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 reoxidizing NADH. The novel enzyme according to the present invention is a 3-hydroxybutyrate oxidase that is derived from a microorganism selected from the group consisting of microorganisms belonging to the genus Achromobacter, microorganisms belonging to the genus Pseudomonas, microorganisms belonging to the genus Trypanosoma, and microorganisms belonging to the genus Nocardia and has a physicochemical property of catalyzing a reaction for producing hydrogen peroxide by acting on D-3-hydroxybutyric acid, or is a 3-hydroxybutyrate oxidase that comprises an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a sequence similar thereto.

Description

3-Hydroxybutyrate Oxidase

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

[0002] As an enzyme using D-3-hydroxybutyric acid as a substrate, 3-hydroxybutyrate dehydrogenase (HBDH: EC 1.1.1.30) is known. HBDH belongs to the short-chain dehydrogenase / reductase (SDR) family and uses NAD + (nicotinamide adenine dinucleotide) as a coenzyme to reversibly catalyze the oxidation reaction of 3-hydroxybutyric acid to acetoacetic acid, and is an enzyme having an optimum pH for the oxidation reaction on the alkaline side of 8 or higher. Hydroxybutyric acid is called a ketone body together with acetoacetic acid and acetone.

[0003] A rapid increase in blood ketone body concentration is known as a cause of diabetic ketoacidosis (DKA), a severe complication of type-1 diabetes. Therefore, in order to prevent complications, it is recommended to monitor the amount of ketone bodies in type-1 diabetic patients. HBDH is an industrially important enzyme used for such ketone body measurement. Specifically, as shown in FIG. 6, in the ketone body measurement system using HBDH, first, the oxidation of 3-hydroxybutyric acid (HB) is catalyzed by HBDH, and the electrons generated during the oxidation are transferred to and from the coenzyme NAD to generate NADH. Next, the generated NADH is re-oxidized via an artificial electron mediator or NADH oxidase to obtain a signal.

[0004] HBDH has been isolated from various microorganisms, and in particular, the HBDHs disclosed in Patent Documents 1 to 3 are also on the market. These HBDHs are currently the standards for enzymes used in ketone body measurement.

[0005] JP-A-11-318438 JP-A-8-70856 JP-A-2003-339385

[0006] Since the HBDH reaction requires the coenzyme NAD, the addition of NAD is essential in ketone body measurement systems. Therefore, ketone body measurement using HBDH inevitably presents challenges due to the need for a coenzyme in addition to HBDH, and because NAD is thermally unstable, strict storage conditions are required.

[0007] Given the industrial importance of enzymes used in ketone body measurement, there is a need for the creation of enzymes with superior usability. Therefore, the present invention aims to provide a novel enzyme that acts in ketone body measurement without requiring NAD, which has low thermal stability like HBDH, and therefore without requiring a substance for the reoxidation of NADH.

[0008] The inventors of this invention, by chance, discovered a novel enzyme that catalyzes the reaction that produces hydrogen peroxide using D-3-hydroxybutyric acid as a substrate, from a library of more than 10,000 bacterial strains. This invention was completed based on this finding.

[0009] That is, the present invention provides the invention in the following embodiments. Item 1. A 3-hydroxybutyrate oxidase derived from a microorganism selected from the group consisting of Achromobacter, Pseudomonas, Trypanosoma, and Nocardia, having the following physicochemical properties: (1) Action: Catalytically reacts with D-3-hydroxybutyrate to produce hydrogen peroxide. Item 2. The 3-hydroxybutyrate oxidase according to Item 1, further having at least one of the following physicochemical properties: (2) Optimal pH: 8.3 to 8.7; (3) pH stability: Stable in the range of pH 8.5 to 9; (4) Action: Reduces electron mediators without the use of coenzymes in the presence of oxygen. Item 3. Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter denitrificans, Achromobacter Pseudomonas fragi, Pseudomonas putida, Pseudomonas aeruginosa; Trypanosoma cruzi; and Nocardia A 3-hydroxybutyrate oxidase according to item 1 or 2, derived from a microorganism selected from the group consisting of brasilensis.Item 4. 3-hydroxybutyrate oxidase comprising a polypeptide shown in any of (I) to (III) below: (I) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; (II) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, added, inserted, or deleted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and having the activity to catalyze a reaction in which D-3-hydroxybutyrate acts to produce hydrogen peroxide; (III) A polypeptide comprising an amino acid sequence with 70% or more sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and having the activity to catalyze a reaction in which D-3-hydroxybutyrate acts to produce hydrogen peroxide. Item 5. An enzyme preparation comprising the 3-hydroxybutyrate oxidase described in any of Items 1 to 4 as an active ingredient. Item 6. The enzyme preparation according to item 5, comprising the intracellular fraction of a microorganism selected from the group consisting of microorganisms of the genera Achromobacter, Pseudomonas, Trypanosoma, and Nocardia. Item 7. Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter denitrificans, Achromobacter aloeverae, Pseudomonas fragi, Pseudomonas putida, Pseudomonas aeruginosa, Trypanosoma cruzi, and Nocardia An enzyme preparation according to claim 5 or 6, comprising an intracellular fraction of a microorganism selected from the group consisting of brasilensis. Claim 8. An enzyme preparation according to any one of claims 5 to 7, used for reducing an electron mediator without the use of a coenzyme in the presence of oxygen. Claim 9. An enzyme preparation according to claim 8, wherein the mediator is 2,6-dichloroindophenol.Item 10. An enzyme preparation according to any one of items 5 to 8, used as an enzyme preparation for measuring ketone bodies. Item 11. A sensor for measuring ketone bodies, comprising 3-hydroxybutyrate oxidase according to any one of items 1 to 4 or an enzyme preparation according to any one of items 5 to 10. Item 12. A kit for measuring ketone bodies, comprising the enzyme preparation according to item 10 or the ketone body sensor according to item 11. Item 13. 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 quantitative step of quantifying the hydrogen peroxide. Item 14. The method according to item 13, wherein the 3-hydroxybutyrate oxidase is the 3-hydroxybutyrate oxidase according to any one of items 1 to 4. Item 15. A method for measuring 3-hydroxybutyrate in a sample, comprising an enzyme treatment step of contacting a sample containing D-3-hydroxybutyrate with a 3-hydroxybutyrate oxidase described in any of items 1 to 4 in the presence of oxygen, in the absence of a coenzyme, and in the presence of an electron mediator, and a quantitative step of quantifying the amount of reduction of the electron mediator. Item 16. A method for producing 3-hydroxybutyrate oxidase, comprising a culture step of culturing a microorganism selected from the group consisting of Achromobacter, Pseudomonas, Trypanosoma, and Nocardia, and a recovery step of recovering a fraction having the activity to catalyze a reaction that acts on D-3-hydroxybutyrate to produce hydrogen peroxide from the culture obtained in the culture step.Section 17. In the culturing step, Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter denitrificans, Achromobacter Pseudomonas fragi, Pseudomonas putida, Pseudomonas aeruginosa; Trypanosoma A method for producing 3-hydroxybutyrate oxidase according to item 16, comprising culturing a microorganism selected from the group consisting of cruzi and Nocardia brasiliensis. Item 18. A method for producing 3-hydroxybutyrate oxidase, comprising the step of culturing a transformant using an expression cassette or recombinant vector containing DNA encoding 3-hydroxybutyrate oxidase according to item 4.

[0010] The present invention provides a novel enzyme that catalyzes the reaction that produces hydrogen peroxide using D-3-hydroxybutyric acid as a substrate.

[0011] A schematic diagram illustrates a preferred example of the ketone body measurement sensor of the present invention. A schematic diagram illustrates a preferred example of the ketone body measurement sensor of the present invention. The results of confirming the 3-hydroxybutyrate oxidase (HBOx) activity in the intracellular fraction of Achromobacter piechaudii NBRC102461 strain and Achromobacter xylosoxidans NBRC15126 strain are shown. The pH stability of the 3-hydroxybutyrate oxidase activity (HBOx) fraction from Achromobacter piechaudii NBRC102461 strain is shown. The pH stability of the 3-hydroxybutyrate oxidase activity (HBOx) fraction from Achromobacter xylosoxidans NBRC15126 strain is shown. This shows the reducing action of the electron mediator DCIP by 3-hydroxybutyrate oxidase activity (HBOx) and 3-hydroxybutyrate dehydrogenase (HBDH) in the presence (a) or absence (b) of the coenzyme (β-NAD). A schematic diagram of a ketone body measurement sensor using 3-hydroxybutyrate dehydrogenase (HBDH) is shown.

[0012] [1] 3-Hydroxybutyrate Oxidase [1-1] Physicochemical Properties The first embodiment of the 3-hydroxybutyrate oxidase of the present invention is derived from a microorganism selected from the group consisting of Achromobacter, Pseudomonas, Trypanosoma, and Nocardia, and has the following physicochemical properties (1), and in a preferred embodiment, has at least one of the following physicochemical properties (2), (3), and (4).

[0013] (1) Mechanism of Action: It catalyzes the reaction in which it acts on D-3-hydroxybutyrate to produce hydrogen peroxide. The specific mechanism of action related to the physicochemical properties (1) described above is presumed to be that the hydrogen of the 3-hydroxyl group of D-3-hydroxybutyrate is transferred to oxygen, thereby producing hydrogen peroxide together with acetoacetic acid. 3-hydroxybutyrate oxidase does not require a coenzyme for this reaction.

[0014] (2) Optimal pH: 8.3 to 8.7, preferably 8.4 to 8.6. The optimal pH for the physicochemical properties (2) described above is the pH at which the relative activity of 3-hydroxybutyrate oxidase exhibiting maximum activity is 80% or more, preferably 90% or more, when reacted with D-3-hydroxybutyrate at various pH levels, 25°C, and for 3 hours, with the activity of 3-hydroxybutyrate oxidase exhibiting maximum activity being taken as 100%.

[0015] (3) pH stability: Stable at pH 8.5 to 9. The pH defined in the pH stability related to the physicochemical properties (3) above is the pH at which the residual activity of 3-hydroxybutyrate oxidase exhibiting maximum residual activity is 80% or more, preferably 90% or more, after storing 3-hydroxybutyrate oxidase under various pH, 4°C, and 16-hour conditions, with the residual activity of 3-hydroxybutyrate oxidase exhibiting maximum residual activity being taken as 100%.

[0016] In preferred embodiments, 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 preferred embodiments, the residual activity may be 15% or more, preferably 50% or more, and more preferably 60% or more, in the pH range of 7 to 9.

[0018] In preferred embodiments, 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] (4) Mechanism of action: Reduces electron mediators in the presence of oxygen without the need for coenzymes. In a preferred embodiment, 3-hydroxybutyrate oxidase has a function not found in general oxidases in that it reduces electron mediators in the presence of oxygen, and a function not found in general enzymes that react electron mediators in the presence of oxygen in that it reduces electron mediators without the need for coenzymes.

[0020] Examples of Achromocacter microorganisms that produce 3-hydroxybutyrate oxidase are not particularly limited and include, for example, Achromocacter ruhlandii, Achromocacter xylosoxidans, Achromocacter cholinophagum, Achromocacter piechaudii, Achromocacter spanius, Achromocacter insolitus, Achromocacter denitrificans, and Achromocacter aloeverae. The Achromocacter microorganisms may be wild-type strains or mutant strains (for example, those obtained by ultraviolet irradiation).

[0021] 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 the NBRC (National Biotechnology Center, National Institute of Technology and Evaluation) and can be obtained by following prescribed procedures.

[0022] In particular, the 3-hydroxybutyrate oxidase derived from Achromobacter piechaudii NBRC102461 strain is preferred because it exhibits a relatively high residual activity over a wide pH range. Specifically, the 3-hydroxybutyrate oxidase derived from Achromobacter piechaudii NBRC102461 strain is stable in the pH range of 8.5 to 9, and exhibits 90% residual activity in the pH range of 8 to 9, 60% or more residual activity in the pH range of 7 to 9, and 55% or more residual activity in the pH range of 5 to 9.

[0023] The 3-hydroxybutyrate oxidase of Achromobacter xylosoxidans strain NBRC15126 is stable in the pH range of 8.5 to 9, and its residual activity is 40% or more in the pH range of 8 to 9.

[0024] 3-hydroxybutyrate oxidase derived from a microorganism of the genus Achromobacter, preferably Achromobacter piechaudii, and more preferably Achromobacter piechaudii strain NBRC102461, is preferred because it is excellent in the action described in (4) above.

[0025] Examples of Pseudomonas microorganisms that produce 3-hydroxybutyrate oxidase are not particularly limited, and include, for example, Pseudomonas fragii, Pseudomonas putida, and Pseudomonas aeruginosa. The Pseudomonas microorganisms may be wild-type strains or mutant strains (for example, those obtained by ultraviolet irradiation).

[0026] The microorganisms of the genus Trypanosoma that produce 3-hydroxybutyrate oxidase are not particularly limited, and for example, Trypanosoma cruzi can be cited. The Trypanosoma microorganisms may be wild-type strains or mutant strains (for example, those obtained by ultraviolet irradiation).

[0027] The Nocardia microorganisms that produce 3-hydroxybutyrate oxidase are not particularly limited, and for example, Nocardia brasilinsis can be cited. The Nocardia microorganisms may be wild-type strains or mutant strains (for example, those obtained by ultraviolet irradiation).

[0028] [1-2] Sequence The second embodiment of the 3-hydroxybutyrate oxidase of the present invention consists of a polypeptide as shown in any of (I) to (III) below: (I) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; (II) A polypeptide consisting of an amino acid sequence in which one or more amino acids are substituted, added, inserted, or deleted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and having the activity to catalyze a reaction in which D-3-hydroxybutyrate acts to produce hydrogen peroxide; (III) A polypeptide consisting of an amino acid sequence with sequence identity of 70% or more to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and having the activity to catalyze a reaction in which D-3-hydroxybutyrate acts to produce hydrogen peroxide.

[0029] Sequence ID 1 is the amino acid sequence of 3-hydroxybutyrate oxidase from Achromobacter piechaudii. Sequence ID 3 is the amino acid sequence of 3-hydroxybutyrate oxidase from Pseudomonas fragii. Sequence ID 4 is the amino acid sequence of 3-hydroxybutyrate oxidase from Pseudomonas putida. Sequence ID 5 is the amino acid sequence of 3-hydroxybutyrate oxidase from Pseudomonas aeruginosa. Sequence ID 6 is the amino acid sequence of 3-hydroxybutyrate oxidase from Trypanosoma cruzzi. Sequence ID 7 is the amino acid sequence of 3-hydroxybutyrate oxidase from Nocardia brasiliensis.

[0030] Polypeptides (II) and (III) are 3-hydroxybutyrate oxidases with a sequence similar to polypeptide (I), with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 as their basic skeleton.

[0031] The modification of amino acids introduced into the polypeptide (II) above may include only one type of modification (e.g., substitution) from among substitution, addition, insertion, and deletion, or it may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide (II) above, the number of amino acids to be substituted, added, inserted, or deleted may be one, more than one, or several. For example, 1 to 79, 1 to 78, 1 to 76, preferably 1 to 53, 1 to 52, 1 to 51, 1 to 39, 1 to 38, or 1 to 26, 1 to 25, more preferably 1 to 13, 1 to 12, preferably 1 to 7, 1 to 5, and particularly preferably 1 or 2 or 1.

[0032] Furthermore, in the polypeptide of (III) above, sequence identity should be 70% or more, but preferably 80% or more, 85% or more, or 90% or more, more preferably 95% or more, 97% or more, 98% or more, and especially preferably 99% or more, or 99.5% or more.

[0033] Here, in the polypeptide of (III) above, the sequence identity with respect to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 is the sequence identity calculated by comparing it with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. Furthermore, "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 National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0034] When amino acid substitutions are introduced into the polypeptides of (II) and (III) above, one type of amino acid substitution is a conservative substitution. Specifically, in polypeptides of (II) and (III), amino acid substitutions introduced into SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 include, for example, substitution of the amino acid before substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution of the amino acid before substitution with another noncharged amino acid if the amino acid before substitution is an acidic amino acid, and substitution of the amino acid before substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0035] When amino acid addition is introduced to the polypeptides of (II) and (III) above, examples of the form of amino acid addition include the addition of a purification tag (e.g., a binding oligopeptide such as oligohistidine).

[0036] The polypeptides in (II) and (III) above include not only polypeptides obtained by artificial mutation, but also polypeptides resulting from naturally occurring mutations (mutants or variants) based on individual differences or species differences in the organism from which the polypeptide originates.

[0037] [1-3] Activity The activity of 3-hydroxybutyrate oxidase is defined as the amount of enzyme that gives an increase in absorbance equivalent to 1 μmol of hydrogen peroxide per minute, using D-3-hydroxybutyrate as the substrate, with 1 unit (1 U) being defined. The oxidase activity is obtained by measurement using a hydrogen peroxide detection system that utilizes the color change produced by 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 condensate.

[0038] [2] Method for producing 3-hydroxybutyrate oxidase [2-1] Method for producing by culturing a producing microorganism The first embodiment of the method for producing 3-hydroxybutyrate oxidase of the present invention includes a culture step of culturing a microorganism selected from the group consisting of Achromobacter, Pseudomonas, Trypanosoma, and Nocardia microorganisms, and a recovery step of recovering a fraction having activity to catalyze the reaction that acts on D-3-hydroxybutyrate to produce hydrogen peroxide (hereinafter also referred to as "3-hydroxybutyrate oxidase activity") from the culture obtained in the culture step. The first embodiment of the method for producing 3-hydroxybutyrate oxidase of the present invention may further include a purification step of purifying the recovered fraction having 3-hydroxybutyrate oxidase activity.

[0039] Microorganisms of the genera Achromobacter, Pseudomonas, Trypanosoma, and Nocardia are 3-hydroxybutyrate oxidase-producing microorganisms, and specific examples thereof are described in "[1] 3-hydroxybutyrate oxidase" above. In the production method of the present invention, one of the microorganisms of the genera Achromobacter, Pseudomonas, Trypanosoma, and Nocardia described in "[1] 3-hydroxybutyrate oxidase" above may be used alone, or multiple species may be used in combination.

[0040] The conditions in the culture process should be set appropriately considering the nutritional and physiological properties of the microorganisms, but liquid culture is preferred. Furthermore, in the case of industrial production, aerated and stirred culture is preferred.

[0041] As the nutrient source of the medium, those necessary for the growth of the above-mentioned microorganism can be appropriately selected by those skilled in the art. Examples of the nutrient source include, for example, carbon sources (such as glucose, sucrose, lactose, maltose, soluble starch, glycerin, dextrin, molasses, pyruvic acid, etc.), nitrogen sources (such as ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, peptone, yeast extract, corn steep liquor, casein hydrolyzate, alkali extract of soybean meal, bran, meat extract, etc.), inorganic salts (such as potassium salts, magnesium salts, sodium salts, manganese salts, iron salts, zinc salts, phosphates, carbonates, sulfates, etc.), etc., and one or more of them are selected. Furthermore, amino acids and / or vitamins for promoting the growth of microorganisms can also be added to the medium.

[0042] The culture temperature is not particularly limited as long as the above-mentioned microorganism can grow and produce 3-hydroxybutyric acid oxidase. Examples of the culture temperature include about 15 to 37°C. The timing of completion of the culture can be appropriately determined in consideration of the time when the 3-hydroxybutyric acid oxidase reaches the maximum yield, and the culture time is usually about 12 to 72 hours.

[0043] In the recovery step, those skilled in the art can appropriately select a method for recovering a fraction having 3-hydroxybutyric acid oxidase activity from the culture obtained in the culture step.

[0044] The fraction having 3-hydroxybutyric acid oxidase activity is preferably the fraction within the cells in the culture. Therefore, in the recovery step, preferably, the culture is subjected to solid-liquid separation (such as centrifugation, etc.) to recover the cells, and the recovered cells are subjected to mechanical methods (such as ultrasonic waves, French press, etc.) and / or enzymatic methods (such as lytic enzymes such as lysozyme), and further, if necessary, solubilized by solubilization methods (such as enzymes such as protease, surfactants such as sodium lauryl sulfate (SDS), etc.), so that a fraction having 3-hydroxybutyric acid oxidase activity can be recovered.

[0045] The fraction having 3-hydroxybutyric acid oxidase activity can be obtained as a water-soluble fraction. The fraction may be obtained in a concentrated form by a concentration treatment for appropriately removing part or all of the water. Examples of the method used in the concentration treatment include vacuum concentration, membrane concentration, salting-out treatment, and fractional precipitation using a hydrophilic organic solvent (e.g., methanol, ethanol, acetone, etc.).

[0046] In the purification step, those skilled in the art can appropriately select any treatment for removing part or all of the unnecessary solid components from the fraction having 3-hydroxybutyric acid oxidase activity obtained in the recovery step. Examples of the treatment used in the purification step include one or a combination of more than one selected from membrane filtration (such as microfiltration), gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.

[0047] The purified 3-hydroxybutyric acid oxidase may be in the form of an aqueous solution or in a dried form obtained by a drying treatment such as freeze drying, vacuum drying, spray drying, etc.

[0048] [2-2] Production method by culturing transformant The second embodiment of the production method of the 3-hydroxybutyric acid oxidase of the present invention includes the step of culturing a transformant with an expression cassette or a recombinant vector containing DNA encoding the second embodiment of the 3-hydroxybutyric acid oxidase shown in the above "[1-2] sequence".

[0049] Those skilled in the art can appropriately prepare and design the DNA encoding the second embodiment of the amino acid sequence shown in the above "[1-2] sequence".

[0050] Examples of the DNA sequences include the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. SEQ ID NO: 2 is a sequence derived from Achromobacter piechaudii that encodes the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 8 is an E. coli codon-optimized sequence of a Pseudomonas fragii sequence that encodes the amino acid sequence of SEQ ID NO: 3. SEQ ID NO: 9 is an E. coli codon-optimized sequence of a Pseudomonas putida sequence that encodes the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 10 is an E. coli codon-optimized sequence of a Pseudomonas aeruginosa sequence that encodes the amino acid sequence of SEQ ID NO: 5. SEQ ID NO: 11 is an E. coli codon-optimized sequence of a Trypanosoma cruzzi sequence that encodes the amino acid sequence of SEQ ID NO: 6. Sequence ID 12 is an E. coli codon-optimized sequence of a Nocardia brasilensis sequence that encodes the amino acid sequence of Sequence ID 7.

[0051] Preferred examples of the DNA of the present invention include the DNA shown in either (i) or (ii) below.

[0052] (i) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; (ii) DNA consisting of the nucleotide sequence of DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12

[0053] DNA (ii) is sequence-similar DNA that uses the base sequence of DNA (i) as its basic framework.

[0054] In the DNA described in (ii) above, "stringent conditions" means conditions in which the DNA is incubated at 50°C to 65°C for 4 hours to overnight in a 6× SSC (1× SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5× Denhartz's [Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficol 400] and 100 μg / ml salmon sperm DNA.

[0055] Hybridization under stringent conditions is specifically carried out by the following method: A nylon membrane immobilizing a DNA library or cDNA library is prepared, and the nylon membrane is blocked at 65°C in a pre-hybridization solution containing 6×SSC, 0.5% SDS, 5×Denharts, and 100 μg / ml salmon sperm DNA. Then, 32 Each probe labeled with P is added and incubated overnight at 65°C. After washing this nylon membrane in 6×SSC for 10 minutes at room temperature, in 2×SSC containing 0.1% SDS for 10 minutes at room temperature, and in 0.2×SSC containing 0.1% SDS at 45°C for 30 minutes, autoradiography can be performed to detect DNA that specifically hybridizes with the probe.

[0056] In the example of DNA in (ii) above, a base sequence encoding a purification tag (for example, a binding oligopeptide such as oligohistidine) is further added to the DNA in (i) above.

[0057] One method for obtaining the DNA in question is the hybridization-based method described below.

[0058] First, DNA obtained from a suitable gene source is linked to a plasmid or phage vector according to standard procedures to create a DNA library. This library is introduced into a suitable 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 immobilized on the membrane. This membrane is then prepared in advance. 32Hybridization is performed in a solution of the above composition containing a probe labeled with P, etc., while maintaining the temperature under the above stringent conditions. As the probe, a polynucleotide encoding any of the polypeptides (I) to (III) above can be used.

[0059] After hybridization is complete, nonspecifically adsorbed probes are washed away, and clones that have formed hybrids with the probes are identified by autoradiography or other methods. This procedure is repeated until hybrid-forming clones can be isolated. Finally, from the obtained clones, the gene encoding the protein with the desired enzyme activity is selected. Gene isolation can be performed by known polynucleotide extraction methods such as the alkaline method.

[0060] This DNA can also be isolated from Achromobacter piechaudii, Pseudomonas fragii, Pseudomonas putida, Pseudomonas aeruginosa, Trypanosoma cruzzi, or Nocardia brasiliensis. For example, using genomic DNA derived from Achromobacter piechaudii, Pseudomonas fragii, Pseudomonas putida, Pseudomonas aeruginosa, Trypanosoma cruzi, or Nocardia brasiliensis as a template, the target DNA can be isolated from the genome of the microorganism by PCR or hybridization using primers or probes designed based on known amino acid sequence information considering gene degeneracy, or primers or probes designed based on known base sequence information.

[0061] This DNA contains various types of DNA derived from codon degeneracy. It is easily possible to artificially create multiple types of DNA encoding the same amino acid sequence using known genetic engineering techniques. For example, in the production of genetically engineered proteins, if the codons used in the original gene encoding the target protein are infrequently used in the host, the protein expression level may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon utilization frequency for the host without altering the encoded amino acid sequence. For example, if E. coli is used as the host, DNA with codon utilization frequency optimized for E. coli is preferable.

[0062] As an indicator of codon utilization frequency, the sum of the host-optimal codon utilization frequencies for each codon can be adopted. An optimal codon is defined as the codon with the highest utilization frequency among codons corresponding to the same amino acid. Codon utilization frequency is not particularly limited as long as it is optimized for the host, but the following are examples of optimal codons in 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).

[0063] Methods for introducing mutations into genes and artificially modifying amino acid sequences include known methods such as the Kunkel method or the Gapped duplex method, and mutation introduction kits utilizing site-directed mutagenesis, such as the QuickChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneArt™ Site-Directed Mutagenesis PLUS System (Invitrogen), and the TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, PrimeSTAR Mutagenesis Basal Kit, etc.: Takara Bio).

[0064] The base sequence of DNA can be confirmed by sequencing using conventional methods. For example, it can be done by dideoxynucleotide chain termination (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be analyzed using a suitable DNA sequencer.

[0065] To confirm whether the obtained DNA encodes the target 3-hydroxybutyrate oxidase, the determined base sequence can be compared with SEQ ID NOs: 2, 8, 9, 10, 11, or 12. Alternatively, the amino acid sequence predicted from the determined base sequence can be compared with SEQ ID NOs: 1, 3, 4, 5, 6, or 7.

[0066] The DNA can be incorporated into an expression cassette or recombinant vector. The expression cassette or recombinant vector can be obtained by ligating a promoter and terminator to the DNA, or by inserting the expression cassette or DNA into an expression vector.

[0067] The expression cassette or recombinant vector of the present invention may, as regulatory factors, include a promoter and a terminator, as well as, if necessary, transcription elements such as enhancers, CCAAT boxes, TATA boxes, and SPI sites. These regulatory factors only need to be operably ligated to the DNA. Operable ligation means that the various regulatory factors that regulate the DNA are ligated to the DNA in a manner that allows them to function within the host cell.

[0068] Preferred expression vectors are those constructed for genetic recombination from phages, plasmids, or viruses that can autonomously proliferate within a host. Such expression vectors are well known, and those skilled in the art can appropriately select and use suitable combinations with host cells. For example, when using microorganisms as hosts, examples include pBluescript (pBS) II SK(-) (manufactured by Stratagene), pSTV vectors (manufactured by Takara Bio), pUC vectors (manufactured by Takara Bio), pET vectors (manufactured by Sigma-Aldrich Japan LLC), pGEX vectors (manufactured by Global Life Science Technologies Japan (Cytiva)), pCold vectors (manufactured by Takara Bio), pHY300PLK (manufactured by Takara Bio), pUB110 (McKenzie, T. et al., 1986, Plasmid 15(2), pp. 93-103), pBR322 (manufactured by Takara Bio), pRS403 (manufactured by Stratagene), and pMW218 / 219 (manufactured by Nippon Gene). Examples of vectors used with algae or microalgae as hosts include pUC19 (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 (Thermo Fisher Scientific Inc.). Examples of vectors used with plant cells as hosts include pRI vectors (Takara Bio Inc.), pBI vectors (Clontech Inc.), and IN3 vectors (Implanta Innovations Inc.).

[0069] A transformant can be obtained by transforming the host using the expression cassette or recombinant vector.

[0070] The host used to produce the transformant is not particularly limited as long as it is capable of gene introduction, autonomous replication, and expression of the gene traits of the present invention. Suitable examples include bacteria belonging to the Escherichia genus such as Escherichia coli, the Bacillus genus such as Bacillus subtilis, the Pseudomonas genus such as Pseudomonas putida, actinomycetes, yeasts, filamentous fungi, etc. Other examples include animal cells, insect cells, plants, etc. Among these, Escherichia coli is particularly preferred.

[0071] The host used for the production of the transformant may be Achromobacter piechaudii, Pseudomonas fragii, Pseudomonas putida, Pseudomonas aeruginosa, Trypanosoma cruzi, or Nocardia brasilensis, which are the source organisms of the second embodiment of the 3-hydroxybutyrate oxidase of the present invention.

[0072] The transformant can be obtained by introducing the expression cassette or recombinant vector into a host. The site where the DNA is introduced is not particularly limited as long as the target gene 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.

[0073] The conditions for introducing the expression cassette or recombinant vector into the host can be appropriately set according to the type of host, etc. If the host is a microorganism, examples include methods using competent cells treated with calcium ions, electroporation, spheroplast, and lithium acetate. If the host is an animal cell, examples include electroporation, calcium phosphate, and lipofection. If the host is an insect cell, examples include calcium phosphate, lipofection, and electroporation. If the host is a plant cell, examples include electroporation, Agrobacterium, particle gun, and PEG.

[0074] A second embodiment of the 3-hydroxybutyrate oxidase of the present invention can be produced by culturing the transformant.

[0075] 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 3-hydroxybutyrate oxidase, and the form of the purified 3-hydroxybutyrate oxidase are as described in "[2-1] Method for production by culturing the producing microorganism" above.

[0076] [3] Enzyme preparation The enzyme preparation of the present invention contains the 3-hydroxybutyrate oxidase described in "[1] 3-hydroxybutyrate oxidase" above as an active ingredient.

[0077] The amount of 3-hydroxybutyrate oxidase contained 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 exerted. Examples of the amount of 3-hydroxybutyrate oxidase in the enzyme preparation of the present invention include, for example, 0.1 to 100,000 U / g.

[0078] The enzyme preparation of the present invention may or may not contain other components in addition to the active ingredient, provided that these components do not affect the effects of the present invention. Examples of other components that may or may not be included include other enzymes other than 3-hydroxybutyrate oxidase, bases or additives, and culture residues generated by the above manufacturing method.

[0079] Other enzymes include, for example, amylases (α-amylase, β-amylase, glucoamylase), glucosidases (α-glucosidase, β-glucosidase), galactosidases (α-galactosidase, β-galactosidase), proteases (acid protease, neutral protease, alkaline protease), peptidases (leucine peptidase, aminopeptidase), lipases, esterases, cellulases, phosphatases (acid phosphatase, alkaline phosphatase), nucleases, deaminases, oxidases, dehydrogenases, glutaminases, pectinases, catalases, dextranases, transglutaminases, protein deamide enzymes, pullulanases, and laccases. These other enzymes may be used individually or in combination.

[0080] Examples of bases or additives include excipients, buffers, suspending agents, stabilizers, preservatives, antimicrobials, physiological saline, and water. 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 antimicrobials include ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. These additives may be used individually or in combination.

[0081] Culture residues include components derived from the culture medium, contaminating proteins, and bacterial cell components.

[0082] The form of the enzyme preparation of the present invention is not particularly limited and can be, for example, liquid or solid (powder, granules, etc.). The enzyme preparation of the present invention can be prepared by generally known methods.

[0083] The enzyme preparation of the present invention is preferably used as an enzyme preparation for measuring ketone bodies. The ketone body to be measured is D-3-hydroxybutyrate. The specific method of using the enzyme preparation of the present invention as an enzyme preparation for measuring ketone bodies will be described in detail later in "[5] Method for measuring 3-hydroxybutyrate in a sample".

[0084] [4] Ketone body measuring sensor The ketone body measuring sensor of the present invention comprises the 3-hydroxybutyrate oxidase described in "[1] 3-hydroxybutyrate oxidase" above or an enzyme preparation containing the 3-hydroxybutyrate oxidase described in "[3] Enzyme preparation" above.

[0085] The ketone body measuring sensor of the present invention may be configured as an object that contains the above-mentioned 3-hydroxybutyrate oxidase or enzyme preparation, and converts the changes and amounts based on the reaction between a substrate (D-3-hydroxybutyrate, which is also a ketone body) and oxygen by 3-hydroxybutyrate oxidase into a readable signal.

[0086] The changes resulting from the reaction between the substrate and oxygen by 3-hydroxybutyrate oxidase include, specifically, substrate consumption, hydrogen peroxide production, and reduction of electron mediators.

[0087] Electrochemical and optical signals are examples of signals that can be used to read the changes resulting from the reaction between a substrate and oxygen by 3-hydroxybutyrate oxidase.

[0088] Electrochemical signals include those obtained by converting a chemical state (e.g., the presence of D-3-hydroxybutyrate) into an electrical signal (e.g., electric current).

[0089] Optical signals include absorption and / or emission based on the generated hydrogen peroxide, and absorption and / or emission based on the electron mediator that decreases upon reduction.

[0090] In the ketone body measurement sensor of the present invention, it is preferable that the 3-hydroxybutyrate oxidase is immobilized on an insoluble support. The method of immobilization of 3-hydroxybutyrate oxidase is not particularly limited and includes physical immobilization by adhesion, adsorption, absorption, swelling, etc., chemical or biochemical immobilization by specific non-covalent bonding, and chemical immobilization by covalent bonding.

[0091] Furthermore, 3-hydroxybutyrate oxidase may be immobilized on an insoluble support in a single form, or it may be immobilized on an insoluble support in the form of a composition together with other components. A specific embodiment of the case where 3-hydroxybutyrate oxidase is immobilized on an insoluble support in the form of a composition is one in which the composition is physically fixed (preferably fixed by adhesion) to the insoluble support.

[0092] The insoluble support is composed of a solid or solid material, at least on its surface, 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 constituting at least the surface of the insoluble support include swellable materials (e.g., gelatin), porous substrates (e.g., cellulose, filter paper), resins, glass, redox polymers (i.e., polymers to which electron mediators are bound), metals, carbon, etc. Examples of electron mediators include 2,6-dichloroindophenol (DCIP), phenazine methosulfate (PMS), and hexacyanoferrate(III) ions.

[0093] The shape of the insoluble carrier is not particularly limited and examples include substrate-like, flake-like, stick-like, and bead-like forms.

[0094] A preferred example of the above-mentioned insoluble carrier is an electrode whose surface is made of the above-mentioned material. Furthermore, such an electrode preferably has 3-hydroxybutyrate oxidase physically immobilized on it, and more preferably has an enzyme preparation containing 3-hydroxybutyrate oxidase physically immobilized (preferably by adhesion).

[0095] Specific embodiments of the ketone body measurement sensor 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, and the like.

[0096] In a specific example of the ketone body measurement sensor of the present invention (referred to as "Embodiment 1"), the sensor can be configured as an object that converts the consumption of a substrate by 3-hydroxybutyrate oxidase into an electrochemical signal. A schematic diagram of Embodiment 1 is shown in Figure 1. The example in Figure 1 utilizes a reaction that proceeds under deoxygenation conditions and forms an enzyme-functional electrode in which the catalytic reaction of the immobilized enzyme and the electrode reaction are integrated. In the enzyme-functional electrode, the electrode is used as an electron acceptor for the enzyme reaction, and when D-3-hydroxybutyrate (HB), the substrate of 3-hydroxybutyrate oxidase (HBOx), is electrochemically oxidized, electrons are transferred to the electrode via an electron mediator that acts as an electron acceptor for 3-hydroxybutyrate oxidase (HBOx). By measuring the flow of electrons per unit time (i.e., current) corresponding to the turnover number of the reaction of 3-hydroxybutyrate oxidase (HBOx), the substrate D-3-hydroxybutyrate (HB) can be quantified from the rate of the enzyme reaction.

[0097] In a specific example of the ketone body measurement sensor of the present invention (referred to as "Embodiment 2"), the sensor can be configured as an object that converts the generation of hydrogen peroxide by 3-hydroxybutyrate oxidase into an electrochemical signal. A schematic diagram of Embodiment 2 is shown in Figure 2. The example in Figure 2 utilizes a reaction that proceeds in the presence of oxygen. The substrate, D-3-hydroxybutyrate (HB), is oxidized by 3-hydroxybutyrate oxidase (HBOx), and the hydrogen peroxide produced is electrochemically oxidized by a hydrogen peroxide electrode (generally, a selective permeable film that allows hydrogen peroxide to pass through to the detection electrode is laminated on its surface), and the substrate, D-3-hydroxybutyrate (HB), can be quantified from the current that flows at this time.

[0098] In a specific example of the ketone body measurement sensor of the present invention (referred to as "Embodiment 3"), the object can be configured to convert the consumption of the substrate by 3-hydroxybutyrate oxidase into an optical or electrochemical signal. When detecting an enzymatic reaction using an optical signal, the object more specifically utilizes a reaction that proceeds in the presence of oxygen without the intervention of a coenzyme, and is configured to measure the absorbance at a predetermined wavelength using an electron mediator that absorbs a predetermined wavelength as an indicator. This allows for the quantification of the substrate, D-3-hydroxybutyrate (HB), from the amount of absorbance reduction corresponding to the reduction of the electron mediator due to electron transfer from 3-hydroxybutyrate oxidase (HBOx) to the electron mediator. When 2,6-dichloroindophenol (DCIP), which has an absorption maximum at 600 nm, is used as the electron mediator, the substrate HB can be quantified from the amount of absorbance reduction at 600 nm corresponding to the reduction of DCIP. When detecting an enzymatic reaction using electrochemical signals, similar to the first embodiment described above, the substrate D-3-hydroxybutyrate (HB) can be quantified from the rate of the enzymatic reaction 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.

[0099] [5] Method for measuring 3-hydroxybutyrate in a sample [5-1] Method by quantitative determination of generated hydrogen peroxide An embodiment of the method for measuring 3-hydroxybutyrate in a sample includes an enzymatic treatment step of contacting a sample containing D-3-hydroxybutyrate with 3-hydroxybutyrate oxidase to generate hydrogen peroxide, and a quantitative step of quantifying the hydrogen peroxide.

[0100] In the enzyme treatment process, the sample is not particularly limited as long as it contains D-3-hydroxybutyric acid, but examples include biological samples. Examples of biological samples include body fluids, excretions, 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.

[0101] As 3-hydroxybutyrate oxidase, the 3-hydroxybutyrate oxidase described in "[1] 3-hydroxybutyrate oxidase" above can be used. The 3-hydroxybutyrate oxidase can be used in the form of "[3] Enzyme preparation" or "[4] Ketone body measurement sensor" above.

[0102] Contact between the sample and 3-hydroxybutyrate oxidase can be achieved, for example, by preparing a mixture containing the sample and the enzyme preparation described in "[3] Enzyme Preparation". Alternatively, contact between the sample and 3-hydroxybutyrate oxidase can be achieved by dropping, placing, flowing, or absorbing the sample onto an insoluble support on which 3-hydroxybutyrate oxidase is immobilized in the ketone body measurement sensor described in "[4] Ketone Body Measurement Sensor", or by immersing the insoluble support in the sample.

[0103] The sample is brought into contact with 3-hydroxybutyrate oxidase, and the enzymatic reaction is carried out under predetermined conditions. The conditions for the enzymatic reaction, such as temperature and pH conditions that produce 3-hydroxybutyrate oxidative activity of 3-hydroxybutyrate oxidase, are appropriately selected by those skilled in the art. For example, the reaction temperature is specifically 20 to 40°C, preferably 23 to 30°C, and more preferably 25 to 30°C. The reaction time is not particularly limited, but for example, it is 5 seconds to 3 hours, preferably 10 seconds to 10 minutes.

[0104] In the quantitative analysis step, hydrogen peroxide is quantified. The amount of hydrogen peroxide generated from the reaction between D-3-hydroxybutyrate and 3-hydroxybutyrate oxidase correlates with the amount of D-3-hydroxybutyrate consumed. Therefore, by quantifying the amount of hydrogen peroxide generated, the amount of D-3-hydroxybutyrate contained in the sample can be quantified.

[0105] As explained in Figure 1 as Embodiment 1 in "[4] Ketone Body Measurement Sensor" above, D-3-hydroxybutyrate can also be measured based on an electrochemical signal derived from the consumption of D-3-hydroxybutyrate, but this measurement system requires an electron mediator (although it does not use a coenzyme with low thermal stability). On the other hand, as explained in Figure 2 as Embodiment 2 in "[4] Ketone Body Measurement Sensor" above, when D-3-hydroxybutyrate is measured based on the generation of hydrogen peroxide, it is possible to construct a measurement system that does not use an electron mediator (not only does it not use a coenzyme with low thermal stability), but it is also possible to construct a system that does not use an electron mediator.

[0106] The method for quantifying hydrogen peroxide is not particularly limited, but examples include methods for quantifying electrical signals or optical signals (absorption, luminescence, etc.) associated with the generation of hydrogen peroxide.

[0107] The method for quantifying the electrical signal associated with the generation of hydrogen peroxide is as described above in "[4] Ketone Body Measurement Sensor" with reference to Figure 2.

[0108] One method for quantifying the optical signal associated with the generation of hydrogen peroxide involves reacting hydrogen peroxide with a pair of oxidative coupling chromogenic pigments in the presence of peroxidase to produce a dye, and then measuring the absorbance of visible light based on the color produced by this dye. Examples of pair of oxidative coupling chromogenic pigments include a coupler and anilines, and a coupler and phenols. Examples of couplers include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinonehydrazine. 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, N-E Examples include 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, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS). Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

[0109] [5-2] Method for determining the amount of reduction of electron mediator Another embodiment of a method for measuring 3-hydroxybutyrate in a sample includes an enzymatic treatment step of contacting a sample containing D-3-hydroxybutyrate with 3-hydroxybutyrate oxidase in the absence of a coenzyme and in the presence of an electron mediator, and a quantitative step of determining the amount of reduction of the electron mediator.

[0110] The sample and 3-hydroxybutyrate oxidase used in the enzyme treatment process are the same as those described in "[5-1] Method for determining the amount of hydrogen peroxide generated" above.

[0111] Contact between the sample and 3-hydroxybutyrate oxidase in the enzyme treatment process is carried out in the presence of oxygen, in the absence of coenzymes, and in the presence of an electron mediator. This contact can be carried out, for example, by preparing a mixture of the sample, the enzyme preparation described in "[3] Enzyme Preparation" above, and an electron mediator, but without coenzymes, in the presence of oxygen. Alternatively, this contact can be carried out by dropping, placing, flowing, or absorbing a sample containing an electron mediator and without coenzymes onto an insoluble support on which 3-hydroxybutyrate oxidase is immobilized in the ketone body measurement sensor described in "[4] Ketone Body Measurement Sensor" above, in the presence of oxygen, or by immersing the insoluble support in a sample containing an electron mediator and without coenzymes, in the presence of oxygen.

[0112] Examples of coenzymes include NAD and FAD.

[0113] Examples of electron mediators include 2,6-dichloroindophenol (DCIP), phenazine methosulfate (PMS), and hexacyanoferrate(III) ions, with DCIP being more preferred.

[0114] The sample and 3-hydroxybutyrate oxidase are brought into contact in the presence of oxygen, in the absence of coenzymes, and in the presence of electron mediators, and the enzymatic reaction is carried out under predetermined conditions. The conditions for the enzymatic reaction, such as temperature and pH conditions that produce 3-hydroxybutyrate oxidase activity, are appropriately selected by those skilled in the art. For example, the reaction temperature can be specifically 20 to 40°C, preferably 25 to 40°C, more preferably 30 to 40°C, and even more preferably 35 to 39°C. The reaction time is not particularly limited, but can be 5 seconds to 3 hours, preferably 10 seconds to 30 minutes, and more preferably 1 to 10 minutes.

[0115] In the quantitative analysis step, the amount of reduction of the electron mediator is quantified. During the reaction between D-3-hydroxybutyrate and 3-hydroxybutyrate oxidase, the electron mediator is reduced by electron transfer from 3-hydroxybutyrate oxidase to the electron mediator. The amount of reduction of the electron mediator correlates with the amount of D-3-hydroxybutyrate consumed. Therefore, by quantifying the amount of reduction of the electron mediator, the amount of D-3-hydroxybutyrate contained in the sample can be quantified.

[0116] As described in the above section "[4] Sensor for Measuring Ketone Bodies" as Embodiment 3, D-3-hydroxybutyrate can be measured based on an optical signal derived from the reduction of an electron mediator. An electron mediator that absorbs a predetermined wavelength loses its absorption characteristics at that wavelength when it is reduced by receiving electrons from 3-hydroxybutyrate oxidase. Therefore, the amount of reduction of the electron mediator can be measured as the decrease in absorbance at the predetermined wavelength. When 2,6-dichloroindophenol (DCIP), which has an absorption maximum at 600 nm, is used as the electron mediator, the amount of reduction of DCIP can be measured as the decrease in absorbance at 600 nm.

[0117] [6] Ketone body measurement kit The ketone body measurement kit of the present invention includes the enzyme preparation for ketone body measurement described in "[3] Enzyme preparation" above or the ketone body measurement sensor described in "[4] Ketone body measurement sensor" above.

[0118] The ketone body measurement kit of the present invention may further include, in addition to the ketone body measurement enzyme preparation or ketone body measurement sensor, other suitable items used for measurement and / or sample collection.

[0119] Examples of such other items include electron mediators, a combination of oxidative coupling colorimetric chromogens, deproteinizing solutions, buffers (e.g., acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate-buffered saline, etc.), and lancet devices. Regarding electron mediators, see the section above, "[5-2] Method for Determining the Reduction Amount of Electron Mediators." The ketone body measurement kit of the present invention may contain one of these other items alone, or a combination of several items.

[0120] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0121] Test Example 1: A screening was conducted using the applicant's strain library (containing more than 10,000 strains) as an indicator, based on oxidase activity (3-hydroxybutyrate oxidative activity) in the presence of D-3-hydroxybutyrate (3-HB). This oxidase activity was confirmed using a hydrogen peroxide detection system that utilizes the color change produced by the oxidative condensation of 4-aminoantipyrine (4-AA) and TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline) in the presence of peroxidase.

[0122] (1) Experimental Method The activity measurement reagents 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 solution shown in Table 1.

[0123] The intracellular fractions stored in the applicant's strain library were used as screening and evaluation samples.

[0124] To 120 μL of the activity measurement reagent, 10 μL of the substrate solution or blank (specifically, the same buffer used to prepare the substrate solution) and 20 μL of the enzyme sample (intracellular fraction) were added and mixed, and 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 (-)), then 3-hydroxybutyrate oxidation activity can be evaluated as being present.

[0125] (2) Results Figure 3 shows the results for intracellular fractions of Achromobacter piechaudii strain 7576 (NBRC102461) and Achromobacter xylosoxidans strain 7577 (NBRC15126) out of more than 10,000 strains. As shown in Figure 3, changes in absorbance in the intracellular fractions of the above two strains in the presence of a substrate, i.e., 3-hydroxybutyrate oxidase activity, were confirmed.

[0126] Furthermore, the intracellular fractions of the two strains described above were heat-treated at 80°C for 20 minutes. The absorbance changes in the intracellular fractions after heat treatment, as shown in (1) above, were examined, confirming the inactivation of 3-hydroxybutyrate oxidase activity due to the heat treatment. In other words, the inactivation of 3-hydroxybutyrate oxidase activity in the intracellular fractions of the two strains described above was confirmed.

[0127] Based on the above, 3-hydroxybutyrate oxidase derived from Achromobacter piechaudii (NBRC102461 strain) and Achromobacter xylosoxidans (NBRC15126 strain) was confirmed.

[0128] Test Example 2: The components shown in Table 2 were dissolved in water to the indicated concentrations, and the culture medium was prepared by autoclaving at 121°C for 20 minutes.

[0129] Three mL of culture medium was placed in a test tube, and the Achromobacter strains shown in Table 3 were inoculated into the medium. Each strain was then cultured with shaking for 48 hours under the culture temperature conditions listed in Table 3. After culturing, the medium was centrifuged to collect the wet cells. 10 mM phosphate buffer was added to the wet cells, and they were then disrupted using a multi-bead shocker. The lysate was centrifuged, and the supernatant was collected to obtain the intracellular fraction.

[0130] 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 calculated by subtracting the absorbance at 550 nm measured in the absence of the substrate 3-hydroxybutyrate (substrate--) from the absorbance measured in the presence of the substrate 3-hydroxybutyrate (substrate-+). Intracellular fractions showing a value of 0.004 or higher were judged to possess 3-hydroxybutyrate oxidase activity. The results are shown in Table 3.

[0131]

[0132] As shown in Table 3 above, 3-hydroxybutyrate oxidase activity was confirmed in the intracellular fraction of all Achromocacter microorganisms, including those showing large absorbance differences, such as Achromocacter piechaudii strain 7576 (NBRC 102461), Achromocacter xylosoxidans strain 7577 (NBRC 15126), Achromocacter aloeverae (NBRC 111463T), and Achromocacter denitrificans (NBRC 15125T).

[0133] In Test Example 3, the pH stability and optimal pH were evaluated using intracellular fractions (the intracellular fractions from Test Example 2) of Achromobacter piechaudii strain 7576 (NBRC102461) and Achromobacter xylosoxidans strain 7577 (NBRC15126).

[0134] (1) Optimal pH Using 40 mM phosphate buffer (pH 6-8) or 40 mM Tris-HCl buffer (pH 8.5) as buffers shown in Table 1, the components shown in Table 1 were mixed to the indicated concentrations to prepare activity assay reagents and substrate solutions adjusted to pH 6, 6.5, 8, or 8.5. Using these activity assay reagents and substrate solutions, the activity of 3-hydroxybutyrate oxidase was evaluated in the same manner as in Test Example 1. When the activity at the pH condition showing maximum activity was set to 100%, the relative amount of activity at each pH condition was calculated as relative activity (%). The results are shown in the table below.

[0135]

[0136] As is clear from Table 4, both strains showed the highest 3-hydroxybutyrate oxidase activity at pH 8.5.

[0137] (2) pH-stable intracellular fractions were dissolved in 40 mM Britton-Robinson buffers (pH 5-9) with different pH levels and incubated (stored) at 4°C for 16 hours. After storage, Tris-HCl buffer (pH 8.5) was added to adjust the pH to 8.5. Using Tris-HCl buffer (pH 8.5) as the buffer shown in Table 1, the components shown in Table 1 were mixed to the indicated concentrations to prepare the activity measurement reagent and substrate solution adjusted to pH 8.5. Using these activity measurement reagents and substrate solutions, the 3-hydroxybutyrate oxidase activity (residual activity) in the intracellular fractions after storage was evaluated in the same manner as in Test Example 1. The relative value (%) of residual activity at each pH was derived, with the highest residual activity value set to 100%.

[0138] 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 Achromobacter microorganisms 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) showed high stability at test pH 8 and 9, and furthermore, it showed more than 60% stability at pH 7, 8, and 9, and more than 55% stability at all test pH levels.

[0139] Test Example 4: In this test example, 3-hydroxybutyrate oxidase was heterologously expressed using DNA (SEQ ID NO: 2) extracted from the Achromobacter piechaudii strain.

[0140] DNA was extracted from Achromobacter piechaudii strain 7576 (NBRC102461) and used as a template for PCR amplification. The PCR-amplified DNA was in fusion-linked to a pET24a(+) vector and transformed with E. coli DH5α. Plasmids were constructed and the inserted sequences were confirmed by sequencing.

[0141] After transformation using E. coli BL21 (DE3), the transformed strains were inoculated into LB medium and pre-cultured at 37°C for 16 hours. 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.

[0142] The obtained culture medium was centrifuged at 15,000 rpm for 10 minutes and glass beads were crushed with 100 mM Tris-HCl (pH 9.0). The supernatant was microfiltration to obtain 3-hydroxybutyrate oxidase (SEQ ID NO: 1). The activity of 3-hydroxybutyrate oxidase was confirmed in the same manner as in Test Example 1 (incubation conditions: 25°C, 3 hours).

[0143] As a result, the absorbance difference obtained by subtracting the absorbance at 550 nm measured in the absence of 3-hydroxybutyrate (substrate--) from the absorbance measured in the presence of the substrate 3-hydroxybutyrate (substrate-+) was 0.077. Furthermore, when the 3-hydroxybutyrate oxidase activity was similarly confirmed, except that the obtained 3-hydroxybutyrate oxidase was treated at 100°C for 10 minutes, it was confirmed that the above absorbance difference disappeared.

[0144] Test Example 5: In this test example, electron transfer of 3-hydroxybutyrate oxidase to the electron mediator (2,6-dichloroindophenol; DCIP) was confirmed.

[0145] Each enzyme shown in Table 5 was prepared. For enzyme A (3-hydroxybutyrate oxidase), we used 3-hydroxybutyrate oxidase derived from the sequence of Achromobacter piechaudii strain 7576, obtained by the method of Test Example 4. For enzymes a and b (3-hydroxybutyrate dehydrogenase), commercially available products were used.

[0146]

[0147] Reaction solutions were prepared to have the compositions shown in Table 6. Specifically, for each of enzymes A, a, and b, a reaction solution containing β-NAD and a reaction solution without β-NAD were prepared. The reaction solutions were incubated at 37°C for 5 minutes, and the absorbance at 600 nm was measured over time using a spectrophotometer to calculate the decrease in absorbance per minute (ΔAbs600nm / min). The decrease in absorbance (ΔAbs600nm / min) corresponds to the reduction amount of the electron mediator DCIP.

[0148]

[0149] Figure 5(a) shows the decrease in absorbance when the reaction solution contains β-NAD, and Figure 5(b) shows the decrease in absorbance when the reaction solution does not contain β-NAD. As shown in these figures, enzyme a is completely dependent on β-NAD for the reduction of DCIP, and enzyme a is substantially dependent on β-NAD for the reduction of DCIP, whereas enzyme A of the present invention, which is a 3-hydroxybutyrate oxidase, was able to reduce DCIP even under conditions without β-NAD.

[0150] From the above, it was found that 3-hydroxybutyrate oxidase has the ability to reduce electron mediators independently of coenzymes. Therefore, it was recognized that 3-hydroxybutyrate oxidase, which has such an effect, can be applied to a method of measuring ketone bodies by measuring the amount of reduction of electron mediators without using coenzymes.

[0151] Generally, oxidases catalyze the oxidation of a substrate and the reduction of oxygen to produce hydrogen peroxide in the presence of oxygen (see, for example, Figure 2), and react with electron mediators under deoxygenated conditions (see, for example, Figure 1). Therefore, in the presence of oxygen, the reaction of oxidases with electron mediators usually hardly occurs. On the other hand, dehydrogenases that require coenzymes (FAD, NAD, etc.) will react with electron mediators in the presence of oxygen, but the mechanism of this reaction requires the intervention of coenzymes (Figure 6). Accordingly, the action of reducing electron mediators in the presence of oxygen without coenzymes, as observed with the 3-hydroxybutyrate oxidase of the present invention, was an unexpected result, both from the common technical knowledge regarding oxidases and from the common technical knowledge regarding the reaction mechanism of enzyme-mediated reduction of electron mediators in the presence of oxygen.

[0152] Test Example 6 The amino acid sequences of SEQ ID NOs. 3-7 and the nucleotide sequences of SEQ ID NOs. 8-12 were obtained or designed using a unique method. Using DNA consisting of the nucleotide sequences of SEQ ID NOs. 8-12, 3-hydroxybutyrate oxidases (enzymes B-F) having the amino acid sequences of SEQ ID NOs. 3-7 were heterologously expressed according to the method of Test Example 4. The 3-hydroxybutyrate oxidase activity of the obtained enzymes B-F and enzyme A obtained in Test Example 4 was confirmed in the same manner as in Test Example 1 (however, the incubation conditions were changed to 37°C and 30 minutes). The absorbance at 550 nm measured in the presence of the substrate 3-hydroxybutyrate (substrate (+)) was subtracted from the absorbance at 550 nm measured in the absence of the substrate 3-hydroxybutyrate (substrate (-)). The intracellular fraction liquid showing a value of 0.004 or higher was judged to have 3-hydroxybutyrate oxidase activity. The results are shown in Table 7.

[0153]

[0154] As shown in Table 7, in addition to enzyme A, 3-hydroxybutyrate oxidase activity was also confirmed for enzymes B to F.

Claims

1. 3-hydroxybutyrate oxidase comprising a polypeptide as shown in any of (I) to (III) below: (I) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 1, or SEQ ID NO: 3; (II) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, added, inserted, or deleted in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 1, or SEQ ID NO: 3, and having the activity to catalyze a reaction in which D-3-hydroxybutyrate acts to produce hydrogen peroxide; (III) A polypeptide comprising an amino acid sequence with 70% or more sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 1, or SEQ ID NO: 3, and having the activity to catalyze a reaction in which D-3-hydroxybutyrate acts to produce hydrogen peroxide.

2. 3-hydroxybutyrate oxidase derived from microorganisms selected from the group consisting of Pseudomonas, Trypanosoma, Nocardia, and Achromobacter, having the following physicochemical properties: (1) Action: Catalytically reacts with D-3-hydroxybutyrate to produce hydrogen peroxide.

3. The 3-hydroxybutyrate oxidase according to claim 1, further having at least one of the following physicochemical properties: (2) Optimal pH: 8.3 to 8.7; (3) pH stability: Stable in the range of pH 8.5 to 9; (4) Action: Reduces electron mediators without the use of coenzymes in the presence of oxygen.

4. Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fragi; Trypanosoma cruzi; Nocardia and Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter The 3-hydroxybutyrate oxidase according to claim 1, derived from a microorganism selected from the group consisting of denitrificans and Achromobacter aloeverae.

5. An enzyme preparation for 3-hydroxybutyrate oxidation, comprising the 3-hydroxybutyrate oxidase described in claim 1 or 2 as an active ingredient.

6. The enzyme preparation according to claim 5, comprising an intracellular fraction of a microorganism selected from the group consisting of microorganisms of the genus Pseudomonas, Trypanosoma, Nocardia, and Achromobacter.

7. Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fragi; Trypanosoma cruzi; Nocardia and Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter The enzyme preparation according to claim 5, comprising an intracellular fraction of a microorganism selected from the group consisting of denitrificans and Achromobacter aloeverae.

8. The enzyme preparation according to claim 5, used for reducing an electron mediator without the use of a coenzyme in the presence of oxygen.

9. The enzyme preparation according to claim 8, wherein the mediator is 2,6-dichloroindophenol.

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

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

12. A kit for measuring ketone bodies, comprising the enzyme preparation described in claim 10.

13. A method for measuring 3-hydroxybutyrate in a sample, comprising an enzymatic treatment step of contacting a sample containing D-3-hydroxybutyrate with 3-hydroxybutyrate oxidase to generate hydrogen peroxide, and a quantitative step of quantifying the hydrogen peroxide.

14. The method according to claim 13, wherein the 3-hydroxybutyrate oxidase is the 3-hydroxybutyrate oxidase described in claim 1 or 2.

15. A method for measuring 3-hydroxybutyrate in a sample, comprising: an enzymatic treatment step of contacting a sample containing D-3-hydroxybutyrate with the 3-hydroxybutyrate oxidase described in claim 1 or 2 in the presence of oxygen, in the absence of a coenzyme, and in the presence of an electron mediator; and a quantitative step of quantifying the amount of reduction of the electron mediator.

16. A method for producing 3-hydroxybutyrate oxidase, comprising: a culture step of culturing a microorganism selected from the group consisting of microorganisms of the genus Pseudomonas, Trypanosoma, Nocardia, and Achromobacter; and a recovery step of recovering a fraction from the culture obtained in the culture step that has the activity to catalyze a reaction in which D-3-hydroxybutyrate acts to produce hydrogen peroxide.

17. In the culturing step, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fragi; Trypanosoma cruzi; Nocardia and Achromobacter ruhlandii, Achromobacter xylosoxidans, Achromobacter cholinophagum, Achromobacter piechaudii, Achromobacter spanius, Achromobacter insolitus, Achromobacter The method for producing the product according to claim 16, comprising culturing a microorganism selected from the group consisting of denitrificans and Achromobacter aloeverae.

18. A method for producing 3-hydroxybutyrate oxidase, comprising the step of culturing a transformant using an expression cassette or recombinant vector containing DNA encoding 3-hydroxybutyrate oxidase as described in claim 1.

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