Novel 3-hydroxybutyrate oxidase

Abstract

The purpose of the present invention is to provide a novel enzyme for ketone body measurement that, unlike 3-hydroxybutyrate dehydrogenase, does not require NAD, which has low thermal stability, and therefore does not require a substance for reoxidation of NADH. The novel enzyme of the present invention is a 3-hydroxybutyrate oxidase derived from a microorganism belonging to the genus Achromobacter, and having a physicochemical property of catalyzing a reaction for producing hydrogen peroxide by acting on D-3-hydroxybutyric acid, or comprising an amino acid sequence represented by SEQ ID NO: 1 or a similar sequence thereof.

Description

Novel 3-hydroxybutyrate oxidase

[0001] The present invention relates to a novel enzyme that catalyzes a reaction to produce hydrogen peroxide using D-3-hydroxybutyric acid as a substrate.

[0002] 3-Hydroxybutyrate dehydrogenase (HBDH: EC 1.1.1.30) is known as an enzyme that uses D-3-hydroxybutyrate as a substrate. HBDH belongs to the short-chain dehydrogenase / reductase (SDR) family and is a NAD + It is an enzyme that reversibly catalyzes the oxidation of 3-hydroxybutyrate to acetoacetate using as a coenzyme, and has an optimal pH for the oxidation reaction on the alkaline side of 8 or higher. 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 ketone body levels in type 1 diabetes patients is recommended to prevent complications. HBDH is an industrially important enzyme used in such ketone body measurement. Specifically, as shown in Figure 5 , in a ketone body measurement system using HBDH, HBDH first catalyzes the oxidation of 3-hydroxybutyrate (HB), and electrons generated during the oxidation are transferred to the coenzyme NAD to generate NADH. The generated NADH is then reoxidized via an artificial electron mediator or NADH oxidase, thereby obtaining a signal.

[0004] HBDH has been isolated from various microorganisms, and in particular, the HBDHs disclosed in Patent Documents 1 to 3 have been put on the market. These HBDHs are currently the standard enzymes for measuring ketone bodies.

[0005] JP-A No. 11-318438 JP-A No. 8-70856 JP-A No. 2003-339385

[0006] Because the HBDH reaction requires the coenzyme NAD, the addition of the coenzyme NAD to the system is essential in ketone body measurement systems. Therefore, ketone body measurement using HBDH involves unavoidable problems in that it requires a coenzyme other than HBDH and that strict storage conditions are imposed because the NAD is thermally unstable.

[0007] In view of the industrial importance of enzymes used in measuring ketone bodies, the creation of enzymes with superior usability is desirable. Therefore, an object of the present invention is to provide a novel enzyme for measuring ketone bodies that does not require NAD, which has low thermostability like HBDH, and therefore does not require a substance for reoxidizing NADH.

[0008] The present inventors have coincidentally discovered, from a library of over 10,000 strains, a novel enzyme that catalyzes the reaction of producing hydrogen peroxide using D-3-hydroxybutyric acid as a substrate. The present invention was completed based on this discovery.

[0009] That is, the present invention provides the following aspects of the invention: Item 1. A 3-hydroxybutyrate oxidase derived from a microorganism of the genus Achromobacter, and having the following physicochemical properties: (1) Action: Acts on D-3-hydroxybutyrate to catalyze a reaction that produces hydrogen peroxide. Item 2. The 3-hydroxybutyrate oxidase according to Item 1, further having the following physicochemical properties: (2) Optimum pH: 8.3 to 8.7; (3) pH stability: Stable in the pH range of 8.5 to 9. Item 3. Item 3. The 3-hydroxybutyrate oxidase according to Item 1 or 2, wherein the microorganism of the genus Achromobacter is Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter denitrificans, or Achromobacter aloeverae. A 3-hydroxybutyrate oxidase consisting of a polypeptide shown in any of the following (I) to (III): (I) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (II) a polypeptide consisting of an amino acid sequence obtained by substituting, adding, inserting or deleting one or more amino acids in SEQ ID NO: 1, and having the activity of acting on D-3-hydroxybutyrate to catalyze a reaction to produce hydrogen peroxide; (III) a polypeptide consisting of an amino acid sequence having 70% or more sequence identity to SEQ ID NO: 1, and having the activity of acting on D-3-hydroxybutyrate to catalyze a reaction to produce hydrogen peroxide. Item 5. An enzyme preparation comprising the 3-hydroxybutyrate oxidase according to any of Items 1 to 4 as an active ingredient. Item 6. The enzyme preparation according to Item 5, which contains an intracellular fraction of a microorganism of the genus Achromobacter. Item 7. The enzyme preparation according to Item 5 or 6, which is used as an enzyme preparation for measuring ketone bodies. Item 8. A sensor for measuring ketone bodies, comprising the 3-hydroxybutyrate oxidase according to any of Items 1 to 4 or the enzyme preparation according to Item 5 or 6. Item 9. A kit for measuring ketone bodies, comprising the enzyme preparation according to Item 7 or the sensor for measuring ketone bodies according to Item 8.Item 10. A method for measuring 3-hydroxybutyrate in a sample, comprising an enzyme treatment step of contacting a sample containing D-3-hydroxybutyrate with 3-hydroxybutyrate oxidase to generate hydrogen peroxide, and a quantification step of quantifying the hydrogen peroxide. Item 11. The method according to Item 10, wherein the 3-hydroxybutyrate oxidase is the 3-hydroxybutyrate oxidase according to any one of Items 1 to 4. Item 12. A method for producing 3-hydroxybutyrate oxidase, comprising a culture step of culturing a microorganism of the genus Achromobacter, and a recovery step of recovering from the culture obtained in the culture step a fraction having the activity of acting on D-3-hydroxybutyrate to catalyze a reaction to generate hydrogen peroxide. Item 13. Item 13. The production method according to Item 12, wherein the microorganism of the genus Achromobacter is selected from the group consisting of Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter denitrificans, and Achromobacter aloeverae. Item 14. The method according to Item 12 or 13, wherein the microorganism of the genus Achromobacter is Achromobacter piechaudii strain NBRC 102461 and / or Achromobacter xylosoxidans strain NBRC 15126. Item 15. A method for producing 3-hydroxybutyrate oxidase, comprising the step of culturing a transformant with an expression cassette or recombinant vector comprising DNA encoding the 3-hydroxybutyrate oxidase according to Item 4.

[0010] According to the present invention, there is provided a novel enzyme that catalyzes the reaction of producing hydrogen peroxide using D-3-hydroxybutyric acid as a substrate.

[0011] Schematic representation of a preferred example of a sensor for measuring ketone bodies of the present invention. Schematic representation of a preferred example of a sensor for measuring ketone bodies of the present invention. Results of confirming 3-hydroxybutyrate oxidase activity in intracellular fractions of Achromobacter piechaudii NBRC102461 strain and Achromobacter xylosoxidans NBRC15126 strain are shown. The pH stability of a 3-hydroxybutyrate oxidase active fraction derived from Achromobacter piechaudii NBRC102461 strain is shown. The pH stability of a 3-hydroxybutyrate oxidase active fraction derived from Achromobacter xylosoxidans NBRC15126 strain is shown. Schematic representation of a sensor for measuring ketone bodies using 3-hydroxybutyrate dehydrogenase (HBDH).

[0012] [1] 3-hydroxybutyrate oxidase [1-1] Physicochemical properties A first embodiment of the 3-hydroxybutyrate oxidase of the present invention is derived from a microorganism of the genus Achromobacter and has the following physicochemical property (1), and in a preferred embodiment, has the following physicochemical properties (2) and (3).

[0013] (1) Action: It acts on D-3-hydroxybutyric acid to catalyze the reaction that produces hydrogen peroxide. The specific mechanism of action related to the above physicochemical property (1) is presumed to be that the hydrogen of the 3-hydroxyl group of D-3-hydroxybutyric acid is transferred to oxygen, thereby producing hydrogen peroxide together with acetoacetic acid. 3-Hydroxybutyric acid oxidase does not require a coenzyme for this reaction.

[0014] (2) Optimum pH: 8.3 to 8.7, preferably 8.4 to 8.6. The optimum pH for the physicochemical property (2) is a pH at which 3-hydroxybutyrate oxidase exhibits a relative activity of 80% or more, preferably 90% or more, when the activity of 3-hydroxybutyrate oxidase exhibiting maximum activity is taken as 100% when 3-hydroxybutyrate oxidase is reacted with D-3-hydroxybutyrate at various pH values ​​at 25°C for 3 hours.

[0015] (3) pH stability: Stable at pH 8.5 to 9. The pH defined by the pH stability in the above physicochemical property (3) is a pH at which the residual activity of 3-hydroxybutyrate oxidase is 80% or more, preferably 90% or more, when the residual activity of 3-hydroxybutyrate oxidase exhibiting the maximum residual activity is taken as 100% after storage of the enzyme at various pH values ​​at 4°C for 16 hours.

[0016] In a preferred embodiment, the residual activity may be 40% or more, preferably 80% or more, and more preferably 90% or more, in the pH range of 8 to 9.

[0017] In a preferred embodiment, the residual activity may be 15% or more, preferably 50% or more, more preferably 60% or more, in the pH range of 7 to 9.

[0018] In a preferred embodiment, the residual activity may be 4% or more, preferably 30% or more, more preferably 50% or more, and even more preferably 55% or more, in the pH range of 5 to 9.

[0019] The Achromobacter microorganism that produces 3-hydroxybutyrate oxidase is not particularly limited, and examples thereof include Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter denitrificans, and Achromobacter aloeverae. The Achromobacter microorganism may be a wild-type strain or a mutant strain (e.g., one obtained by ultraviolet irradiation).

[0020] More specific examples of bacteria that produce 3-hydroxybutyrate oxidase include Achromobacter piechaudii strain NBRC102461 and Achromobacter xylosoxidans strain NBRC15126. These strains are stored at NBRC (National Institute of Technology and Evaluation, Biotechnology Center) and can be purchased by following the prescribed procedures.

[0021] In particular, the 3-hydroxybutyrate oxidase derived from Achromobacter piechaudii strain NBRC102461 is preferred because it exhibits a relatively high residual activity over a wide pH range. Specifically, the 3-hydroxybutyrate oxidase derived from Achromobacter piechaudii strain NBRC102461 is stable in the pH range of 8.5 to 9, and exhibits a residual activity of 90% even in the pH range of 8 to 9, 60% or more in the pH range of 7 to 9, and 55% or more in the pH range of 5 to 9.

[0022] The 3-hydroxybutyrate oxidase of Achromobacter xylosoxidans NBRC15126 strain is stable in the pH range of 8.5 to 9, and the residual activity is 40% or more in the pH range of 8 to 9.

[0023] [1-2] Sequence A second embodiment of the 3-hydroxybutyrate oxidase of the present invention comprises a polypeptide shown in any one of the following (I) to (III): (I) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (II) a polypeptide consisting of an amino acid sequence obtained by substituting, adding, inserting or deleting one or more amino acids in SEQ ID NO: 1, and having the activity of acting on D-3-hydroxybutyrate to catalyze a reaction that produces hydrogen peroxide; (III) a polypeptide consisting of an amino acid sequence that has 70% or more sequence identity to SEQ ID NO: 1, and having the activity of acting on D-3-hydroxybutyrate to catalyze a reaction that produces hydrogen peroxide.

[0024] SEQ ID NO: 1 is the amino acid sequence of 3-hydroxybutyrate oxidase from Achromobacter piechaudii.

[0025] The polypeptides (II) and (III) are 3-hydroxybutyrate oxidases having the amino acid sequence of SEQ ID NO: 1 as a backbone and similar in sequence to the polypeptide (I).

[0026] The amino acid modification introduced into the polypeptide of (II) may include only one type of modification (e.g., substitution) from among 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 acids to be substituted, added, inserted, or deleted may be one or more or several, for example, 1 to 78, preferably 1 to 52, 1 to 39, or 1 to 26, more preferably 1 to 13, preferably 1 to 7, 1 to 5, and particularly preferably 1, 2, or 1.

[0027] Furthermore, in the polypeptide (III), the sequence identity may be 70% or more, preferably 80% or more, 85% or more, or 90% or more, more preferably 95% or more, 97% or more, or 98% or more, and particularly preferably 99% or more, or 99.5% or more.

[0028] Here, in the polypeptide (III), the sequence identity to SEQ ID NO: 1 refers to the sequence identity calculated by comparing the amino acid sequence with SEQ ID NO: 1. Furthermore, the term "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLAST PACKAGE [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.

[0029] When amino acid substitutions are introduced into the polypeptides (II) and (III), examples of the amino acid substitutions include conservative substitutions. That is, in the polypeptides (II) and (III), examples of the amino acid substitutions introduced into SEQ ID NO: 1 include, for example, substitution of a nonpolar amino acid with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution of a noncharged amino acid with another uncharged amino acid if the amino acid before substitution is an acidic amino acid, and substitution of a basic amino acid with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0030] When an amino acid is added to the polypeptides (II) and (III) above, the form of the amino acid addition may be, for example, the addition of a purification tag (for example, a binding oligopeptide such as oligohistidine).

[0031] The polypeptides (II) and (III) include not only polypeptides obtained by artificial mutation, but also polypeptides resulting from naturally occurring mutations (mutants or variants) based on individual or species differences in the organisms from which the polypeptides are derived.

[0032] [1-3] Activity Regarding the activity of 3-hydroxybutyrate oxidase, one unit (1 U) is the amount of enzyme that increases the absorbance equivalent to 1 μmol of hydrogen peroxide per minute using D-3-hydroxybutyrate as a substrate. The oxidase activity is measured using a hydrogen peroxide detection system that utilizes color development due to the oxidative condensation of 4-aminoantipyrine (4-AA) and TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline) in the presence of peroxidase. The amount of hydrogen peroxide produced by the reaction can be calculated based on the change in absorbance measured per minute and the molar extinction coefficient of the 4-AA / TOOS oxidative condensation product.

[0033] [2] Method for producing 3-hydroxybutyrate oxidase [2-1] Production method by culturing a microorganism that produces the enzyme A first embodiment of the method for producing 3-hydroxybutyrate oxidase of the present invention comprises a culturing step of culturing a microorganism of the genus Achromobacter, and a recovery step of recovering, from the culture obtained in the culturing step, a fraction having the activity of catalyzing a reaction that acts on D-3-hydroxybutyrate to produce hydrogen peroxide (hereinafter also referred to as "3-hydroxybutyrate oxidase activity"). The first embodiment of the method for producing 3-hydroxybutyrate oxidase of the present invention can further comprise a purification step of purifying the recovered fraction having 3-hydroxybutyrate oxidase activity.

[0034] Achromobacter microorganisms are bacteria that produce 3-hydroxybutyrate oxidase, and details thereof are as described above in "[1] 3-hydroxybutyrate oxidase." In the production method of the present invention, one of the Achromobacter microorganisms described above in "[1] 3-hydroxybutyrate oxidase" may be used alone, or two or more of them may be used in combination.

[0035] The conditions for the culture step may be appropriately set taking into consideration the nutritional and physiological properties of the microorganism, but liquid culture is preferred. For industrial production, aeration and agitation culture is preferred.

[0036] Those skilled in the art can appropriately select the nutrient sources for the medium based on those required for the growth of the microorganisms. Examples of nutrient sources include carbon sources (glucose, sucrose, lactose, maltose, soluble starch, glycerin, dextrin, molasses, pyruvic acid, etc.), nitrogen sources (ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, peptone, yeast extract, corn steep liquor, casein hydrolysate, alkaline extract of soybean meal, wheat bran, meat extract, etc.), and inorganic salts (potassium salt, magnesium salt, sodium salt, manganese salt, iron salt, zinc salt, phosphate, carbonate, sulfate, etc.). Furthermore, amino acids and / or vitamins for promoting the growth of the microorganisms can be added to the medium.

[0037] The culture temperature is not particularly limited as long as the microorganism can grow and produce 3-hydroxybutyrate oxidase. An example of the culture temperature is about 15 to 37°C. The timing for completing the culture may be appropriately determined based on the time when the 3-hydroxybutyrate oxidase reaches its maximum yield, and the culture time is usually about 12 to 72 hours.

[0038] In the recovery step, a method for recovering a fraction having 3-hydroxybutyrate oxidase activity from the culture obtained in the culture step can be appropriately selected by those skilled in the art.

[0039] The fraction having 3-hydroxybutyrate oxidase activity is preferably an intracellular fraction of the culture. Therefore, in the recovery step, the culture is preferably subjected to solid-liquid separation (e.g., centrifugation) to recover the bacterial cells, and the recovered bacterial cells are then subjected to a mechanical method (e.g., ultrasound, French press, etc.) and / or an enzymatic method (e.g., a lytic enzyme such as lysozyme), and further solubilized, if necessary, by a solubilization method (e.g., an enzyme such as protease, a surfactant such as sodium lauryl sulfate (SDS), etc.), thereby allowing the recovery of a fraction having 3-hydroxybutyrate oxidase activity.

[0040] The fraction having 3-hydroxybutyrate oxidase activity can be obtained as a water-soluble fraction. The fraction may be obtained in a concentrated form by a concentration treatment to remove some or all of the water, as appropriate. Techniques used for the concentration treatment include vacuum concentration, membrane concentration, salting out, and fractional precipitation using a hydrophilic organic solvent (e.g., methanol, ethanol, acetone, etc.).

[0041] In the purification step, a person skilled in the art can appropriately select any treatment for removing part or all of unnecessary solid components from the fraction having 3-hydroxybutyrate oxidase activity obtained in the recovery step. The treatment used in the purification step can be one or a combination of several selected from membrane filtration (microfiltration, etc.), gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.

[0042] The purified 3-hydroxybutyrate oxidase may be in the form of an aqueous solution, or may be in the form of a dried product obtained by a drying process such as freeze drying, vacuum drying, or spray drying.

[0043] [2-2] Production method by culturing a transformant A second embodiment of the method for producing 3-hydroxybutyrate oxidase of the present invention comprises the step of culturing a transformant with an expression cassette or recombinant vector containing DNA encoding the second embodiment of 3-hydroxybutyrate oxidase shown in the above-mentioned "[1-2] Sequence".

[0044] A DNA encoding the second embodiment of 3-hydroxybutyrate oxidase can be appropriately prepared and designed by a person skilled in the art according to the amino acid sequence shown in the above "(1-2) Sequence."

[0045] An example of the DNA sequence is the base sequence shown in SEQ ID NO: 2. SEQ ID NO: 2 is a sequence derived from Achromobacter piechaudii that encodes the amino acid sequence of SEQ ID NO: 1.

[0046] Preferred examples of the DNA of the present invention include DNAs shown in either (i) or (ii) below.

[0047] (i) DNA consisting of the base sequence shown in SEQ ID NO: 2; (ii) DNA consisting of the base sequence of a DNA that hybridizes under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 2;

[0048] The DNA (ii) is a DNA having a similar base sequence to that of the DNA (i).

[0049] 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.

[0050] 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.

[0051] An example of the DNA (ii) above is the DNA (i) above to which a base sequence encoding a tag for purification (for example, a binding oligopeptide such as oligohistidine) has been further added.

[0052] The DNA can be obtained by the hybridization method described below.

[0053] First, DNA obtained from an appropriate gene source is ligated to a plasmid or phage vector according to a standard method to prepare a DNA library. This library is introduced into an appropriate host, and the resulting transformants are cultured on a plate. The grown colonies or plaques are transferred to a nitrocellulose or nylon membrane, and after denaturation treatment, the DNA is fixed to the membrane. This membrane is pre- 32 Hybridization is carried out by incubating under the stringent conditions described above in a solution of the above composition containing a probe labeled with P or the like. A polynucleotide encoding any one of the polypeptides (I) to (III) described above can be used as the probe.

[0054] After hybridization, nonspecifically adsorbed probes are washed away, and clones that hybridize with the probe are identified by autoradiography or other methods. This procedure is repeated until hybrid clones are isolated. Finally, a gene encoding a protein with the desired enzymatic activity is selected from the resulting clones. Gene isolation can be performed by known polynucleotide extraction methods, such as the alkaline method.

[0055] The DNA can also be isolated from Achromobacter piechaudii. For example, the target DNA can be isolated from the genome of the microorganism by PCR or hybridization using genomic DNA derived from Achromobacter piechaudii as a template and primers or probes designed from known amino acid sequence information taking gene degeneracy into consideration, or primers or probes designed based on known nucleotide sequence information.

[0056] This DNA includes 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. For example, when Escherichia coli is used as the host, DNA with a codon usage frequency optimized for E. coli is preferred.

[0057] 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).

[0058] Methods for introducing mutations into genes and artificially modifying amino acid sequences include known techniques such as the Kunkel method or the Gapped duplex method, and mutagenesis kits using site-directed mutagenesis, such as QuikChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneArt™ Site-Directed Mutagenesis PLUS System (Invitrogen), and TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, PrimeSTAR Mutagenesis Basal Kit, etc.: Takara Bio Inc.).

[0059] The DNA base sequence can be confirmed by sequencing using a conventional method, such as the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be analyzed using an appropriate DNA sequencer.

[0060] Whether the obtained DNA is a DNA encoding the desired 3-hydroxybutyrate oxidase can be confirmed by comparing the determined nucleotide sequence with SEQ ID NO: 2. Alternatively, the amino acid sequence deduced from the determined nucleotide sequence can be compared with SEQ ID NO: 1.

[0061] The DNA can be incorporated into an expression cassette or a recombinant vector, which can be obtained by linking a promoter and a terminator to the DNA, or by inserting the expression cassette or the DNA into an expression vector.

[0062] 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, and an SPI site, as necessary. These control elements may be operably linked to the DNA. "Operably linked" means that the DNA is linked to various control elements that regulate the DNA in a state that allows it to operate in a host cell.

[0063] The expression vector is preferably one constructed for genetic recombination from a phage, plasmid, or virus that can autonomously replicate in a host. Such expression vectors are known, and those skilled in the art can select and use an appropriate combination with a host cell. For example, when a microorganism is used as a host, examples of suitable vectors include pBluescript (pBS) II SK(-) (Stratagene), pSTV-based vectors (Takara Bio), pUC-based vectors (Takara Bio), pET-based vectors (Sigma-Aldrich Japan LLC), pGEX-based vectors (Global Life Science Technologies Japan (Cytiva)), pCold-based vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2), pp. 93-103), pBR322 (Takara Bio), pRS403 (Stratagene), and pMW218 / 219 (Nippon Gene). When algae or microalgae are used as hosts, examples of vectors include pUC19 (manufactured by Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (see Yangmin Gong, et al., Journal of Basic Microbiology, 2011, vol. 51, pp. 666-672), or pJET1 (manufactured by Thermo Fisher Scientific). When plant cells are used as hosts, examples of vectors include pRI-based vectors (manufactured by Takara Bio Inc.), pBI-based vectors (manufactured by Clontech), and IN3-based vectors (manufactured by Inplanta Innovations).

[0064] A transformant can be obtained by transforming a host with the expression cassette or recombinant vector.

[0065] The host used to produce the transformant is not particularly limited as long as it can be introduced with a gene, is capable of autonomous proliferation, and is capable of expressing the traits of the gene of the present invention, and suitable examples include microorganisms such as bacteria belonging to the genus Escherichia, such as Escherichia coli, the genus Bacillus, such as Bacillus subtilis, and the genus Pseudomonas, such as Pseudomonas putida; actinomycetes; yeast; and filamentous fungi, but other microorganisms may also be used, such as animal cells, insect cells, and plants. Of these, Escherichia coli is particularly preferred.

[0066] The host used for producing the transformant may be Achromobacter piechaudii, which is the source of the 3-hydroxybutyrate oxidase according to the second embodiment of the present invention.

[0067] The transformant can be obtained by introducing the expression cassette or recombinant vector into a host. The location of the DNA introduction 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 or recombinant vector of the present invention include, for example, recombinant vector methods and genome editing methods.

[0068] Conditions for introducing the expression cassette or recombinant vector into the host may be appropriately set depending on the type of host, etc. When the host is a microorganism, examples of such methods include a method using competent cells treated with calcium ions, electroporation, the spheroplast method, and the lithium acetate method. When the host is an animal cell, examples of such methods include electroporation, the calcium phosphate method, and the lipofection method. When the host is an insect cell, examples of such methods include the calcium phosphate method, the lipofection method, and the electroporation method. When the host is a plant cell, examples of such methods include the electroporation method, the Agrobacterium method, the particle gun method, and the PEG method.

[0069] The 3-hydroxybutyrate oxidase of the second embodiment of the present invention can be produced by culturing the transformant. The 3-hydroxybutyrate oxidase of the second embodiment of the present invention can also be produced by culturing Achromobacter piechaudii itself (untransformed), which is the microorganism that produces the enzyme.

[0070] The culture conditions for the transformant or the producing microorganism, the recovery of the fraction having 3-hydroxybutyrate oxidase activity, the concentration of the fraction, the purification of the 3-hydroxybutyrate oxidase, and the form of the purified 3-hydroxybutyrate oxidase are as described above in "[2-1] Production method by culturing a producing microorganism."

[0071] [3] Enzyme Preparation The enzymatic preparation of the present invention contains the 3-hydroxybutyrate oxidase described above in "[1] 3-hydroxybutyrate oxidase" as an active ingredient.

[0072] The content of 3-hydroxybutyrate oxidase in the enzyme preparation of the present invention is appropriately set within a range in which the 3-hydroxybutyrate oxidase activity of the active ingredient is exhibited. Examples of the content of 3-hydroxybutyrate oxidase in the enzyme preparation of the present invention include 0.1 to 100,000 U / g.

[0073] The enzyme preparation of the present invention may or may not contain other components in addition to the active ingredient, to the extent that the effects of the present invention are not affected. Examples of other components that may or may not be contained include enzymes other than 3-hydroxybutyrate oxidase, bases or additives, and culture residues generated in the above-mentioned production method.

[0074] 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, phosphatases (acid phosphatases, alkaline phosphatases), nucleases, deaminases, oxidases, dehydrogenases, glutaminase, pectinases, catalases, dextranases, transglutaminase, protein deamidating enzymes, pullulanases, laccases, etc. These other enzymes may be used singly or in combination.

[0075] Examples of bases or additives include excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, water, etc. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, etc. Examples of buffers include phosphates, citrates, acetates, etc. Examples of stabilizers include propylene glycol, ascorbic acid, etc. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. Examples of preservatives include ethanol, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. These additives may be used alone or in combination.

[0076] Culture residues include components derived from the culture medium, contaminating proteins, bacterial components, and the like.

[0077] 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 of the present invention can be prepared by a generally known method.

[0078] The enzymatic preparation of the present invention is preferably used as an enzymatic preparation for measuring ketone bodies. The ketone body to be measured is D-3-hydroxybutyric acid. Specific methods for using the enzymatic preparation of the present invention when used as an enzymatic preparation for measuring ketone bodies will be described in detail below in "[5] Method for measuring 3-hydroxybutyric acid in a sample."

[0079] [4] Sensor for measuring ketone bodies The sensor for measuring ketone bodies of the present invention comprises an enzyme preparation containing the 3-hydroxybutyrate oxidase described in "[1] 3-hydroxybutyrate oxidase" above or the 3-hydroxybutyrate oxidase described in "[3] Enzyme preparation" above.

[0080] The sensor for measuring ketone bodies of the present invention contains the above-mentioned 3-hydroxybutyrate oxidase or enzyme agent, and can be configured as an object that converts the change and the amount thereof due to the reaction of the substrate (D-3-hydroxybutyrate, which is also a ketone body) with oxygen by 3-hydroxybutyrate oxidase into a readable signal.

[0081] Specific examples of changes resulting from the reaction of a substrate with oxygen by 3-hydroxybutyrate oxidase include consumption of the substrate and production of hydrogen peroxide.

[0082] Signals that can read the amount of change due to the reaction of the substrate with oxygen by 3-hydroxybutyrate oxidase include electrochemical signals and optical signals.

[0083] Electrochemical signals include those in which a chemical state (eg, the presence of D-3-hydroxybutyric acid) is converted into an electrical signal (eg, electric current).

[0084] The optical signal includes light absorption and / or light emission based on the hydrogen peroxide produced.

[0085] In the ketone body measurement sensor of the present invention, the 3-hydroxybutyrate oxidase is preferably immobilized on an insoluble support. The manner of immobilization of the 3-hydroxybutyrate oxidase 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.

[0086] Furthermore, the 3-hydroxybutyrate oxidase may be immobilized on an insoluble support in the form of a single 3-hydroxybutyrate oxidase, or may be immobilized on an insoluble support in the form of a composition together with other components. A specific embodiment in which the 3-hydroxybutyrate oxidase 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.

[0087] At least the surface of the insoluble support is composed of a solid or solid material that does not dissolve in the reaction system between 3-hydroxybutyrate oxidase and 3-hydroxybutyrate and that can immobilize 3-hydroxybutyrate oxidase. 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 to which an electron mediator is bound), metals, carbon, etc. Examples of electron mediators include 2,6-dichloroindophenol (DCIP), phenazine methosulfate (PMS), and hexacyanoferrate(III) ion.

[0088] The shape of the insoluble carrier is not particularly limited, and examples thereof include substrate-like, flake-like, stick-like, and bead-like shapes.

[0089] A preferred example of the insoluble carrier is an electrode whose surface is made of the above-mentioned material. 3-hydroxybutyrate oxidase is preferably physically immobilized on such an electrode, and more preferably, an enzyme agent containing 3-hydroxybutyrate oxidase is physically immobilized (preferably by adhesion) on such an electrode.

[0090] 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.

[0091] A preferred embodiment of the ketone body measurement sensor of the present invention can be configured as an object that converts a signal into an electrochemical signal based on the consumption of a substrate by 3-hydroxybutyrate oxidase. A schematic diagram of this example is shown in FIG. 1. In the example shown in FIG. 1, an enzyme-functional electrode is formed, in which the catalytic reaction of an immobilized enzyme and the electrode reaction are integrated. In an enzyme-functional electrode, the electrode is used as an electron acceptor for the enzymatic reaction. When electrochemically oxidizing D-3-hydroxybutyrate (HB), the substrate of 3-hydroxybutyrate oxidase (HBOx), electrons are transferred to the electrode via an electron mediator that acts as an electron acceptor for 3-hydroxybutyrate oxidase (HBOx). The amount of D-3-hydroxybutyrate (HB), the substrate, can be quantified from the enzymatic reaction rate obtained by measuring the flow of electrons per unit time (i.e., current) corresponding to the turnover number of the 3-hydroxybutyrate oxidase (HBOx) reaction.

[0092] A preferred embodiment of the ketone body measurement sensor of the present invention can be configured as an object that converts an electrochemical signal based on the production of hydrogen peroxide by 3-hydroxybutyrate oxidase. A schematic diagram of this example is shown in Figure 2. In the example of Figure 2, the substrate D-3-hydroxybutyrate (HB) is oxidized by 3-hydroxybutyrate oxidase (HBOx), and the hydrogen peroxide generated in this process is electrochemically oxidized at a hydrogen peroxide electrode (generally, the surface of which is laminated with a selectively permeable membrane that allows hydrogen peroxide to pass toward the sensing electrode). The substrate D-3-hydroxybutyrate (HB) can be quantified from the current that flows during this process.

[0093] [5] Method for measuring 3-hydroxybutyric acid in a sample A method for measuring 3-hydroxybutyric acid in a sample includes an enzyme treatment step of contacting a sample containing D-3-hydroxybutyric acid with 3-hydroxybutyrate oxidase to generate hydrogen peroxide, and a quantification step of quantifying the hydrogen peroxide.

[0094] In the enzyme treatment step, the sample is not particularly limited as long as it contains D-3-hydroxybutyric acid, and examples thereof include biological samples. Examples of biological samples include body fluids, excrement, tissues, cells, etc. More specifically, examples include blood samples (whole blood, serum, plasma), urine, semen, prostatic fluid, tears, saliva, sweat, ascites, cerebrospinal fluid, milk, lymph, tissue extracts, etc. Examples of organisms from which biological samples are derived include mammals such as humans.

[0095] The 3-hydroxybutyrate oxidase may be the 3-hydroxybutyrate oxidase described in the above section "[1] 3-hydroxybutyrate oxidase." The 3-hydroxybutyrate oxidase may be in the form of the above section "[3] Enzyme preparation" or the above section "[4] Sensor for measuring ketone bodies."

[0096] The sample can be contacted with 3-hydroxybutyrate oxidase by, for example, preparing a mixed solution of the sample and the enzyme preparation described above in "[3] Enzyme Preparation." Alternatively, the sample can be contacted with 3-hydroxybutyrate oxidase by, for example, dropping, placing, pouring, or absorbing the sample onto an insoluble support on which 3-hydroxybutyrate oxidase is immobilized in the ketone body measurement sensor described above in "[4] Ketone body measurement sensor," or by, for example, immersing the insoluble support in the sample.

[0097] The sample is contacted with 3-hydroxybutyrate oxidase, and the enzymatic reaction is allowed to proceed under predetermined conditions. The enzymatic reaction conditions, such as temperature and pH, that induce the 3-hydroxybutyrate oxidizing activity of 3-hydroxybutyrate oxidase can be appropriately selected by those skilled in the art. For example, the reaction temperature can be 20 to 40°C, preferably 23 to 30°C, and more preferably 25 to 30°C. The reaction time is not particularly limited, but can be, for example, 5 seconds to 3 hours, and preferably 10 seconds to 10 minutes.

[0098] In the quantification step, hydrogen peroxide is quantified. The amount of hydrogen peroxide generated by the reaction between D-3-hydroxybutyric acid and 3-hydroxybutyrate oxidase correlates with the amount of D-3-hydroxybutyric acid consumed. Therefore, by quantifying the amount of hydrogen peroxide generated, the amount of D-3-hydroxybutyric acid contained in the sample can be quantified.

[0099] As explained with reference to FIG. 1 in the above section "[4] Ketone body measurement sensor," D-3-hydroxybutyric acid can also be measured based on an electrochemical signal resulting from the consumption of D-3-hydroxybutyric acid. However, this measurement system requires an electron mediator (although it does not use a coenzyme with low thermostability). On the other hand, when D-3-hydroxybutyric acid is measured based on the production of hydrogen peroxide, it becomes possible to construct a measurement system that not only does not use a coenzyme with low thermostability, but also does not require an electron mediator.

[0100] The method for quantifying hydrogen peroxide is not particularly limited, but examples thereof include a method for quantifying an electrical signal or an optical signal (absorption, emission, etc.) accompanying the production of hydrogen peroxide.

[0101] The method for quantifying the electrical signal accompanying the production of hydrogen peroxide is as explained above in “[4] Sensor for measuring ketone bodies” with reference to FIG. 2 .

[0102] One method for quantifying the optical signal accompanying the generation of hydrogen peroxide involves reacting hydrogen peroxide with a pair of oxidative coupling color-forming chromogens in the presence of peroxidase to produce a dye, and then measuring the color produced by this dye by measuring the absorbance of visible light. Examples of combinations of a pair of oxidative coupling color-forming chromogens include a combination of a coupler and an aniline, and a combination of a coupler and a phenol. Examples of couplers include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine. Examples of anilines include N-(3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-dimethyl-3-methylaniline, N,N-di(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, and N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline. N-ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS), and the like. Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

[0103] [6] Ketone body measurement kit The ketone body measurement kit of the present invention includes the enzyme preparation for ketone body measurement described in the above “[3] Enzyme preparation” or the sensor for ketone body measurement described in the above “[4] Sensor for ketone body measurement.”

[0104] 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.

[0105] Examples of such other items include electron mediators, combinations of a pair of oxidative coupling color-developing chromogens, deproteinized solutions, buffer solutions (e.g., acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate-buffered saline, etc.), 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 two or more of them.

[0106] 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.

[0107] Test Example 1 Screening was performed from the applicant's strain library (containing more than 10,000 strains) using oxidase activity (3-hydroxybutyrate oxidation activity) in the presence of D-3-hydroxybutyrate (3-HB) as an indicator. The oxidase activity was confirmed using a hydrogen peroxide detection system that utilizes color development due to oxidative condensation of 4-aminoantipyrine (4-AA) and TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline) in the presence of peroxidase.

[0108] (1) Experimental Procedure The activity measurement reagent solutions and substrate solutions shown in Table 1 below were prepared. In this test example, 40 mM Tris-HCl buffer (pH 8.5) was used as the buffer shown in Table 1.

[0109] The intracellular fraction stored in the applicant's strain library was used as a sample for screening evaluation.

[0110] To 120 μL of the activity measurement test solution, 10 μL of the substrate solution or blank (specifically, the same buffer as that used to prepare the substrate solution) and 20 μL of the enzyme sample (intracellular fraction solution) were added and mixed, and the mixture was incubated at 25°C for 3 hours. After incubation, the absorbance at 550 nm was measured using a microplate reader. If the absorbance measured in the presence of the substrate (substrate (+)) is higher than the absorbance measured in the absence of the substrate (substrate (-)), it can be evaluated as having 3-hydroxybutyrate oxidizing activity.

[0111] (2) Results Of the more than 10,000 strains, the results for the intracellular fractions of Achromobacter piechaudii strain 7576 (NBRC102461 strain) and Achromobacter xylosoxidans strain 7577 (NBRC15126 strain) are shown in Figure 3. As shown in Figure 3, the intracellular fractions of the above two strains showed a change in absorbance in the presence of a substrate, i.e., 3-hydroxybutyrate oxidase activity.

[0112] Furthermore, the intracellular fractions of the two strains were heat-treated at 80°C for 20 minutes. The change in absorbance of the intracellular fractions after heat treatment, as described in (1) above, was examined, confirming the inactivation of 3-hydroxybutyrate oxidase activity due to heat treatment. In other words, the inactivation of 3-hydroxybutyrate oxidase activity in the intracellular fractions of the two strains was confirmed.

[0113] From the above, the presence of 3-hydroxybutyrate oxidase derived from Achromobacter piechaudii (NBRC102461 strain) and Achromobacter xylosoxidans (NBRC15126 strain) was confirmed.

[0114] Test Example 2 The components shown in Table 2 were dissolved in water to the indicated concentrations, and the solution was autoclaved at 121°C for 20 minutes to prepare a medium.

[0115] 3 mL of medium was placed in a test tube, and an Achromobacter strain shown in Table 3 was inoculated into the medium. Shaking culture was performed for 48 hours under the culture temperature conditions for each strain shown in Table 3. After culture, the medium was centrifuged to recover wet bacterial cells. 10 mM phosphate buffer was added to the wet bacterial cells, which were then disrupted using a multi-beads shocker. The disrupted bacterial cell solution was centrifuged, and the supernatant was recovered to obtain an intracellular fraction.

[0116] The 3-hydroxybutyrate oxidase activity of the obtained intracellular fraction was confirmed in the same manner as in Test Example 1. Furthermore, the absorbance at 550 nm measured in the absence of the substrate 3-hydroxybutyrate (substrate (-)) was subtracted from the absorbance at 550 nm measured in the presence of the substrate 3-hydroxybutyrate (substrate (+)) to determine a value. Intracellular fractions showing a value of 0.004 or higher were determined to have 3-hydroxybutyrate oxidase activity. The results are shown in Table 3.

[0117]

[0118] As shown in Table 3 above, 3-hydroxybutyrate oxidase activity was confirmed in the intracellular fractions of all Achromobacter microorganisms, including those for which large differences in absorbance were confirmed, such as Achromobacter piechaudii strain 7576 (NBRC102461 strain), Achromobacter xylosoxidans strain 7577 (NBRC15126 strain), Achromobacter aloeverae (NBRC 111463T strain), and Achromobacter denitrificans (NBRC 15125T strain).

[0119] Test Example 3 The pH stability and optimum pH were evaluated for the intracellular fractions (intracellular fractions of Test Example 2) of Achromobacter piechaudii strain 7576 (NBRC102461 strain) and Achromobacter xylosoxidans strain 7577 (NBRC15126 strain).

[0120] (1) Optimum pH As the buffer solution shown in Table 1, 40 mM phosphate buffer (pH 6-8) or 40 mM Tris-HCl buffer (pH 8.5) was used, and the components shown in Table 1 were mixed to the indicated concentrations to prepare activity measurement reagent solutions and substrate solutions adjusted to pH 6, 6.5, 8, or 8.5. Using these activity measurement reagent solutions and substrate solutions, 3-hydroxybutyrate oxidase activity was evaluated in the same manner as in Test Example 1. The activity under the pH condition that showed the maximum activity was taken as 100%, and the relative amount of activity under each pH condition was calculated as relative activity (%). The results are shown in the table below.

[0121]

[0122] As is clear from Table 4, the highest 3-hydroxybutyrate oxidase activity was observed at pH 8.5 for both strains.

[0123] (2) pH Stability The intracellular fraction solution was dissolved in 40 mM Britton-Robinson buffer solutions (pH 5 to 9) of different pH values ​​and incubated (stored) at 4°C for 16 hours. After storage, the pH was adjusted to 8.5 by adding Tris-HCl buffer solution (pH 8.5). The components shown in Table 1 were mixed in Tris-HCl buffer solution (pH 8.5) to the indicated concentrations to prepare activity measurement reagent solutions and substrate solutions adjusted to pH 8.5. Using these activity measurement reagent solutions and substrate solutions, the 3-hydroxybutyrate oxidase activity (residual activity) of the intracellular fraction solution after storage was evaluated in the same manner as in Test Example 1. The relative value (%) of the residual activity at each pH was calculated, with the highest residual activity value designated as 100%.

[0124] The results are shown in Figures 4(A) and 4(B). As shown in Figures 4(A) and 4(B), the activity of 3-hydroxybutyrate oxidase obtained from microorganisms of the genus Achromobacter was stable in the alkaline range. In particular, as shown in Figure 4(A), the activity of 3-hydroxybutyrate oxidase obtained from Achromobacter piechaudii strain 7576 (NBRC102461 strain) was highly stable at test pHs of 8 and 9, and furthermore, was stable at 60% or more at pHs of 7, 8, and 9, and was stable at 55% or more at all test pHs.

[0125] Test Example 4 In this test example, 3-hydroxybutyrate oxidase was heterologously expressed using DNA (SEQ ID NO: 2) extracted from an Achromobacter piechaudii strain.

[0126] DNA was extracted from Achromobacter piechaudii strain 7576 (NBRC102461) and PCR amplified using this as a template. The PCR-amplified DNA was ligated to the pET24a(+) vector via In Fusion and transformed into Escherichia coli DH5α. A plasmid was constructed, and the insert sequence was confirmed by sequencing.

[0127] After transformation using Escherichia coli BL21 (DE3), the transformed strain was inoculated into LB medium and pre-cultured for 16 hours at 37° C. The pre-culture solution was inoculated into TB medium and cultured at 37° C. for 4 hours, after which IPTG was added to a final concentration of 0.5 mM and cultured at 25° C. for 18 hours.

[0128] The resulting culture medium was centrifuged at 15,000 rpm for 10 minutes and crushed with glass beads in 100 mM Tris-HCl (pH 9.0). The supernatant was microfiltrated to obtain 3-hydroxybutyrate oxidase (SEQ ID NO: 1). 3-hydroxybutyrate oxidase activity was confirmed in the same manner as in Test Example 1.

[0129] As a result, the absorbance difference (absorbance at 550 nm measured in the absence of the substrate 3-hydroxybutyrate (substrate (-)) minus the absorbance at 550 nm measured in the presence of the substrate 3-hydroxybutyrate (substrate (+))) was 0.077. Furthermore, the 3-hydroxybutyrate oxidase activity was confirmed in the same manner, except that the obtained 3-hydroxybutyrate oxidase was treated at 100°C for 10 minutes. As a result, it was confirmed that the above-mentioned absorbance difference had disappeared.

Claims

1. 3-hydroxybutyrate oxidase derived from a microorganism of the genus Achromobacter and having the following physicochemical properties: (1) Action: It acts on D-3-hydroxybutyrate to catalyze the reaction that produces hydrogen peroxide.

2. The 3-hydroxybutyrate oxidase according to claim 1, further having the following physicochemical properties: (2) optimum pH: 8.3 to 8.7; (3) pH stability: stable in the pH range of 8.5 to 9.

3. The 3-hydroxybutyrate oxidase according to claim 1, wherein the Achromobacter microorganism is Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter denitrificans, or Achromobacter aloeverae.

4. A 3-hydroxybutyrate oxidase consisting of a polypeptide shown in any one of (I) to (III) below: (I) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (II) a polypeptide consisting of an amino acid sequence in which one or more amino acids in SEQ ID NO: 1 have been substituted, added, inserted or deleted, and which has the activity of acting on D-3-hydroxybutyrate to catalyze a reaction that produces hydrogen peroxide; (III) a polypeptide consisting of an amino acid sequence that has a sequence identity of 70% or more to SEQ ID NO: 1, and which has the activity of acting on D-3-hydroxybutyrate to catalyze a reaction that produces hydrogen peroxide.

5. An enzyme preparation for oxidizing 3-hydroxybutyrate, comprising the 3-hydroxybutyrate oxidase according to claim 1 or 4 as an active ingredient.

6. The enzyme preparation according to claim 5, which contains an intracellular fraction of the microorganism belonging to the genus Achromobacter.

7. The enzyme preparation according to claim 5, which is used as an enzyme preparation for measuring ketone bodies.

8. A sensor for measuring ketone bodies, comprising the 3-hydroxybutyrate oxidase according to claim 1 or 4 or the enzyme preparation according to claim 5.

9. A kit for measuring ketone bodies, comprising the enzyme preparation according to claim 7 or the sensor for measuring ketone bodies according to claim 8.

10. A method for measuring 3-hydroxybutyric acid in a sample, comprising an enzyme treatment step of contacting the sample containing D-3-hydroxybutyric acid with 3-hydroxybutyric acid oxidase to generate hydrogen peroxide, and a quantification step of quantifying the hydrogen peroxide.

11. The method according to claim 10, wherein the 3-hydroxybutyrate oxidase is the 3-hydroxybutyrate oxidase according to claim 1 or 4.

12. A method for producing 3-hydroxybutyrate oxidase, comprising: a culturing step of culturing a microorganism of the genus Achromobacter; and a recovery step of recovering from the culture obtained in the culturing step a fraction having the activity of catalyzing a reaction that acts on D-3-hydroxybutyrate to produce hydrogen peroxide.

13. The production method according to claim 12, wherein the Achromobacter microorganism is selected from the group consisting of Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter denitrificans, and Achromobacter aloeverae.

14. The production method according to claim 12, wherein the microorganism of the genus Achromobacter is Achromobacter piechaudii NBRC102461 strain and / or Achromobacter xylosoxidans NBRC15126 strain.

15. A method for producing 3-hydroxybutyrate oxidase, comprising the step of culturing a transformant with an expression cassette or recombinant vector containing DNA encoding the 3-hydroxybutyrate oxidase according to claim 4.