Dried formulations of 3-hydroxybutyrate dehydrogenase

Abstract

Provided is a 3-hydroxybutyrate dehydrogenase desiccant having a composition that maintains the function of a target protein, the powder shape, the solubility of the dried preparation, and the clarity of a protein solution. By optimizing the selection of a stabilizer for the target protein and / or the addition amount thereof, it is possible to produce a dried 3-hydroxybutyrate dehydrogenase preparation having excellent powder shape, solubility of the dried preparation and clarity of a protein solution while maintaining the function of the target protein in the powder step.

Description

Technical Field

[0001] The present invention relates to a dry preparation of 3-hydroxybutyrate dehydrogenase. Background Art

[0002] Ketone bodies in the blood are used as metabolic indicators that reflect the degree of insufficiency of insulin action in diabetic patients and are therefore important markers in the field of clinical examinations. Ketone bodies in the blood are mainly produced as metabolites in the oxidation process of fatty acids in the liver and are also an indicator of whether carbohydrates are properly used as energy sources. Ketone bodies are a general term for acetoacetic acid, 3-hydroxybutyric acid and acetone. Most of the ketone bodies in the blood are occupied by acetoacetic acid and 3-hydroxybutyric acid. As an enzyme used in the quantification of ketone bodies, 3-hydroxybutyrate dehydrogenase (hereinafter also recorded as "HBDH") is an industrially useful enzyme.

[0003] HBDH (E.C1.1.1.30) is an enzyme that reversibly catalyzes the reaction of oxidizing 3-hydroxybutyrate to produce acetoacetate and reduced NAD in the presence of nicotinamide adenine dinucleotide (NAD). Microbial-derived enzymes are also known to exist in Rhodospirillum rubrum (Non-Patent Document 1) and / or Pseudomonas lemoignei (Non-Patent Document 2), Rhizobium meliloti (Non-Patent Document 3), Alcaligenes faecalis (Patent Document 1), and Rhodobacter phaeroides (Patent Document 2).

[0004] Ketone bodies in blood are primarily measured using liquid reagents and / or ketone sensors. Enzymes used in reagents and / or sensors are often distributed as dried products (hereinafter referred to as "dried preparations"). There are various methods for drying enzymes. For example, there are spray drying, which involves spraying an enzyme-containing solution and drying it with hot air; and freeze drying, which involves freezing an enzyme-containing solution, reducing pressure, and drying it.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 8-7085

[0008] Patent Document 2: Japanese Patent Application Laid-Open No. 11-318438

[0009] Non-patent literature

[0010] Non-patent literature 1: J. Biol. Chem. 1962, 237: 603-607.

[0011] Non-patent document 2: J. Biol. Chem. 1965, 240: 4023-4028.

[0012] Non-patent literature 3: J. Bacteriol. 1999, 181(3): 849-857. Summary of the Invention

[0013] Problems to be solved by the invention

[0014] In any method, drying the enzyme can sometimes cause problems such as loss of activity due to modification and / or formation of turbidity upon redissolution. In such cases, drying the enzyme protein alone is avoided, and stabilizers are often added. Conventional techniques involve coexisting HBDH with bovine serum albumin and freeze-drying it. However, from the perspective of bovine transmissible spongiform encephalopathy safety, a composition that does not contain bovine-derived raw materials, even among animal-derived raw materials, is ideal.

[0015] The present inventors have studied the composition of dry preparations, particularly for applications in ketone sensors. In ketone sensors, the hygroscopicity of the dry preparation consisting of HBDH and a stabilizer leads to deterioration in sensor performance. Specifically, it is believed that amino acids such as serine and / or glutamine have hydrophilic groups such as amino and / or carboxyl groups, which adsorb water and are therefore hygroscopic. Furthermore, salts such as calcium chloride are known to be deliquescent.

[0016] On the other hand, in ketone sensors, the enzyme immobilized on the surface of the sensor chip is dissolved by a very small amount of blood liquid, thereby initiating an enzymatic reaction. Therefore, moderate hydrophilicity is required to dissolve the enzyme even with a small amount of water in the sample.

[0017] One object of the present invention is to provide a dry preparation of 3-hydroxybutyrate dehydrogenase suitable for ketone sensors and the like.

[0018] Solutions for solving problems

[0019] The present invention includes the following aspects.

[0020] Item 1.

[0021] A dry preparation of 3-hydroxybutyrate dehydrogenase comprises 3-hydroxybutyrate dehydrogenase, a hydrophilic polymer and sugars (one or more sugars).

[0022] Item 2.

[0023] The dry preparation of 3-hydroxybutyrate dehydrogenase according to item 1, wherein the saccharide is a disaccharide and / or a sugar alcohol.

[0024] Item 3.

[0025] The dry preparation of 3-hydroxybutyrate dehydrogenase according to item 1 or 2, wherein the concentration of the hydrophilic polymer is 1 to 30% of the concentration of the total protein, or the content of the hydrophilic polymer is 1 to 30% relative to the protein.

[0026] Item 4.

[0027] The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of items 1 to 3, wherein the content of the saccharide (if two or more types, each) is 1 to 60% of the total protein content, or the content of the saccharide is 1 to 60% relative to the protein.

[0028] Item 5.

[0029] The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of items 1 to 4, wherein the concentration of the hydrophilic polymer is 1 to 30% of the concentration of the total protein, and the concentration of the saccharide (if two or more types are used, each of them) is 1 to 60% of the concentration of the total protein, or

[0030] The content of the hydrophilic polymer is 1 to 30% relative to the protein, and the content of the carbohydrate is 1 to 60% relative to the protein.

[0031] Item 6.

[0032] The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of items 1 to 5, wherein the concentration of the hydrophilic polymer is 1 to 20% of the total protein concentration, and the concentration of the saccharide (if two or more types are used, each of them) is 1 to 40% of the total protein concentration, or

[0033] The content of the hydrophilic polymer is 1 to 20% relative to the protein, and the content of the carbohydrate is 1 to 40% relative to the protein.

[0034] Item 7.

[0035] The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of items 1 to 6, wherein the content of the hydrophilic polymer is 1 to 10% of the content of the total protein, and the content of the saccharide (in the case of two or more saccharides, each saccharide) is 1 to 20% of the content of the total protein, or

[0036] The content of the hydrophilic polymer is 1 to 10% relative to the protein, and the content of the sugar is 1 to 20% relative to the protein.

[0037] Item 8.

[0038] The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of Items 1 to 7, wherein the hydrophilic polymer is a nonionic polymer.

[0039] Item 9.

[0040] The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of items 1 to 8, wherein the hydrophilic polymer is polyvinyl pyrrolidone.

[0041] Item 10.

[0042] The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of items 1 to 9, wherein the saccharide contains disaccharides and sugar alcohols.

[0043] Item 11.

[0044] The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of items 2 to 10, wherein the disaccharide is sucrose and the sugar alcohol is mannitol.

[0045] Effects of the Invention

[0046] According to the present invention, a dry preparation of 3-hydroxybutyrate dehydrogenase suitable for ketone sensors and the like can be provided. For example, by optimizing the type and amount of stabilizer added to the target protein, a dry preparation of 3-hydroxybutyrate dehydrogenase can be produced that has excellent powder shape, solubility of the dry preparation, and clarity of the protein solution while maintaining the function of the target protein during the powdering process. DETAILED DESCRIPTION

[0047] The present invention is described in detail below. HBDH used in the present invention is an enzyme that acts on 3-hydroxybutyrate in the presence of nicotinamide adenine dinucleotide (NAD), reversibly catalyzing the oxidation of 3-hydroxybutyrate to produce acetoacetate and reduced NAD. The source of HBDH used in the present invention is not particularly limited and can be produced from genetically modified organisms.

[0048] In the present invention, a dried preparation refers to a preparation obtained by drying a composition containing HBDH using a drying method commonly used by those skilled in the art, such as freeze-drying and / or air-drying. While the drying method is not particularly limited, freeze-drying is particularly preferred from the perspective of minimizing the loss of enzyme activity.

[0049] The dry preparation of 3-hydroxybutyrate dehydrogenase of the present invention is characterized by containing a hydrophilic polymer and saccharides (one or more saccharides) together.

[0050] A hydrophilic polymer refers to a polymer that exhibits water solubility, for example, by containing polar or charged functional groups, and is capable of interacting with water and / or other polar substances and dissolving therein. Specifically, nonionic polymers such as polyvinyl pyrrolidone, polyethylene glycol, polyethyleneimine, and polyvinyl alcohol can be cited. Polyvinyl pyrrolidone is particularly preferred. The average molecular weight of the hydrophilic polymer is preferably 10,000 to 40,000, more preferably 15,000 to 35,000, and even more preferably 20,000 to 30,000. The average molecular weight can be measured by conventional methods, such as HPLC.

[0051] Examples of sugars include monosaccharides, disaccharides, polysaccharides, and sugar alcohols. Disaccharides and / or sugar alcohols are preferred. Examples of disaccharides include sucrose, lactulose, maltose, and melibiose. On the other hand, examples of sugar alcohols include mannitol, erythritol, lactitol, maltitol, sorbitol, and xylitol. Disaccharides and sugar alcohols may be used alone or in combination. The combination of sucrose and mannitol is particularly preferred.

[0052] The purpose of adding these stabilizers is to suppress the loss of HBDH enzymatic activity during the freeze-drying process and to improve the powder shape, solubility, and clarity of the resulting dry preparation. Therefore, the amount added can be appropriately adjusted within a range that achieves these objectives. Thus, while not particularly limited, the concentration of the hydrophilic polymer is, for example, 1-35% of the concentration of HBDH or, when containing proteins other than HBDH, the total protein concentration (at an absorbance of 1 at 280 nm of 1 mg / mL), preferably 1-30%, more preferably 1-25%, even more preferably 1-20%, even more preferably 1-15%, and particularly preferably 1-10%. The concentration of the saccharide (if two or more types are present, each) is, for example, 1-65% of the concentration of HBDH or, when containing proteins other than HBDH, the total protein concentration (at an absorbance of 1 at 280 nm of 1 mg / mL), preferably 1-60%, more preferably 1-50%, even more preferably 1-40%, even more preferably 1-30%, and particularly preferably 1-20%. Throughout this specification, "concentration" may be referred to as "content" and / or "solid content." In one embodiment, when polyvinyl pyrrolidone, sucrose, and mannitol are used, the concentration of polyvinyl pyrrolidone relative to the solid content of HBDH, or relative to the solid content of total protein when containing proteins other than HBDH, is 1-30%, sucrose relative to the solid content of total protein. More preferably, the concentrations are 1-20%, 1-40%, and 1-40% respectively. Even more preferably, the concentrations are 1-10%, 1-20%, and 1-20% respectively. The concentration of HBDH in the dry preparation relative to the total protein concentration is 50% or higher, preferably 70%, 80%, 90%, 95%, or 99% or higher. In one embodiment, the dry preparation may contain only HBDH.

[0053] In the dry preparation of 3-hydroxybutyrate dehydrogenase of the present invention, in addition to the above, any component can be contained as needed, and its composition is not particularly limited. As an arbitrary component, for example, a buffer can be mentioned. As a buffer, a buffer having a buffering capacity in the range of pH 4 to 9 can be appropriately added, for example, a buffer such as boric acid, Tris hydrochloric acid, potassium phosphate and / or a Good's buffer such as ACES, BES, Bis-Tris, CHES, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TES, Tricine can be mentioned. In addition, a buffer based on dicarboxylic acids such as phthalic acid, maleic acid, and glutaric acid can also be listed. Only one of these buffers can be used, or two or more can be used. Furthermore, it can also be a composite composition containing one or more substances other than the above.

[0054] In addition, as needed, chelating agents such as EDTA and / or surfactants such as polyoxyethylene (10) octylphenyl ether (Triton X-100) and polyoxyethylene sorbitan monolaurate (Tween 20) may also be contained. In addition, as for their added concentration, there is no particular limitation as long as it is within the range of buffering capacity, and the preferred upper limit is 20 mM or less, more preferably 10 mM or less. The preferred lower limit is 1 mM or more. In the dry preparation of 3-hydroxybutyrate dehydrogenase of the present invention, the content of the buffer is not particularly limited, and is preferably used in the range of 0.1% (mass ratio) or more, particularly preferably 0.5 to 2% (mass ratio).

[0055] The concentration of the enzyme solution used in the drying step is adjusted so that the protein concentration, A280 (A280 = 1, 1 mg / mL), is preferably 25 ± 8, more preferably 25 ± 4, and even more preferably 25 ± 2. When the enzyme concentration used in the drying step is within this range, the recovery rate is generally not reduced during the drying step, and the resulting dried product is in a shape that is easy to handle. Furthermore, drying does not take time.

[0056] One preferred embodiment of the present invention is a dry preparation of 3-hydroxybutyrate dehydrogenase, characterized by containing polyvinyl pyrrolidone, sucrose, and mannitol. The dry preparation of 3-hydroxybutyrate dehydrogenase can be produced by the above-described method, for example, in the form of a dry powder and / or a freeze-dried preparation.

[0057] According to the present invention, the hygroscopicity of a dry preparation of 3-hydroxybutyrate dehydrogenase can be reduced. Low hygroscopicity, as used in the present invention, means that after the dry preparation is stored at 70% humidity and 25°C for 6 hours, the powder does not become clay-like or adhere to the spatula when mixed with a spatula or the like.

[0058] According to the present invention, the solubility of a dry preparation of 3-hydroxybutyrate dehydrogenase can be improved. High solubility in the present invention means that when the dry preparation is dissolved in, for example, PBS buffer to an enzyme concentration of approximately 1 kU / mL, the protein dissolves rapidly, without becoming insoluble due to aggregation or other factors.

[0059] According to the present invention, the clarity of a dry preparation of 3-hydroxybutyrate dehydrogenase can be improved. High clarity, as used herein, means that when the dry preparation is dissolved in, for example, PBS buffer to an enzyme concentration of approximately 1 KU / mL, no suspended matter and / or turbidity is produced in the solution.

[0060] According to the present invention, the stability of a dried 3-hydroxybutyrate dehydrogenase formulation can be improved. High stability, as used herein, means that after the dried formulation is stored at 37°C for one week, the residual HBDH activity (%) is increased or at least maintained compared to when no stabilizer is added.

[0061] Specifically, whether the stability is improved can be evaluated as follows.

[0062] In the activity assay described in the HBDH activity assay method described below, the HBDH activity value (a) per unit mass of the dried product after drying and the HBDH activity value (b) per unit mass of the dried product after storage at a constant temperature for a certain period of time are measured. The relative value of the measured value (b) relative to the measured value (a) set to 100 is calculated ((b) / (a) × 100). This calculated relative value is defined as the residual activity rate. The addition of the compound is then compared with the absence of the addition of the compound. If the residual activity rate increases with the addition of the compound, it is determined that the stability has improved.

[0063] Determination of HBDH activity

[0064] Reagents

[0065] 100 mM Tris-HCl buffer (pH 8.5; 25°C)

[0066] 158mM 3-hydroxybutyric acid solution

[0067] 27.9mM NAD + solution

[0068] The above Tris-HCl buffer 2.3mL, 3-hydroxybutyric acid solution 0.5mL, NAD + 0.2 mL of the solution was mixed to prepare a reaction reagent.

[0069] Measurement principle

[0070] D-3-Hydroxybutyrate + NAD+ →Acetoacetate + NADH + H +

[0071] The increase in NADH generated by the above reaction was evaluated by measuring absorbance at 340 nm.

[0072] Definition of enzyme activity

[0073] Under the following reaction conditions, the amount of enzyme that catalyzes the production of 1 μmol of NADH per minute is defined as 1 U.

[0074] (U = μmol / min).

[0075] Determination method

[0076] Specifically, HBDH activity can be measured as follows.

[0077] The mixture contained 0.1M Tris-HCl (pH 8.5), 25mM sodium DL-3-hydroxybutyrate, 1.8mM NAD + 3 mL of the reaction solution was taken into a quartz cuvette with an optical path length of 1 cm and preheated at 37°C for 5 minutes. 100 μL of sample was added thereto and mixed slowly, and then the absorbance at 340 nm was recorded for 2-3 minutes using a spectrophotometer controlled at 37°C. The absorbance change (ΔOD) per minute was calculated from the part showing a linear increase in absorbance, and the HBDH activity was calculated based on formula (1). The sample was appropriately diluted with enzyme dilution buffer (0.1 M Tris-HCl (pH 8.5) containing 0.1% BSA) in such a way that the HBDH activity was 0.15 to 0.5 U / mL. Blind testing can be carried out using enzyme dilution buffer instead of the sample.

[0078] HBDH (U / mL) = (ΔOD test -ΔOD blank )×3.1×dilution ratio / (6.22×1.0×0.1)…(1)

[0079] Here, 3.1 is the volume of the reaction solution (mL), and 6.22 is the millimolar molecular absorption coefficient of NADH at 340 nm (cm 2 / μmol), 1.0 is the optical path length of the absorbance cell (cm), and 0.1 is the volume of the enzyme solution (mL).

[0080] Example

[0081] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not particularly limited to the following Examples.

[0082] Cultivation of HBDH-producing Escherichia coli

[0083] The plasmid pET-24b(+)-HBDH containing the HBDH gene was added to the chemically competent cells of the Escherichia coli JM109 (DE3) strain and transformed by the heat shock method. After recovery culture in SOC medium, it was inoculated into LB agar medium containing 0.5% glucose and 100 mg / L of kanamycin sulfate, and cultured overnight at 37°C to obtain colonies of transformants. Scrape several obtained colonies and inoculate them into 50 mL of LB medium containing 0.5% glucose and 100 mg / L of kanamycin sulfate, and shaken at 30°C for 16 hours for seed culture. Then, 70 mL of seed culture solution was added to 7 L of culture medium (the composition is shown in Table 1) in a 10 L capacity fermenter, and the main culture was carried out at 30°C. When OD660 reached 7 (about 8 hours after the start of culture), IPTG was added to a final concentration of 0.4 mM and cultured for another 16 hours. Samples were taken at the end of the culture and the HBDH activity of the bacterial cell fragment was analyzed.

[0084] [Table 1]

[0085] Element Concentration (g / L) Yeast peptone 14 yeast extract 18 glycerin 8 sorbitol 4 glucose 5 Potassium dihydrogen phosphate 12.5 Dipotassium hydrogen phosphate 2.3 ADEKANOL LG-126 0.2 Kanamycin sulfate 0.1

[0086] Purification of HBDH

[0087] The resulting bacterial cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.5), and disrupted in a French press (Niro Soavi) at a flow rate of 160 mL / min at 700-800 bar. After nucleic acid removal, the supernatant was centrifuged. Ammonium sulfate (Sumitomo Chemical Co., Ltd.) was slowly added to the supernatant to a saturation of 0.6. After stirring at room temperature for 30 minutes, the target protein was precipitated. The precipitate collected by centrifugation was redissolved in 50 mM phosphate buffer (pH 7.5). Then, gel filtration using a Sephadex G-25 column, anion exchange chromatography using a DEAE-agarose gel column (elution conditions were all applied with a sodium chloride concentration gradient of 0 to 1 M and peak components were extracted), and hydrophobic chromatography using a phenyl-agarose gel column (elution conditions were all applied with an ammonium sulfate concentration gradient of 25% saturation to 0% and peak components were extracted), and the ammonium sulfate was further removed by gel filtration using a G-25 agarose gel column to obtain an HBDH solution.

[0088] Preparation of dry preparations

[0089] Solutions containing various additives were prepared in the HBDH solution obtained in this manner. The concentration of the HBDH solution was adjusted to an A280 (absorbance at 280 nm) of 25. The concentrations of various stabilizers were calculated relative to the enzyme concentration (assuming an A280 of 1 is 1 mg / mL). Polyvinylpyrrolidone (MW 24,500), sucrose, and mannitol were added at concentrations of 30%, 60%, and 60% of the enzyme concentration (by mass). Specifically, since the enzyme concentration had an A280 of 25, the various stabilizers were added to achieve final concentrations of 7.5 mg / mL, 15 mg / mL, and 15 mg / mL, respectively. After addition, the solution was filtered through a filter (pore size: 0.2 μm) and accurately dispensed into sample vials in 2 mL portions. A control solution without any stabilizers was also prepared. This solution was freeze-dried in a vacuum to completely evaporate the water, and then pulverized with a spatula to form a powder. A plurality of dry preparations with different stabilizer concentrations were prepared by the same method as above (each level is shown in Table 2) and used in the following tests.

[0090] [Table 2]

[0091]

[0092] FDR yield

[0093] The total HBDH activity before freeze-drying was measured (the activity value at this time is recorded as (a). Next, the total HBDH activity after freeze-drying was measured (the activity value at this time is recorded as (b), and is shown as the powder titer in Table 3). The residual activity rate was calculated as the relative value of the measured value (b) relative to the measured value (a) set to 100% ((b) / (a)×100), and this relative value was used as the residual activity rate (FDR yield).

[0094] Powder shape test

[0095] About 10 mg of the crushed powder preparation was accurately weighed and placed in a paper wrapper, and the evaluation was performed based on the following criteria.

[0096] ++: No solid matter larger than 1 mm exists

[0097] +: There are 1 or more but less than 10 solid objects larger than 1 mm in size.

[0098] -: There are 10 or more solid objects larger than 1 mm in size.

[0099] Solubility test

[0100] About 10 mg of the powdered preparation was accurately weighed and placed in a beaker, and PBS buffer was added to give a concentration of about 0.25 KU / mL. The time required for complete dissolution was measured and determined based on the following criteria.

[0101] ++: Complete dissolution time is less than 5 seconds

[0102] +: The time for complete dissolution is more than 5 seconds and less than 10 seconds

[0103] -: The time for complete dissolution exceeds 10 seconds

[0104] Clarification test

[0105] About 10 mg of the powdered preparation was accurately weighed and placed in a beaker, and PBS buffer was added to give a concentration of about 0.25 KU / mL. After slow stirring at room temperature for about 1 hour, OD660 was measured with a spectrophotometer and determined based on the following criteria.

[0106] ++: OD660 is less than 10

[0107] +: OD660 is more than 10 and less than 70

[0108] -: OD660 more than 70

[0109] Hygroscopicity test

[0110] About 10 mg of the powder preparation was accurately weighed and placed in a centrifuge tube. The tube was stored at a humidity of 70% and 25°C for 7 hours, and then mixed with a spatula. The sample was evaluated based on the following criteria.

[0111] ++: Same shape as before moisture absorption

[0112] +: Different from before absorbing moisture, but not becoming clay-like and not adsorbed to the scraper

[0113] -: Becomes clay-like or sticks to the scraper

[0114] Stability test

[0115] The HBDH activity per unit mass of the dried product (a) after drying and the HBDH activity per unit mass of the dried product after storage at 37°C for one week (b) were measured. The relative value of the measured value (b) relative to the measured value (a) set to 100 was calculated ((b) / (a) × 100). This calculated relative value was used as the residual activity rate. The addition of the compound was then compared with the absence of the compound. If the residual activity rate increased due to the addition, it was determined that the stability had improved.

[0116] Table 3 shows the results of HBDH activity (powder titer) and FDR yield of the freeze-dried powder preparations. Powder titers exceeded 300 U / mg at all levels, a value well within the practical range. Regarding FDR yield, the addition of polyvinyl pyrrolidone, sucrose, and mannitol improved the residual activity rate at all levels compared to the absence of any additives.

[0117] [Table 3]

[0118] level Powder potency (U / mg) FDR yield (%) 1 696 85 2 349 93 3 419 95 4 527 99

[0119] The results of the powder shape test are shown in Table 4. Regarding the powder shape, no solid components larger than 1 mm were observed in the case where nothing was added and in level 4. Although more solid matter was present in the other levels compared to the case where nothing was added, it was determined that these levels were not problematic for practical use.

[0120] [Table 4]

[0121] level Powder shape 1 ++ 2 + 3 + 4 ++

[0122] The results of the solubility test are shown in Table 5. Regarding solubility, Levels 3 and 4 dissolved in a short time, indicating improved solubility. Level 2 dissolved in the same time as when nothing was added.

[0123] [Table 5]

[0124] level Solubility 1 - 2 - 3 + 4 ++

[0125] The results of the clarification test are shown in Table 6. Regarding clarification, Level 4 had the lowest OD660 value, and no suspended matter and / or aggregates were generated. Levels 2 and 3 had lower OD660 values ​​than the case where nothing was added.

[0126] [Table 6]

[0127] level OD660 Clarification 1 85 - 2 63 + 3 18 ++ 4 5 ++

[0128] The results of the hygroscopicity test are shown in Table 7. Regarding hygroscopicity, Level 4 showed the same shape as before hygroscopicity. When nothing was added, the product adhered to the spatula and became clay-like. Levels 2 and 3 showed no clay-like appearance and no adhesion to the spatula, unlike before hygroscopicity.

[0129] [Table 7]

[0130] level Hygroscopicity 1 - 2 + 3 + 4 ++

[0131] The results of the stability test are shown in Table 8. Compared to the case where nothing was added, the addition of polyvinyl pyrrolidone, sucrose, and mannitol improved the stability at all levels.

[0132] [Table 8]

[0133] level stability(%) 1 65 2 95 3 93 4 98

[0134] Industrial applicability

[0135] The dry 3-hydroxybutyrate dehydrogenase preparation of the present invention is particularly useful as a reagent and / or sensor for measuring ketone bodies, and is therefore expected to be widely used in industries in the life science field, including clinical testing and diagnostic medicine.

Claims

1. A dry preparation of 3-hydroxybutyrate dehydrogenase, comprising 3-hydroxybutyrate dehydrogenase, a hydrophilic polymer and a carbohydrate.

2. The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein The sugars are disaccharides and / or sugar alcohols.

3. The dry preparation of 3-hydroxybutyrate dehydrogenase according to claim 1, wherein The concentration of the hydrophilic polymer is 1 to 30% of the total protein concentration.

4. The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein The content of the sugars is 1 to 60% of the content of the total protein. When there are two or more types of sugars, the content of the sugars is the content of each of them.

5. The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein The concentration of the hydrophilic polymer is 1 to 30% of the total protein concentration, and the concentration of the sugar is 1 to 60% of the total protein concentration. When there are two or more sugars, the concentrations of the sugars are the concentrations of each of them.

6. The dry preparation of 3-hydroxybutyrate dehydrogenase according to claim 1, wherein The concentration of the hydrophilic polymer is 1 to 20% of the total protein concentration, and the concentration of the sugar is 1 to 40% of the total protein concentration. When there are two or more sugars, the concentrations of the sugars are the concentrations of each of them.

7. The dry preparation of 3-hydroxybutyrate dehydrogenase according to claim 1, wherein The concentration of the hydrophilic polymer is 1 to 10% of the total protein concentration, and the concentration of the sugar is 1 to 20% of the total protein concentration. When there are two or more sugars, the concentrations of the sugars are the concentrations of each of them.

8. The dry preparation of 3-hydroxybutyrate dehydrogenase according to claim 1, wherein The hydrophilic polymer is a nonionic polymer.

9. The dry preparation of 3-hydroxybutyrate dehydrogenase according to claim 1, wherein The hydrophilic polymer is polyvinylpyrrolidone.

10. The dry preparation of 3-hydroxybutyrate dehydrogenase according to claim 1, wherein Sugars include disaccharides and sugar alcohols.

11. The dry preparation of 3-hydroxybutyrate dehydrogenase according to claim 2 or 10, wherein The disaccharide is sucrose and the sugar alcohol is mannitol.

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