and 180 rpm. 5 mL of HBDH_2 production medium (LB medium containing 0.2% glycerol, 0.02% D-glucose, 0.08% D-lactose, 50 mM potassium dihydrogen phosphate, 50 mM disodium hydrogen phosphate, 25 mM ammonium sulfate, 1 mM magnesium sulfate, and 100 μg / mL ampicillin) was prepared, and 25 μL of seed culture was inoculated therein. The mixture was incubated at 25°C and 180 rpm for 16 hours. After incubation, the culture medium was centrifuged at 15,000 rpm for 1 minute to obtain a pellet, which was then resuspended in 1 mL of 25 mM potassium phosphate buffer (PPB) pH 7.5. After sonication of the bacterial suspension, the supernatant obtained by centrifuging at 15,000 rpm for 10 minutes was collected and used as a crude enzyme solution containing HBDH_2.

[0114] (1-3: Heat resistance test of HBDH_2) The crude enzyme solution containing HBDH_2 was diluted with 10 mM PPB (pH 6.5) to a final concentration of HBDH_2 of 0.8 U / mL. Subsequently, the crude enzyme solution containing HBDH_2 and 250 mM PPB (pH 6.5) were mixed in a volume ratio of 3:2 and heated in a water bath maintained at 50°C for 15 minutes. Immediately after heating, the crude enzyme solution containing HBDH_2 was cooled on ice, and the HBDH_2 activity was measured according to the method described in "1-4: Measurement of HBDH_2 activity" below. The residual activity of the sample after heating was calculated, with the activity of the HBDH_2 sample cooled on ice without heating set as 100%.

[0115] (1-4: Activity measurement of HBDH_2) β-NAD oxidation occurs simultaneously with the oxidation of D-3-hydroxybutyrate ＋The amount of β-NADH produced by the reduction reaction was measured by optical quantification using a spectrophotometer (Gallery Plus Enzyme Master enzyme analyzer (Thermo Fisher Scientific) or U-3900 (Hitachi High-Tech Science)) to determine the activity of HBDH_2.

[0116] For the 150 μL of activity measurement reagents shown in Table 3, 145 μL of the reagent before adding the crude enzyme solution containing HBDH_2 was incubated at 37°C for 2 minutes, and then 5 μL of the crude enzyme solution containing HBDH_2 was added and mixed. The crude enzyme solution containing HBDH_2 was used diluted as needed with 100 mM PPB (pH 8.5) containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich). After adding the crude enzyme solution containing HBDH_2, the mixture was incubated at 37°C for 40 seconds, and the absorbance of light at a wavelength of 340 nm (A340) was measured for 120 seconds. The increase in A340 per minute (ΔA) was then measured. Ｓ The following was calculated: Measurements were also performed with 12.5 μL of deionized water added instead of the substrate (D-3-hydroxybutyrate) solution, and the increase in A340 per minute (ΔA) was calculated. ０ The following was calculated. When using the U-3900 spectrophotometer, five times the amount of activity assay reagent was used per measurement.

[0117]

[0118] The HBDH₂ activity (U / mL) was calculated based on the following formula. 1 U is defined as the amount of enzyme that produces 1 μmol of β-NADH per minute at 37°C and pH 8.5. In the formula, "6.22" represents the millimolar extinction coefficient (mM) of β-NADH for light at a wavelength of 340 nm. －１ cm －１ (Formula) df indicates the dilution ratio of the crude enzyme solution containing HBDH_2. (Formula) HBDH_2 activity (U / mL) = (ΔA Ｓ -ΔA ０ )×df×150 / (6.22×5)=(ΔA Ｓ -ΔA ０ ) × df × 4.82

[0119] As a result, the residual activity rate of HBDH_2 was 80%. It is known that the HBDH content of commercial product A is less than 10% when incubated at 50°C for 10 minutes in a pH 8.5 solution, and the HBDH content of commercial product B is less than 20% when incubated at 50°C for 15 minutes in a pH 6.5 solution (as stated in the catalogs for commercial products A and B). HBDH_2 exhibited superior heat resistance compared to the HBDH content of commercial products A and B.

[0120] [Test Example 2: Search for useful mutations in 3-hydroxybutyrate dehydrogenase] <2-1: HBDH_2 / L162F> A mutation (L162F) was introduced into the amino acid sequence (SEQ ID NO: 1) of HBDH_2, in which the 162nd leucine residue was replaced with a phenylalanine residue. This mutation was introduced into HBDH_2 (amino acid sequence: SEQ ID NO: 1, also called "HBDH_2 / L162F") and a heat resistance test was performed in the same manner as in Test Example 1. The specific procedure is as follows.

[0121] (2-1-1: Construction of a plasmid for modified HBDH expression) The L162F mutation described above was introduced in a site-specific manner using pETBA-HBDH_2 as a template to obtain a plasmid containing the gene encoding modified HBDH_2 (SEQ ID NO: 3). The PCR reaction mixture was prepared by mixing 10 μL of KOD one PCR Master Mix (Toyobo), 1.2 μL of 5 μM Fw primer (SEQ ID NO: 8), 1.2 μL of 5 μM Rv primer (SEQ ID NO: 9), 0.5 μL of 40 μg / mL template DNA, and 7.1 μL of deionized water. The PCR reaction conditions were "98°C for 10 seconds → 55°C for 5 seconds → 68°C for 15 seconds" and this cycle was repeated 15 times.

[0122] After PCR, 1 μl of DpnI was added to the solution and treated at 37°C for 1 hour to degrade the template DNA. The resulting solution was used to transform E. coli JM109 strain. The resulting transformants were cultured, and the plasmid extracted was confirmed to have the desired sequence by DNA sequencing analysis.

[0123] (2-1-2: Heat Resistance Test) Using the plasmid obtained in 2-1-1, recombinant production was carried out in the same manner as in 1-2 above to prepare a crude enzyme solution containing HBDH_2 / L162F. A crude enzyme solution containing HBDH_2 was also prepared in the same manner. Furthermore, a heat resistance test was performed in the same manner as in Test Example 1, except that the solutions were heated in a water bath at 50°C or 55°C for 15 minutes, and either the crude enzyme solution containing HBDH_2 or the crude enzyme solution containing HBDH_2 / L162F was used. The results are shown in Table 4.

[0124]

[0125] The crude enzyme solution containing HBDH_2 / L162F showed a higher residual activity rate compared to the crude enzyme solution containing HBDH_2, even when incubated in a pH 8.5 solution at 50°C or 55°C for 15 minutes.

[0126] <2-2: HBDH_2 / V33I, HBDH_2 / V47I, HBDH_2 / V52I, HBDH_2 / V54I, HBDH_2 / V96I> A heat resistance test was performed on HBDH_2 (amino acid sequences: SEQ ID NOs: 10, 11, 12, 13, or 14, also known as "HBDH_2 / V33I", "HBDH_2 / V47I", "HBDH_2 / V52I", "HBDH_2 / V54I", or "HBDH_2 / V96I") into which mutations (V33I, V47I, V52I, V54I, or V96I) were introduced in which the valine residue at positions 33, 47, 52, 54, or 96 in the amino acid sequence of HBDH_2 (SEQ ID NO: 2) were replaced with an isoleucine residue. The specific procedure is as follows.

[0127] (2-2-1: Construction of HBDH expression plasmid) A reaction solution similar to that shown in Table 1 was prepared, except that primers SEQ ID NO: 15 and SEQ ID NO: 16 were used, and pKK223-3 was used instead of pETBA as the template DNA. The steps of 98°C for 10 seconds, 60°C for 5 seconds, and 68°C for 25 seconds were repeated for 35 cycles to prepare a fragment of the pKK223-3 plasmid by PCR.

[0128] A reaction solution similar to that shown in Table 1 was prepared, except that primers for Sequence ID No. 17 and Sequence ID No. 18 were used, and pETBA-HBDH_2 was used instead of pETBA as the template DNA. The HBDH_2 gene fragment was prepared by PCR by repeating the steps of 98°C for 10 seconds, 60°C for 5 seconds, and 68°C for 5 seconds for 35 cycles.

[0129] After PCR, 1.0 μL of DpnI (New England BioLabs) was added to the solution and treated at 37°C for 1 hour to degrade the template plasmid. The PCR products were then purified.

[0130] The above PCR products were subjected to an in-fusion reaction at 50°C for 15 minutes with the composition shown in Table 5 to obtain an HBDH_2 expression plasmid (pKK223-3-HBDH_2). Subsequently, the Escherichiacol JM109 strain was transformed with the obtained plasmid. The resulting transformants were cultured, and the base sequence of the extracted plasmid was confirmed to be the desired sequence by DNA sequencing analysis.

[0131]

[0132] Using pKK223-3-HBDH_2 as a template, the V33I mutation was introduced in a site-specific manner to obtain a plasmid containing the gene encoding the modified HBDH_2. The PCR reaction mixture was prepared by mixing 10 μL of KOD one PCR Master Mix (Toyobo), 1.2 μL of 5 μM Fw primer (SEQ ID NO: 19), 1.2 μL of 5 μM Rv primer (SEQ ID NO: 20), 0.5 μL of 40 μg / mL template DNA, and 7.1 μL of deionized water. The PCR reaction conditions were "98°C for 10 seconds → 55°C for 5 seconds → 68°C for 25 seconds" and this cycle was repeated 15 times.

[0133] Except for using the primer sets of SEQ ID NOs. 21 and 22, PCR was performed using the same procedure as when introducing the V33I mutation, thereby introducing the V47I mutation in a site-specific manner and obtaining a plasmid containing the gene encoding the modified HBDH_2.

[0134] Except for using the primer sets of SEQ ID NOs. 23 and 24, PCR was performed using the same procedure as when introducing the V33I mutation, thereby introducing the V52I mutation in a site-specific manner and obtaining a plasmid containing the gene encoding the modified HBDH_2.

[0135] PCR was performed using the same procedure as when introducing the V33I mutation, except for using the primer sets of SEQ ID NOs. The V54I mutation was introduced in a site-specific manner, yielding a plasmid containing the gene encoding the modified HBDH_2.

[0136] PCR was performed using the same procedure as when introducing the V33I mutation, except for using the primer sets of SEQ ID NOs. The V96I mutation was introduced in a site-specific manner, yielding a plasmid containing the gene encoding the modified HBDH_2.

[0137] After PCR, 1 μl of DpnI was added to the solution and treated at 37°C for 1 hour to degrade the template DNA. The resulting solution was used to transform E. coli JM109 strain. The resulting transformants were cultured, and the plasmid extracted was confirmed to have the desired sequence by DNA sequencing analysis.

[0138] (2-2-2: Recombinant production of modified HBDH_2) The production of modified HBDH_2 (HBDH_2 / V33I, HBDH_2 / V47I, HBDH_2 / V52I, HBDH_2 / V54I, or HBDH_2 / V96I) by introducing mutations V33I, V47I, V52I, V54I, or V96I was carried out as follows.

[0139] A modified HBDH_2 producing strain obtained by transforming E. coli JM109 was inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 μg / mL) in a test tube and cultured overnight at 37°C and 180 rpm. 25 μL of the seed culture was inoculated into 5 mL of LB-amp medium and cultured at 25°C and 180 rpm for 16 hours. After the culture was complete, the culture was centrifuged at 15,000 rpm for 1 minute to obtain a pellet, which was then resuspended in 1 mL of 25 mM potassium phosphate buffer (PPB) pH 7.5. After sonication of the bacterial suspension, the supernatant obtained by centrifugation at 15,000 rpm for 10 minutes was collected and used as a crude enzyme solution containing HBDH_2.

[0140] (2-2-3: Heat Resistance Test) A heat resistance test was conducted in the same manner as in Test Example 1, except that a crude enzyme solution containing HBDH_2 / V33I, HBDH_2 / V47I, HBDH_2 / V52I, HBDH_2 / V54I, or HBDH_2 / V96I was used, and the crude enzyme solution containing the modified HBDH_2 was heated in a water bath at 50°C or 55°C for 15 minutes. The results are shown in Table 6.

[0141]

[0142] Crude enzyme solutions containing HBDH_2 / V33I, HBDH_2 / V47I, HBDH_2 / V52I, HBDH_2 / V54I, and HBDH_2 / V96I showed higher residual activity compared to crude enzyme solutions containing HBDH_2, even when incubated at 50°C or 55°C for 15 minutes in a pH 8.5 solution.

[0143] [Test Example 3: Characterization of Purified 3-Hydroxybutyrate Dehydrogenase] HBDH_2 and HBDH_2 / L162F were purified, and their enzyme properties were evaluated. The specific procedure is as follows.

[0144] (3-1: Purification of HBDH_2 and HBDH_2 / L162F) A crude enzyme solution containing HBDH_2 or HBDH_2 / L162F was applied to HiScreen Q Sepharose Fast Flow (Cytiva, Column Volume 4.7 mL) equilibrated with 10 mM PPB pH 7.5 and bound to an anion exchange resin. The resin was then washed with 10 mM PPB (pH 7.5) containing 47 mL (10 CV) of 150 mM NaCl and 1 mM EDTA, and the NaCl concentration in the 10 mM PPB (pH 7.5) was linearly increased at a rate of 10 mM / CV while the solution was delivered, eluting the HBDH_2 or HBDH_2 / L162F bound to the resin. The purity of each eluted fraction was evaluated by SDS-PAGE, and fractions free of contaminating proteins were collected and used as purified standards for HBDH_2 or HBDH_2 / L162F. The purified standards for HBDH_2 or HBDH_2 / L162F were concentrated and dialyzed using Amicon Ultra Ultracel-10K (Merck) as needed, and used for evaluating enzyme properties.

[0145] (3-2: Evaluation of Substrate Specificity of HBDH_2 and HBDH_2 / L162F) The activity of purified HBDH_2 and purified HBDH_2 / L162F was measured using D-3-hydroxybutyric acid and various hydroxy acids (shown in Figure 1), except that purified HBDH_2 and purified HBDH_2 / L162F were used, in accordance with the method described in "1-3. Measurement of HBDH_2 Activity". The relative activity (%) when using various hydroxy acids was calculated, with the activity when using D-3-hydroxybutyric acid set as 100%. Compounds whose names begin with "DL-" indicate that the activity was measured using a racemic mixture of the D and L forms. The results are shown in Figure 1.

[0146] The relative activity (%) for hydroxy acids other than D-3-hydroxybutyrate was highest for L-3-hydroxybutyrate at 0.32%, followed by L-threonine at 0.07%. This revealed that HBDH_2 / L162F exhibits extremely strict substrate specificity.

[0147] (3-3: Heat resistance evaluation of HBDH_2 and HBDH_2 / L162F) The residual activity of purified HBDH_2 and purified HBDH_2 / L162F was measured using the same method as in Test Example 1, except that purified HBDH_2 and purified HBDH_2 / L162F were used, and the heat resistance of these HBDHs was evaluated. The results are shown in Table 7.

[0148]

[0149] HBDH_2 / L162F showed higher residual activity compared to HBDH_2, and HBDH_2 / L162F exhibited greater heat resistance than HBDH_2. The L162F mutation (a mutation in which the 162nd leucine residue in the amino acid sequence of HBDH_2 is replaced with phenylalanine) was found to improve the thermal stability of HBDH_2. Even after treatment at 55°C for 15 minutes, when HBDH_2 is almost completely inactivated, HBDH_2 / L162F retained 61% of its activity.

[0150] As mentioned above, it is known that the HBDH content of commercial product A is less than 10% when incubated at 50°C for 10 minutes in a pH 8.5 solution, and the HBDH content of commercial product B is less than 20% when incubated at 50°C for 15 minutes in a pH 6.5 solution. HBDH_2 / L162F showed significantly higher heat resistance compared to these commercial HBDH products.

[0151] (3-4: Evaluation of coenzyme specificity of HBDH_2 / L162F) β-NAD ＋ Instead, the same concentration of β-NADP ＋ Using (Oriental Yeast Co., Ltd.), the HBDH_2 / L162F activity was measured by the method described in "1-3. Measurement of HBDH activity", and β-NAD ＋ Taking the activity when using as 100%, β-NADP ＋ The relative activity (%) was calculated when using [the enzyme]. Purified HBDH_2 / L162F was used as the enzyme.

[0152] As a result, β-NADP of HBDH_2 / L162F ＋ The relative activity when using [the specified enzyme] was 0.65%, clearly demonstrating extremely strict coenzyme specificity.

Claims

1. A protein having 3-hydroxybutyrate dehydrogenase activity and containing an amino acid sequence selected from (1) to (6) below. (1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, wherein the amino acid residue corresponding to the 162nd position in the amino acid sequence shown in Sequence ID No. 1 is an amino acid residue other than a leucine residue. (2) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 10, wherein the amino acid residue corresponding to the 33rd position in the amino acid sequence shown in Sequence ID No. 10 is an amino acid residue other than a valine residue. (3) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 11, wherein the amino acid residue corresponding to the 47th position in the amino acid sequence shown in Sequence ID No. 11 is an amino acid residue other than a valine residue. (4) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 12, wherein the amino acid residue corresponding to the 52nd position in the amino acid sequence shown in Sequence ID No. 12 is an amino acid residue other than a valine residue. (6) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 14, wherein the amino acid residue corresponding to the 96th position in the amino acid sequence shown in Sequence ID No. 14 is an amino acid residue other than a valine residue.

2. A protein having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, wherein the amino acid residue corresponding to the 162nd position in the amino acid sequence shown in Sequence ID No. 1 is an amino acid residue other than a leucine residue, and possessing 3-hydroxybutyrate dehydrogenase activity.

3. The protein according to claim 1, wherein the amino acid residues other than the leucine residue are amino acid residues that have a larger side chain volume compared to the leucine residue.

4. The protein according to claim 3, wherein the amino acid residue having a larger side chain volume compared to the leucine residue is an amino acid residue selected from the group consisting of phenylalanine residue, tryptophan residue, tyrosine residue, methionine residue, arginine residue, and lysine residue.

5. The protein according to claim 1, wherein the amino acid residues other than the leucine residue are hydrophobic amino acid residues.

6. The protein according to claim 5, wherein the hydrophobic amino acid residue is an amino acid residue that has a greater degree of hydrophobicity compared to the leucine residue.

7. The protein according to claim 1, wherein the amino acid residue other than the leucine residue is a phenylalanine residue.

8. The protein according to claim 1, wherein the amino acid residues other than the valine residue are amino acid residues that have a larger side chain volume compared to the valine residue.

9. The protein according to claim 8, wherein the amino acid residue having a larger side chain volume compared to the valine residue is an amino acid residue selected from the group consisting of leucine residue, isoleucine residue, phenylalanine residue, tryptophan residue, tyrosine residue, methionine residue, glutamine residue, glutamic acid residue, histidine residue, arginine residue, and lysine residue.

10. The protein according to claim 1, wherein the amino acid residues other than the valine residue are hydrophobic amino acid residues.

11. The protein according to claim 10, wherein the hydrophobic amino acid residue is an amino acid residue that has a greater degree of hydrophobicity compared to the valine residue.

12. The protein according to claim 1, wherein the amino acid residues other than the valine residue are isoleucine residues.

13. A nucleic acid comprising a base sequence encoding a protein according to any one of claims 1 to 12.

14. An expression vector comprising the nucleic acid described in claim 13.

15. A host cell comprising the nucleic acid described in claim 13.

16. A composition for measuring D-3-hydroxybutyric acid, comprising the protein described in any one of claims 1 to 12.

17. A kit for measuring D-3-hydroxybutyrate, comprising a composition containing the protein described in any one of claims 1 to 12.

18. A sensor for measuring D-3-hydroxybutyrate, comprising an electrode on which the protein according to any one of claims 1 to 12 is immobilized.

19. Sample containing D-3-hydroxybutyric acid and β-NAD ＋ A method for measuring D-3-hydroxybutyric acid, comprising the steps of: contacting a protein according to any one of claims 1 to 12 with a protein to produce acetoacetic acid and β-NADH; and calculating the amount of D-3-hydroxybutyric acid based on a signal caused by the β-NADH.

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