Modified enzyme

Abstract

The purpose of the present invention is to provide an HBDH of which the enzymatic activity after being treated at least at one of prescribed temperatures in a temperature range of 40°C or higher is improved compared with the enzymatic activity of an enzyme comprising the amino acid sequence represented by SEQ ID NO: 1 after being treated at the given temperature.　Provided is a modified HBDH comprising an amino acid sequence having such a structure that a substitution with a specific amino acid residue is introduced into at least any one position selected from position-47, position-48, position-49, position-53, position-54, position-55, position-63, position-81, position-147, position-210, position-240, position-241, position-250, position-251 and position-252 in the amino acid sequence represented by SEQ ID NO: 1. The modified HBDH is improved in terms of HBDH activity after being treated at least at one of prescribed temperatures in a temperature range of 40°C or higher compared with the HBDH activity of an enzyme comprising the amino acid sequence represented by SEQ ID NO: 1 after being treated at the same temperature.

Description

Modified enzymes

[0001] The present invention relates to modified enzymes.

[0002] D-3-hydroxybutyrate dehydrogenase (HBDH: EC 1.1.1.30) belongs to the short-chain dehydrogenase / reductase (SDR) family and is a hydroxylase that converts NAD + It is an enzyme that reversibly catalyzes the oxidation of 3-hydroxybutyrate to acetoacetate using the coenzyme α. Hydroxybutyrate, together with acetoacetate and acetone, is called a ketone body.

[0003] A rapid increase in blood ketone body concentrations is known to cause ketoacidosis (DKA), a severe complication of type 1 diabetes. Therefore, monitoring the ketone body levels of type 1 diabetes patients is recommended to prevent complications. HBDH is an industrially important enzyme used for measuring ketone bodies.

[0004] HBDH has been isolated from a variety of microorganisms. For example, Patent Document 1 discloses HBDH derived from Rhodobacter sphaeroides, Non-Patent Document 1 discloses HBDH derived from Paracoccus denitrificans, Non-Patent Document 2 discloses HBDH derived from Ralstonia pickettii T1, Non-Patent Document 3 discloses HBDH derived from Mycobacterium phlei ATCC354, Non-Patent Document 4 discloses HBDH derived from Staphylococcus xylosus, Non-Patent Document 5 discloses HBDH derived from Pseudomonas fragii, Non-Patent Document 6 discloses HBDH derived from Alcaligenes faecalis, and Non-Patent Document 7 discloses HBDH derived from Acidovorax sp. Non-patent document 8 discloses HBDH derived from Bacillus cereus T, and Non-patent document 9 discloses HBDH derived from Zoogloea ramigera I-16-M.

[0005] Although HBDH derived from microorganisms is unstable to heat, Patent Documents 2 and 3 disclose that HBDH derived from Alcaligenes faecalis and HBDH derived from Pseudomonas species, respectively, are HBDHs with excellent thermostability, stable even at 37°C.

[0006] JP-A No. 11-318438 JP-A No. 8-70856 JP-A No. 2003-339385

[0007] Biochim. Biophys. Acta 871, 302-309 (1986)J. Biosci. Bioeng. 101, 501-507 (2006)J. Gen. Microbiol. 104, 123-126 (1978)Acta Microbiol. Pol. 43, 33-45 (1994)J. Mol. Biol. 355, 722-733 (2006)Acta Crystallogr. Sect. F 65, 331-335 (2009)J. Biosci. Bioeng. 97, 78-81 (2004)Can. (1981)

[0008] Considering the industrial importance of HBDH, it is desirable to create a thermostable HBDH that exhibits improved activity after heat treatment compared to the wild type.

[0009] Therefore, the present invention aims to provide an HBDH whose enzymatic activity after treatment at at least any predetermined temperature in a temperature range of 40°C or higher is improved compared to the enzymatic activity of wild-type HBDH after treatment at the predetermined temperature.

[0010] The present inventors attempted to modify HBDH derived from Ralstonia pickettii to impart heat resistance so as to enhance activity after heat treatment. However, despite creating many mutants, few mutants possessed such heat resistance. Among these, the inventors fortuitously discovered that a few mutants possessed heat resistance such that the enzymatic activity after treatment at at least one predetermined temperature in the temperature range of 40°C or higher was improved compared to the enzymatic activity of wild-type HBDH after treatment at said predetermined temperature. That is, the present invention provides the following aspects.

[0011] Item 1. A modified enzyme consisting of a polypeptide shown in any one of (I) to (III) below: (I) In the amino acid sequence shown in SEQ ID NO: 1, [A] substitution of the amino acid residue at position 47 with a methionine residue, [B] substitution of the amino acid residue at position 48 with a histidine residue, [C] substitution of the amino acid residue at position 49 with a leucine residue, [D] substitution of the amino acid residue at position 53 with a phenylalanine residue, a leucine residue, a proline residue, or a glutamic acid residue, [E] substitution of the amino acid residue at position 54 with a leucine residue, an alanine residue, or a phenylalanine residue, [F] substitution of the amino acid residue at position 55 with a glycine residue, a glutamic acid residue, a methionine residue, or a phenylalanine residue, [G] substitution of the amino acid residue at position 63 with a leucine residue, [H] substitution of the amino acid residue at position 81 with an isoleucine residue, [I] substitution of the amino acid residue at position 147 with a glycine residue, [J] substitution of the amino acid residue at position 210 with a valine residue. (II) A polypeptide having an amino acid sequence into which at least one of the following substitutions has been introduced: [K] substitution of the amino acid residue at position 240 with an alanine residue, [L] substitution of the amino acid residue at position 241 with a phenylalanine residue, [M] substitution of the amino acid residue at position 250 with a valine residue, [N] substitution of the amino acid residue at position 251 with a proline residue, and [O] substitution of the amino acid residue at position 252 with an isoleucine residue; (II) A polypeptide having an amino acid sequence into which at least one of the substitutions shown in [A] to [O] has been introduced, in which one or several amino acid residues other than the substituted amino acid residue have been substituted, added, inserted, or deleted, and which has an improved 3-hydroxybutyrate dehydrogenase activity after at least one predetermined temperature treatment in a temperature range of 40°C or higher compared to the activity of a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 after the predetermined temperature treatment;(III) A polypeptide having an amino acid sequence into which at least one of the substitutions shown in [A] to [O] has been introduced, wherein the sequence identity of the portion excluding the substituted amino acid residue is 70% or more, and wherein the 3-hydroxybutyrate dehydrogenase activity after at least one predetermined temperature treatment in a temperature range of 40°C or higher is improved compared to the activity after the predetermined temperature treatment of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. Item 2. DNA encoding the modified enzyme according to Item 1. Item 3. An expression cassette or recombinant vector comprising the DNA according to Item 2. Item 4. A transformant obtained by transforming a host with the expression cassette or recombinant vector according to Item 3. Item 5. A method for producing the modified enzyme, comprising the step of culturing the transformant according to Item 4. Item 6. An enzyme preparation comprising the modified enzyme according to Item 1. Item 7. A sensor for measuring ketone bodies, comprising the modified enzyme according to Item 1. Item 8. A kit for measuring ketone bodies, comprising the modified enzyme according to Item 1. Item 9. Item 1. A method for measuring ketone bodies in a sample, comprising the step of contacting the sample in which ketone bodies are to be measured with the modified enzyme under conditions for the activity of the modified enzyme according to Item 1.

[0012] According to the present invention, a modified HBDH is provided which has improved enzymatic activity after treatment at at least any predetermined temperature in a temperature range of 40°C or higher compared to the enzymatic activity of wild-type HBDH after treatment at the predetermined temperature.

[0013] The present invention will be described in detail below. The 20 types of amino acid residues in an amino acid sequence may be expressed by single-letter abbreviations: G for glycine (Gly), A for alanine (Ala), V for valine (Val), L for leucine (Leu), I for isoleucine (Ile), F for phenylalanine (Phe), Y for tyrosine (Tyr), W for tryptophan (Trp), S for serine (Ser), T for threonine (Thr), C for cysteine ​​(Cys), M for methionine (Met), D for aspartic acid (Asp), E for glutamic acid (Glu), N for asparagine (Asn), Q for glutamine (Gln), K for lysine (Lys), R for arginine (Arg), H for histidine (His), and P for proline (Pro).

[0014] As used herein, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0015] As used herein, the term "substitution" refers not only to cases where an amino acid residue substitution is artificially introduced, but also to cases where an amino acid residue substitution is naturally introduced, i.e., cases where the amino acid residue is originally different. As used herein, the amino acid residue substitution may be either an artificial substitution or a natural substitution, with artificial substitution being preferred.

[0016] 1. Modified 3-hydroxybutyrate dehydrogenase (modified HBDH) The modified enzyme of the present invention, ie, modified 3-hydroxybutyrate dehydrogenase, comprises a polypeptide shown in any one of (I) to (III) below.

[0017] (I) In the amino acid sequence shown in SEQ ID NO: 1, [A] substitution of the amino acid residue at position 47 with a methionine residue, [B] substitution of the amino acid residue at position 48 with a histidine residue, [C] substitution of the amino acid residue at position 49 with a leucine residue, [D] substitution of the amino acid residue at position 53 with a phenylalanine residue, a leucine residue, a proline residue, or a glutamic acid residue, [E] substitution of the amino acid residue at position 54 with a leucine residue, an alanine residue, or a phenylalanine residue, [F] substitution of the amino acid residue at position 55 with a glycine residue, a glutamic acid residue, a methionine residue, or a phenylalanine residue, [G] substitution of the amino acid residue at position 63 with a leucine residue, [H] substitution of the amino acid residue at position 81 with an isoleucine residue, [I] substitution of the amino acid residue at position 147 with a glycine residue, [J] substitution of the amino acid residue at position 210 with a valine residue, [K] a substitution of the amino acid residue at position 240 with an alanine residue, [L] a substitution of the amino acid residue at position 241 with a phenylalanine residue, [M] a substitution of the amino acid residue at position 250 with a valine residue, [N] a substitution of the amino acid residue at position 251 with a proline residue, and [O] a substitution of the amino acid residue at position 252 with an isoleucine residue. (II) A polypeptide having an amino acid sequence having at least one of the substitutions shown in [A] to [O] introduced therein, in which one or several amino acid residues other than the substituted amino acid residue have been substituted, added, inserted or deleted, and which has an improved 3-hydroxybutyrate dehydrogenase activity after at least one predetermined temperature treatment in the temperature range of 40°C or higher compared to the activity of a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 after the predetermined temperature treatment.(III) A polypeptide in which the sequence identity of the portion excluding the substituted amino acid residue in the amino acid sequence of at least one of [A] to [O] is 70% or more, and the 3-hydroxybutyrate dehydrogenase activity after treatment at least at any predetermined temperature in the temperature range of 40°C or higher is improved compared to the activity after treatment at the predetermined temperature of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0018] The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of wild-type 3-hydroxybutyrate dehydrogenase (HBDH) derived from Ralstonia pickettii.

[0019] The polypeptides (I) to (III) may be polypeptides containing any one of the above substitutions [A] to [O] alone (single-substituted variants of Ralstonia picketii-derived HBDH), or may be polypeptides containing two or more of them in combination (multiple-substituted variants of Ralstonia picketii-derived HBDH).

[0020] Among the polypeptides (I) to (III), preferred examples include polypeptides containing at least one of the substitutions [A] to [O] shown in the "Type of substitution" column of Table 1. In Table 1, each preferred substitution may be represented by the amino acid residue position and the substituted amino acid residue, as shown in the "Abbreviation of substitution" column. Specific sequences of polypeptides (I) containing each preferred substitution are shown in the "Sequence of polypeptide (I)" column of Table 1.

[0021]

[0022] In the polypeptide of (II), the amino acid modification introduced may include only one type of modification (e.g., substitution only) from substitution, addition, insertion, and deletion, or may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide of (II), the number of amino acid differences in the portion other than the amino acid residue into which at least one of the substitutions shown in [A] to [O] has been introduced may be one or several, for example, 1 to 77, 1 to 60, 1 to 50, 1 to 40, or 1 to 30, preferably 1 to 25, 1 to 20, 1 to 15, more preferably 1 to 10, even more preferably 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, and particularly preferably 1, 2, or 1.

[0023] In the polypeptide (III), the amino acid sequence identity in the portion other than the amino acid residues into which at least one of the substitutions [A] to [O] has been introduced may be 70% or more with respect to the corresponding portion of the amino acid sequence shown in SEQ ID NO: 1, but is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, still more preferably 95% or more, 96% or more, 97% or more, 98% or more, and particularly preferably 99% or more or 99.5% or more.

[0024] Here, in the polypeptide (III), the term "sequence identity" refers to the amino acid sequence identity value obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters are set as follows: Gap insertion cost value: 11, Gap extension cost value: 1.

[0025] In the polypeptides (II) and (III), the amino acids at positions 16 (I), 154 (Y), 158 (K), 184 (P), 187 (V), and 189 (T) in the amino acid sequence shown in SEQ ID NO: 1 are thought to contribute to NAD (nicotinamide adenine dinucleotide) binding, and the amino acids at positions 93 (Q), 141 (S), 143 (H), 151 (K), and 154 (Y) are thought to contribute to substrate binding. All of these are thought to contribute to 3-hydroxybutyrate dehydrogenase activity, so it is desirable not to introduce substitutions or deletions into these positions.

[0026] In the polypeptides (II) and (III), when an amino acid substitution is introduced into a portion other than the amino acid residue into which at least one of the substitutions [A] to [O] has been introduced, a conservative substitution is a suitable embodiment of the amino acid substitution to be introduced. That is, examples of conservative substitutions in the polypeptides (II) and (III) include substitution of a nonpolar amino acid with another nonpolar amino acid, substitution of an uncharged amino acid with another uncharged amino acid, substitution of an acidic amino acid with another acidic amino acid, and substitution of a basic amino acid with another basic amino acid.

[0027] The "heat resistance improved over that of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1" possessed by the polypeptides (II) and (III) refers to a temperature characteristic in which the HBDH activity after treatment at at least one predetermined temperature in a temperature range of 40°C or higher is higher than the HBDH activity of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 after treatment at said predetermined temperature. The improved heat resistance possessed by the polypeptides (II) and (III) may be observed at at least one predetermined temperature in a temperature range of 40°C or higher (the time spent at said predetermined temperature is 15 minutes), and the temperature range is preferably 45°C or higher. The heat resistance of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is significantly reduced, particularly at temperatures above 50°C, and therefore the temperature range is more preferably 50°C or higher. The upper limit of the temperature range is not particularly limited, but may be, for example, 60°C or lower, preferably 55°C or lower. Specific examples of the temperature range include 40 to 60°C, 45 to 60°C, 50 to 60°C, and 50 to 55°C.

[0028] 2. DNA The DNA of the present invention is a DNA encoding the above-mentioned "1. Modified 3-hydroxybutyrate dehydrogenase (modified HBDH)."

[0029] The DNA of the present invention is not particularly limited as long as it has a nucleotide sequence encoding a modified HBDH consisting of the polypeptides shown in (I) to (III) described in "1. Modified 3-hydroxybutyrate dehydrogenase (modified HBDH)" above. An example of a nucleotide sequence of DNA encoding the amino acid sequence shown in SEQ ID NO: 1 (wild-type HBDH derived from Ralstonia picketii), which is the reference sequence for the polypeptides shown in (I) to (III), is SEQ ID NO: 13. Therefore, the DNA of the present invention can be appropriately designed by those skilled in the art using SEQ ID NO: 13 as the reference sequence.

[0030] Examples of the DNA of the present invention include DNAs shown in any of the following (i) to (iii):

[0031] (i) DNA consisting of a base sequence shown in SEQ ID NO: 13, into which at least one of the substitutions [a] to [o] shown in the table below has been introduced: [a] substitution of the 139th to 141st positions with a nucleotide sequence encoding a methionine residue; [b] substitution of the 142nd to 144th amino acid residues with a nucleotide sequence encoding a histidine residue; [c] substitution of the 145th to 147th amino acid residues with a nucleotide sequence encoding a leucine residue; [d] substitution of the 157th to 159th amino acid residues with a nucleotide sequence encoding a phenylalanine residue, a leucine residue, a proline residue, or a glutamic acid residue; [e] substitution of the 160th to 162nd amino acid residues with a nucleotide sequence encoding a leucine residue, an alanine residue, or a phenylalanine residue; [f] substitution of the 163rd to 165th amino acid residues with a nucleotide sequence encoding a glycine residue, a glutamic acid residue, a methionine residue, or a phenylalanine residue; [g] substitution of amino acid residues at positions 187 to 189 with a nucleotide sequence encoding a leucine residue, [h] substitution of amino acid residues at positions 241 to 243 with a nucleotide sequence encoding an isoleucine residue, [i] substitution of amino acid residues at positions 439 to 441 with a nucleotide sequence encoding a glycine residue, [j] substitution of amino acid residues at positions 628 to 630 with a nucleotide sequence encoding a valine residue, [k] substitution of amino acid residues at positions 718 to 720 with a nucleotide sequence encoding an alanine residue, [l] substitution of amino acid residues at positions 721 to 723 with a nucleotide sequence encoding a phenylalanine residue, [m] substitution of amino acid residues at positions 748 to 750 with a nucleotide sequence encoding a valine residue, [n] substitution of amino acid residues at positions 751 to 753 with a nucleotide sequence encoding a proline residue, and [o] substitution of amino acid residues at positions 754 to 756 with a nucleotide sequence encoding an isoleucine residue. (ii) DNA encoding a polypeptide having improved heat resistance compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, which hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA shown in (i) above.(iii) DNA encoding a polypeptide having improved heat resistance compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, which DNA has 70% or more homology with the DNA shown in (i) above.

[0032] Specific examples of the DNA of (i) above can be easily determined by those skilled in the art based on the nucleotide sequence of SEQ ID NO: 13. Specific nucleotide sequences of DNA encoding the polypeptides shown in SEQ ID NOs: 2 to 12 in Table 1 include the nucleotide sequences of SEQ ID NOs: 14 to 24, respectively.

[0033] With regard to the DNA of (ii) above, "under stringent conditions" refers to conditions in which the DNA is incubated at 50°C to 65°C for 4 hours to overnight in 6xSSC (1xSSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400) and 100 μg / ml salmon sperm DNA.

[0034] Specifically, hybridization under stringent conditions is carried out by the following method: a nylon membrane onto which a DNA library or cDNA library is immobilized is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6x SSC, 0.5% SDS, 5x Denhardt's buffer, and 100 µg / ml salmon sperm DNA. 32 Each P-labeled probe is added and incubated overnight at 65° C. The nylon membrane is washed in 6×SSC at room temperature for 10 minutes, in 2×SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2×SSC containing 0.1% SDS at 45° C. for 30 minutes, and then autoradiography is performed to detect DNA that has specifically hybridized with the probe.

[0035] For the DNA of (iii), the homology may be 70% or more, preferably 80% or more, or 85% or more, more preferably 90% or more, or 93% or more, even more preferably 95% or more, still more preferably 98% or more, even more preferably 98.5% or more, or 99% or more, and particularly preferably 99.3% or more, or 99.5% or more.

[0036] Here, DNA "homology" is calculated using publicly available or commercially available software with an algorithm for comparing a reference sequence with a query sequence. Specifically, BLAST, FASTA, GENETYX (manufactured by Genetics Corporation), or the like can be used, and these may be used with default parameters.

[0037] The DNA of the present invention can be obtained, for example, by introducing at least one of the substitutions [a] to [o] above into DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (wild-type HBDH derived from Ralstonia picketii). The DNA of the present invention can also be artificially synthesized by total gene synthesis.

[0038] For DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the base sequence portion can be obtained by a standard method using PCR from a nucleic acid construct such as a plasmid incorporating the base sequence shown in SEQ ID NO: 13.

[0039] Methods for introducing mutations into genes and artificially modifying amino acid sequences include known methods such as the Kunkel method and the Gapped duplex method, and site-directed mutagenesis kits such as QuikChange. TM Site-Directed Mutagenesis Kit (Stratagene), GeneTailor TM Site-Directed Mutagenesis System (Invitrogen), Takara Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio Inc.), etc. can be used.

[0040] The DNA of the present invention encompasses various types of DNA resulting from codon degeneracy. Various types of DNA encoding the same amino acid sequence can be artificially produced easily using known genetic engineering techniques. For example, in the production of a protein by genetic engineering, if the codons used in the original gene encoding the target protein are used infrequently in the host, the expression level of the protein may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon usage frequency for the host without changing the encoded amino acid sequence.

[0041] The sum of the host-optimal codon usage frequencies for each codon can be used as an index of codon usage frequency. An optimal codon is defined as the codon with the highest usage frequency among codons corresponding to the same amino acid. The codon usage frequency is not particularly limited as long as it is optimized for the host. For example, the following is an example of an optimal codon for E. coli: F: phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: histidine (cat), Q: glutamine (cag), N: asparagine (aat), K: lysine (aaa), D: aspartic acid (gat), E: glutamic acid (gaa), S: serine (agc), P: proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine ​​(tgc), W: tryptophan (tgg), R: arginine (cgc), G: glycine (ggc).

[0042] The base sequence of DNA into which a mutation has been introduced can be confirmed by conventional sequencing. Specific sequencing methods include the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463) and sequence analysis using an appropriate DNA sequencer. Methods for confirming whether a DNA encodes a polypeptide of interest include comparing the determined base sequence with an unsubstituted base sequence such as the base sequence shown in SEQ ID NO: 13, or comparing the amino acid sequence deduced from the determined base sequence with an unsubstituted amino acid sequence such as the amino acid sequence shown in SEQ ID NO: 1.

[0043] 3. Expression Cassette or Recombinant Vector The expression cassette or recombinant vector of the present invention comprises the DNA of the present invention described above in "2. DNA." The expression cassette or recombinant vector of the present invention can be obtained by linking a promoter and a terminator to the DNA of the present invention, or by inserting the expression cassette of the present invention or the DNA of the present invention into an expression vector.

[0044] The expression cassette or recombinant vector of the present invention may contain, as control elements, a promoter and a terminator, as well as transcription elements such as an enhancer, a CCAAT box, a TATA box, or an SPI site, as necessary. These control elements may be operably linked to the DNA of the present invention. "Operably linked" means that the DNA of the present invention is linked to various control elements that regulate the DNA of the present invention in a state that allows it to operate in a host cell.

[0045] Regarding the recombinant vector of the present invention, an expression vector constructed for genetic recombination from a phage, plasmid, or virus capable of autonomously replicating in a host is preferred. Such expression vectors are known, and commercially available expression vectors include pQE-based vectors (Qiagen, Inc.), pTrc99A, pDR540, and pRIT2T (GE Healthcare Biosciences, Inc.), and pET-based vectors (Merck & Co., Inc.). The expression vector may be used in an appropriate combination with the host cell. For example, when Escherichia coli is used as the host cell, examples include a combination of a pET-based vector and a DH5α E. coli strain, a combination of a pET-based vector and a BL21(DE3) E. coli strain, or a combination of a pDR540 vector and a JM109 E. coli strain.

[0046] 4. Transformant The transformant of the present invention is obtained by transforming a host with the expression cassette or recombinant vector of the present invention described above in "3. Expression cassette or recombinant vector."

[0047] The host used for producing the transformant of the present invention is not particularly limited, as long as it allows gene introduction, the expression cassette or recombinant vector is stable, is capable of autonomous replication, and is capable of expressing the traits of the gene comprising the DNA of the present invention. Suitable examples include bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida, and the genus Chryseobacterium such as Chryseobacterium proteolyticum; yeast; and the like. Other suitable host cells include animal cells, insect cells, and plant cells.

[0048] The transformant of the present invention can be obtained by introducing the expression cassette of the present invention or the recombinant vector of the present invention into a host. The location of introduction of the DNA of the present invention is not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the expression cassette of the present invention or the recombinant vector of the present invention include, for example, a recombinant vector method and a genome editing method. Conditions for introducing the expression cassette or recombinant vector into a host may be appropriately determined depending on the type of host, etc. When the host is a bacterium, examples of methods include a method using competent cells treated with calcium ions and an electroporation method. When the host is a yeast, examples of methods include electroporation, the spheroplast method, and the lithium acetate method. When the host is an animal cell, examples of methods include electroporation, the calcium phosphate method, and the lipofection method. When the host is an insect cell, examples of methods include the calcium phosphate method, the lipofection method, and the electroporation method. When the host is a plant cell, examples of methods include electroporation, the Agrobacterium method, the particle gun method, and the PEG method.

[0049] Whether or not the expression cassette of the present invention or the recombinant vector of the present invention has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like.

[0050] When confirming by PCR whether the expression cassette of the present invention or the recombinant vector of the present invention has been incorporated into a host, for example, genomic DNA, the expression cassette, or the recombinant vector may be isolated and purified from the transformant.

[0051] For example, when the host is a bacterium, the expression cassette or recombinant vector is isolated and purified using a lysate obtained by lysing the bacterium. Lysis can be achieved by treating the bacterium with a lytic enzyme such as lysozyme, optionally in combination with a protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS).

[0052] Furthermore, physical disruption methods such as freeze-thawing and French press treatment may be combined. DNA can be separated and purified from the lysate by, for example, an appropriate combination of deproteinization treatments using phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and commercially available kits.

[0053] DNA can be cleaved using conventional methods, for example, restriction enzyme treatment. For example, a type II restriction enzyme that acts on a specific nucleotide sequence can be used. DNA can be ligated to an expression cassette or expression vector using, for example, DNA ligase.

[0054] Then, PCR is performed using the isolated and purified DNA as a template and primers specific to the DNA of the present invention. The PCR amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like, and stained with ethidium bromide and SYBR Green solution, etc., and the amplification product is detected as a band, thereby confirming transformation.

[0055] Alternatively, PCR may be performed using primers pre-labeled with a fluorescent dye or the like to detect the amplified product. Furthermore, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and the amplified product is confirmed by fluorescence, enzyme reaction, or the like.

[0056] 5. Method for Producing Modified 3-Hydroxybutyrate Dehydrogenase (Modified HBDH) The method for producing the modified 3-hydroxybutyrate dehydrogenase of the present invention is a method for producing the enzyme described in "1. Modified 3-hydroxybutyrate dehydrogenase (modified HBDH)" above, and includes a step of culturing the transformant of the present invention. Note that when any of the substitutions [A] to [O] contained in the modified HBDH is naturally introduced, the modified HBDH can be obtained by a production method including a step of culturing a microorganism that produces the modified HBDH.

[0057] The culture conditions may be appropriately set taking into consideration the nutritional and physiological properties of the transformant or microorganism, but liquid culture is preferred. For industrial production, aeration and agitation culture is preferred. The nutrient source for the medium may be any nutrient required for the growth of the transformant or microorganism. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source may be any assimilable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or alkaline extract of soybean meal. In addition to the carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, as well as specific amino acids and specific vitamins, may also be used as needed.

[0058] The culture temperature can be appropriately set within a range in which the transformant or microorganism of the present invention can grow and the transformant or microorganism can produce the modified HBDH, and is preferably about 15 to 45° C., more preferably about 25 to 39° C. The culture may be completed at an appropriate time when the modified HBDH reaches its maximum yield, and the culture time is usually about 12 to 48 hours.

[0059] After culturing the transformant or the microorganism, the culture medium can be subjected to a separation method such as centrifugation to recover the culture supernatant and / or bacterial cells. The bacterial cells can be solubilized by mechanical methods such as ultrasonication or French press treatment, or by treatment with a lytic enzyme such as lysozyme, and optionally by the use of an enzyme such as protease or a surfactant such as sodium lauryl sulfate (SDS), to obtain a water-soluble fraction containing the desired modified HBDH. Furthermore, by selecting an appropriate expression cassette or expression vector and host, the expressed modified HBDH can be secreted into the culture medium.

[0060] The water-soluble fraction containing the modified HBDH obtained as described above may be subjected to a purification treatment as it is, or the modified HBDH in the water-soluble fraction may be concentrated and then subjected to a purification treatment by, for example, vacuum concentration, membrane concentration, salting out, fractional precipitation with a hydrophilic organic solvent (e.g., methanol, ethanol, and / or acetone), etc.

[0061] The modified HBDH can be purified by, for example, an appropriate combination of methods such as gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc. The purified modified HBDH may be powdered, if necessary, by freeze-drying, vacuum drying, spray drying, etc.

[0062] The modified HBDH can be provided in the form of an enzyme preparation. Accordingly, the present invention also provides an enzyme preparation containing the modified HBDH described in "1. Modified 3-hydroxybutyrate dehydrogenase (modified HBDH)" above as an active ingredient.

[0063] The content of the modified HBDH in the enzyme preparation of the present invention is not particularly limited as long as the activity of the modified HBDH as an active ingredient is exhibited when the enzyme preparation is used.

[0064] The enzyme preparation of the present invention may or may not contain other components in addition to the modified HBDH, as long as the effects of the present invention are obtained. Examples of other components include other enzymes other than the modified HBDH, additives, and culture residues generated in the above-mentioned production method.

[0065] Examples of other enzymes include amylases (α-amylase, β-amylase, glucoamylase), glucosidases (α-glucosidase, β-glucosidase), galactosidases (α-galactosidase, β-galactosidase), proteases (acid proteases, neutral proteases, alkaline proteases), peptidases (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase (other than the above-mentioned modified HBDH), glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deamidase, pullulanase, etc. These other enzymes may be contained alone or in combination of two or more.

[0066] Examples of additives include excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers include phosphates, citrates, and acetates. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives include ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. These additives may be contained alone or in combination.

[0067] Culture residues include components derived from the culture medium, contaminating proteins, bacterial components, and the like.

[0068] The form of the enzyme preparation of the present invention is not particularly limited, and examples thereof include liquid and solid forms (powder, granules, etc.) The enzyme preparation in the above forms can be prepared by a generally known method.

[0069] The use of the enzyme preparation of the present invention is not particularly limited as long as it is an application that generally utilizes the activity of HBDH. A preferred application is the measurement of ketone bodies. In this application, the ketone body to be measured is 3-hydroxybutyric acid (R-3-hydroxybutyric acid). Ketone body measurement will be described in detail in "9. Method for measuring ketone bodies in a sample."

[0070] 7. Ketone Body Measurement Sensor The modified HBDH can be used as a sensing means in a ketone body measurement sensor. Therefore, the present invention also provides a ketone body measurement sensor containing the modified HBDH described in "1. Modified 3-hydroxybutyrate dehydrogenase (modified HBDH)" above.

[0071] The sensor for measuring ketone bodies of the present invention includes the above-mentioned modified HBDH, and is capable of converting 3-hydroxybutyric acid (R-3-hydroxybutyric acid) and NAD by the modified HBDH. + The substance can be configured to convert the change and amount thereof resulting from the reaction with nicotinamide adenine dinucleotide and / or its derivatives into a readable signal.

[0072] The modified HBDH produces 3-hydroxybutyrate (R-3-hydroxybutyrate) and NAD + and / or its derivatives, specifically, the changes due to the reaction with 3-hydroxybutyric acid, NAD + and / or a change in the redox state of its derivatives.

[0073] The modified HBDH produces 3-hydroxybutyrate (R-3-hydroxybutyrate) and NAD + The signal that can read the change and the amount thereof based on the reaction with the derivative thereof includes an electrochemical signal and an optical signal.

[0074] Electrochemical signals include those in which a chemical state (such as the presence of 3-hydroxybutyric acid) is converted into an electrical signal (such as an electric current). Optical signals include those in which NAD + and / or its derivative redox state (e.g., NAD +and / or its derivatives, and / or NADH and / or its derivatives).

[0075] In the ketone body measurement sensor of the present invention, the modified HBDH is preferably immobilized on an insoluble support. The manner of immobilization of the modified HBDH is not particularly limited, and examples thereof include physical immobilization by adhesion, adsorption, absorption, swelling, etc., chemical or biochemical immobilization by specific non-covalent bonding, and chemical immobilization by covalent bonding.

[0076] Furthermore, the modified HBDH may be immobilized on an insoluble support either alone or in the form of a composition together with other components. A specific embodiment in which the modified HBDH is immobilized on an insoluble support in the form of a composition is one in which the composition is physically immobilized (preferably by adhesion) to the insoluble support.

[0077] At least the surface of the insoluble support is composed of a solid or solid material that does not dissolve in the reaction system of the modified HBDH and 3-hydroxybutyric acid and that can immobilize the modified HBDH. Specific examples of materials that compose at least the surface of the insoluble support include swellable materials (e.g., gelatin, etc.), porous substrates (e.g., cellulose, filter paper, etc.), resins, glass, redox polymers (i.e., polymers bound to redox mediators), metals, carbon, etc.

[0078] The shape of the insoluble carrier is not particularly limited, and examples thereof include substrate-like, flake-like, stick-like, and bead-like shapes.

[0079] A preferred example of the insoluble carrier is an electrode whose surface is made of the above-mentioned material. Preferably, the modified HBDH is physically immobilized on such an electrode, and more preferably, a composition containing the modified HBDH is physically immobilized (preferably by attachment) on such an electrode.

[0080] Specific embodiments of the sensor for measuring ketone bodies of the present invention may be any form used as a biosensor, such as a sensor chip, a microtiter plate, a test strip, an electrochemical flow cell, etc.

[0081] 8. Ketone body measurement kit The ketone body measurement kit of the present invention includes an enzyme preparation described in "6. Enzyme preparation" above that is used for ketone body measurement (hereinafter also referred to as "enzyme preparation for ketone body measurement") or a ketone body measurement sensor described in "7. Ketone body measurement sensor" above.

[0082] The kit for measuring ketone bodies of the present invention may further include other suitable items used for measurement and / or sample collection in addition to the enzyme preparation for measuring ketone bodies or the sensor for measuring ketone bodies.

[0083] Examples of such other items include coenzymes, reducing reagents, redox mediators, buffer solutions, fluorescent probes, deproteinized solutions, lancet devices, etc. The kit for measuring ketone bodies of the present invention may contain one of these other items alone or a combination of multiple items.

[0084] Examples of coenzymes include NAD + (nicotinamide adenine dinucleotide), NAD + Derivatives (e.g., nicotinamide adenine dinucleotide phosphate (NADP) + ), thionicotinamide-NAD, pyridine aldehyde-NAD, acetylpyridine-NAD, carba-NAD, etc.), FAD + These coenzymes may be used singly or in combination of two or more. Among these coenzymes, NAD is preferred. + , NADP + Examples include:

[0085] Examples of reducing reagents include Nitrotetrazolium Blue, Tetrazolium Blue, etc. These reducing reagents may be used alone or in combination of two or more.

[0086] Examples of redox mediators include 1-methoxy-5-methylphenazinium methylsulfur, 1-methoxy-phenazine methosulfate, 1-methoxy-phenazinium ethylsulfate, thiol-reactive PES, and amine-reactive PES. These redox mediators may be used alone or in combination. Of these redox mediators, 1-methoxy-5-methylphenazinium methylsulfur is preferred.

[0087] Examples of the buffer solution include acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, and GOOD's buffer. Examples of GOOD's buffer include PIPES, MES, and MOPS. These buffer solutions may be used alone or in combination. Among these buffer solutions, GOOD's buffer solution is preferred, and PIPES is more preferred.

[0088] 9. Method for Measuring Ketone Bodies in a Sample The method for measuring ketone bodies in a sample of the present invention comprises the step of contacting the sample in which ketone bodies are to be measured with the modified HBDH described above in "1. Modified 3-hydroxybutyrate dehydrogenase (HBDH)" under conditions that allow 3-hydroxybutyrate dehydrogenase activity.

[0089] The method of the present invention for measuring ketone bodies in a sample is based on the following reaction catalyzed by modified HBDH.

[0090]

[0091] In a specific embodiment of the method of the present invention for measuring ketone bodies in a sample, 3-hydroxybutyric acid in the sample is quantified. In a more specific embodiment, the method of the present invention for measuring ketone bodies in a sample comprises the steps of: (a) contacting a sample containing 3-hydroxybutyric acid with the modified HBDH; (b) converting 3-hydroxybutyric acid and NADPH in the sample by the modified HBDH; + (nicotinamide adenine dinucleotide) and / or a derivative thereof; and (c) reacting 3-hydroxybutyric acid (R-3-hydroxybutyric acid) with NAD + and / or a step of detecting or quantifying a change resulting from the reaction with a derivative thereof. Furthermore, the method may further comprise a step of inactivating the modified HBDH after step (b) and before step (c).

[0092] In step (a), the sample is not particularly limited as long as it is a sample in which ketone bodies are to be measured, and examples thereof include biological samples derived from subjects in which ketone bodies are to be measured. Subjects in which ketone bodies are to be measured include mammals such as humans. Biological samples include body fluids, excrement, and tissues, and specific examples thereof include blood samples (whole blood, serum, plasma), urine, semen, prostatic fluid, tears, saliva, sweat, ascites, cerebrospinal fluid, milk, lymph, and tissue extracts.

[0093] In step (a), the modified HBDH can be used in an amount of, for example, 0.1 μg or more, preferably 1.0 μg or more, more preferably 5.0 μg or more, even more preferably 10 μg or more, and particularly preferably 15 μg or more per gram of 3-hydroxybutyric acid in the sample. The upper limit of the amount used is not particularly limited, but examples include amounts that are, for example, 10,000 μg or less, preferably 1,000 μg or less, more preferably 500 μg or less, even more preferably 100 μg or less, and particularly preferably 50 μg or less per gram of 3-hydroxybutyric acid in the sample. These amounts can be determined through preliminary experiments.

[0094] To bring a sample into contact with the modified HBDH, a coexistence system of the sample and the modified HBDH can be established. For example, (a1) a mixed solution of the sample and an enzyme preparation used for ketone body measurement (enzyme preparation for ketone body measurement) among those described in "6. Enzyme Preparations" above can be prepared, or (a2) a coexistence system of the sample and the modified HBDH can be established by dropping, placing, pouring, or absorbing the sample onto an insoluble support on which the active ingredient (modified HBDH) is immobilized in the ketone body measurement sensor described in "7. Sensor for Ketone Body Measurement" above, or by immersing the insoluble support in the sample.

[0095] The coexistence system of the sample and modified HBDH may further contain the coenzyme, reducing reagent, redox mediator, and / or buffer solution described above in "8. Ketone body measurement kit." The amount of the coenzyme used per 1 g of modified HBDH is, for example, 0.01 to 100 mol, preferably 0.1 to 10 mol, more preferably 0.1 to 10 mol, even more preferably 0.5 to 5.0 mol, and even more preferably 0.8 to 3.0 mol. The amount of the reducing reagent used per 1 g of modified HBDH is, for example, 0.01 to 100 mol, preferably 0.1 to 50 mol, more preferably 0.1 to 10 mol, even more preferably 0.5 to 5.0 mol, and even more preferably 0.8 to 3.0 mol. The amount of the redox mediator used per 1 g of modified HBDH is, for example, 0.01 to 100 mol, preferably 0.1 to 50 mol, more preferably 0.1 to 10 mol, even more preferably 0.5 to 5.0 mol, and still more preferably 0.8 to 3.0 mol.

[0096] In step (b), the reaction conditions, such as temperature and pH, that induce the 3-hydroxybutyrate dehydrogenase activity of the modified HBDH can be appropriately selected by those skilled in the art. For example, specific reaction temperatures include 5 to 80°C, preferably 10 to 70°C, more preferably 15 to 60°C, even more preferably 20 to 55°C, still more preferably 25 to 50°C, and even more preferably 30 to 50°C. The reaction pH can be based on the optimal pH of the modified HBDH, and specifically includes 3 to 9, preferably 5 to 8, and more preferably 6 to 7.5. The reaction time is not particularly limited, but includes, for example, 10 seconds to 12 hours, preferably 30 seconds to 1 hour, and more preferably 1 minute to 30 minutes. The optimal reaction conditions can be determined through preliminary experiments.

[0097] The steps (a) and (b) may be carried out in this order or simultaneously.

[0098] In step (c), NAD + Derivatives of NAD + The basic structure is NAD + Similarly, the substance is not particularly limited as long as it converts 3-hydroxybutyrate into acetoacetate by the modified HBDH and simultaneously generates a reduced form, and examples thereof include nicotinamide adenine dinucleotide phosphate, thionicotinamide-NAD, pyridine aldehyde-NAD, acetylpyridine-NAD, carba-NAD, etc.

[0099] 3-hydroxybutyrate and NAD + The changes resulting from the reaction with 3-hydroxybutyric acid and / or its derivatives are as described above in "7. Sensor for measuring ketone bodies." + The change resulting from the reaction with 3-hydroxybutyric acid and / or its derivatives correlates with the amount of 3-hydroxybutyric acid contained in the sample. Therefore, by detecting or quantifying the change resulting from the reaction, the 3-hydroxybutyric acid contained in the sample can be detected or quantified.

[0100] In a preferred embodiment, the amount of NADH and / or its derivatives produced and the amount of NAD consumed are +and / or its derivatives correlate with the amount of 3-hydroxybutyric acid present in the sample, + The amount of the compound and / or its derivatives and / or the ratio of these amounts can be measured as the amount of change due to the above reaction. Methods for calculating these amounts and ratios can be determined appropriately by those skilled in the art.

[0101] The present invention will be specifically described below with reference to examples, but the present invention should not be construed as being limited to the following examples.

[0102] [Screening of modified HBDH] Genomic DNA was extracted from Ralstonia pickettii. Using the gene sequence of 3-hydroxybutyrate dehydrogenase (HBDH) (SEQ ID NO: 13) as a template, saturation mutagenesis was performed to obtain a group of mutant gene products. Each of the obtained mutant gene products was introduced into pTrc99A, and E. coli JM109 was transformed by standard methods. The obtained transformants were cultured with shaking at 37°C for 16 hours in LB-Lennox medium (1% Bacto Trypton: Thermo Fisher Scientific, 0.5% Bacto Yeast Extract: Thermo Fisher Scientific, 0.5% sodium chloride: Fujifilm Wako). The cells were then subcultured in Terrific Broth (1.2% Bacto Trypton: Thermo Fisher Scientific, 2.4% Bacto Yeast Extract: Thermo Fisher Scientific, 0.8% glycerol: Fuji Film Wako, 0.94% dipotassium hydrogen phosphate: Fuji Film Wako, 0.22% dipotassium hydrogen phosphate: Fuji Film Wako) and cultured with shaking at 37°C for 20 hours. The cells were recovered from the culture medium and disrupted using a bead shocker (Yasui Kikai) in the usual manner, followed by centrifugation at 15,000 rpm for 10 minutes. The recovered supernatant was used as a crude enzyme solution.

[0103] The crude enzyme solution was treated at 40°C for 15 minutes, after which HBDH activity was measured and residual activity was calculated. 1,500 mutant strains were evaluated, but only 11 strains were confirmed to have improved heat resistance compared to the wild type. For the strains in which improved heat resistance was confirmed, sequence analysis was performed to confirm the substituted amino acids. As a result, the amino acid sequences of SEQ ID NOs: 2 to 12 were identified. The crude enzyme solution from the strains in which heat resistance was confirmed was purified by standard methods such as adsorption chromatography and ultrafiltration to obtain purified enzyme samples of modified HBDH having the amino acid sequences of SEQ ID NOs: 2 to 12, respectively.

[0104] Test Example 1: Evaluation of heat resistance of modified HBDH - 1 Comparative Example 1 (purified enzyme sample of wild-type HBDH) and Examples 1 to 5 (purified enzyme samples of modified HBDH) in Table 2a were subjected to the following heat resistance evaluation.

[0105] The protein concentration of the prepared purified enzyme sample was measured using Biorad Protein Assay Reagent (Biorad). The purified enzyme sample was diluted to 0.1 mg / ml with 50 mM phosphate buffer (pH 7.5) to equalize the protein concentration. 100 μL of each was placed in a PCR tube (BM Equipment) and heated for 15 minutes at 40°C, 45°C, 50°C, 55°C, or 60°C in a thermal cycler (Applied Biosystems). The sample was then quickly cooled on ice, and the resulting sample solution was assayed for HBDH activity as follows.

[0106] To 0.1 mol / L Tris-HCl buffer (pH 8.5) containing 145 mmol / L (final concentration) of 3-hydroxybutyric acid, 5.8 mmol / L (final concentration) of hydrazine sulfate, 0.1 g / dL (final concentration) of Triton X-100, and 13 mmol / L (final concentration) of β-NAD, 10 μL of a solution prepared by diluting the sample solution (0.1 mg / mL) at a predetermined ratio was added at 25°C, and the change in absorbance at 340 nm was measured. HBDH activity was measured based on the following equation:

[0107]

[0108] Table 2a shows the relative HBDH activity (%) of each Example, when the HBDH activity after each temperature treatment in Comparative Example 1 is taken as 100%. Table 2b shows the relative HBDH activity (%) of each Example, when the HBDH activity at 4°C is taken as 100%.

[0109]

[0110] Test Example 2: Evaluation of heat resistance of modified HBDH - 2 Comparative Example 1 (purified enzyme sample of wild-type HBDH) and Examples 4 to 11 (purified enzyme samples of modified HBDH) in Table 3a were subjected to the following heat resistance evaluation.

[0111] The protein concentration of the prepared purified enzyme sample was measured using Biorad Protein Assay Reagent (Biorad). The purified enzyme sample was diluted to 0.1 mg / ml with 50 mM phosphate buffer (pH 7.5) to equalize the protein concentration. 100 μL of each was placed in a PCR tube (BM Equipment) and heated for 15 minutes at 40°C, 45°C, 50°C, 55°C, or 60°C in a thermal cycler (Applied Biosystems). The sample was then quickly cooled on ice, and the resulting sample solution was assayed for HBDH activity as follows.

[0112] A substrate solution was prepared using 100 mmol / L PIPES-NaOH buffer (pH 7.0) containing 600 mmol / L 3-hydroxybutyric acid, 60 mmol / L β-NAD, 6.6 mmol / L 1-methoxy-5-methylphenazinium methylsulfur, 6.6 mmol / L nitrotetrazolium blue chloride, and 0.1 g / dL (final concentration) of Triton X-100. 10 μL of sample solution diluted to a predetermined ratio was added to 200 μL of the substrate solution at 37°C, and the change in absorbance at 570 nm was measured.

[0113]

[0114] Table 3a shows the relative HBDH activity (%) of each Example, when the HBDH activity after each temperature treatment in Comparative Example 1 is taken as 100%. Table 3b shows the relative HBDH activity (%) of each Example, when the HBDH activity at 4°C is taken as 100%.

[0115]

[0116]

Claims

1. A modified enzyme consisting of a polypeptide shown in any one of (I) to (III) below: (I) In the amino acid sequence shown in SEQ ID NO: 1, [A] substitution of the amino acid residue at position 47 with a methionine residue, [B] substitution of the amino acid residue at position 48 with a histidine residue, [C] substitution of the amino acid residue at position 49 with a leucine residue, [D] substitution of the amino acid residue at position 53 with a phenylalanine residue, a leucine residue, a proline residue, or a glutamic acid residue, [E] substitution of the amino acid residue at position 54 with a leucine residue, an alanine residue, or a phenylalanine residue, [F] substitution of the amino acid residue at position 55 with a glycine residue, a glutamic acid residue, a methionine residue, or a phenylalanine residue, [G] substitution of the amino acid residue at position 63 with a leucine residue, [H] substitution of the amino acid residue at position 81 with an isoleucine residue, [I] substitution of the amino acid residue at position 147 with a glycine residue, (J) a polypeptide having an amino acid sequence into which at least any one of the following substitutions has been introduced: [J] substitution of the amino acid residue at position 210 with a valine residue, [K] substitution of the amino acid residue at position 240 with an alanine residue, [L] substitution of the amino acid residue at position 241 with a phenylalanine residue, [M] substitution of the amino acid residue at position 250 with a valine residue, [N] substitution of the amino acid residue at position 251 with a proline residue, and [O] substitution of the amino acid residue at position 252 with an isoleucine residue; (II) a polypeptide having an amino acid sequence into which at least any one of the substitutions shown in [A] to [O] has been introduced, in which one or several amino acid residues other than the substituted amino acid residue have been substituted, added, inserted or deleted, and which has a 3-hydroxybutyrate dehydrogenase activity after at least any one of predetermined temperature treatments in a temperature range of 40° C. or higher that is improved compared to the activity after the predetermined temperature treatment of a polypeptide having the amino acid sequence shown in SEQ ID NO: 1;(III) A polypeptide having an amino acid sequence into which at least one of the substitutions shown in [A] to [O] has a sequence identity of 70% or more in the portion excluding the substituted amino acid residues, and has a 3-hydroxybutyrate dehydrogenase activity after at least one predetermined temperature treatment in a temperature range of 40°C or higher that is improved compared to the activity of a polypeptide having the amino acid sequence shown in SEQ ID NO:1 after the predetermined temperature treatment.

2. A DNA encoding the modified enzyme of claim 1.

3. An expression cassette or recombinant vector comprising the DNA of claim 2.

4. A transformant obtained by transforming a host with the expression cassette or recombinant vector described in claim 3.

5. A method for producing a modified enzyme, comprising the step of culturing the transformant according to claim 4.

6. An enzyme preparation comprising the modified enzyme according to claim 1.

7. A sensor for measuring ketone bodies, comprising the modified enzyme according to claim 1.

8. A kit for measuring ketone bodies, comprising the modified enzyme described in claim 1.

9. A method for measuring ketone bodies in a sample, comprising a step of contacting the sample in which ketone bodies are to be measured with the modified enzyme under modified enzyme activity conditions described in claim 1.