WO2024180926A1

WO2024180926A1 - 3-hydroxybutyrate dehydrogenase dry preparation

Info

Publication number: WO2024180926A1
Application number: PCT/JP2024/000683
Authority: WO
Inventors: 裕川南
Original assignee: 東洋紡株式会社
Priority date: 2023-02-28
Filing date: 2024-01-12
Publication date: 2024-09-06

Abstract

Provided is a formulation of 3-hydroxybutyrate dehydrogenase dry preparation, the formulation maintaining the function of a protein of interest and being excellent in a powder shape, solubility of the dry preparation, and clarity of a protein solution. The present invention selects a stabilizer for a protein of interest and/or optimizes the added amount thereof, thereby enabling manufacture of a 3-hydroxybutyrate dehydrogenase dry preparation that is excellent in a powder shape, solubility of the dry preparation, and clarity of a protein solution, while maintaining the function of the protein of interest in a powdering step.

Description

3-Hydroxybutyrate dehydrogenase dry preparation

The present invention relates to a dry preparation of 3-hydroxybutyrate dehydrogenase.

Ketone bodies in blood are used as a metabolic indicator that reflects the degree of insulin deficiency in diabetic patients, and are therefore an important marker in the field of clinical testing. Blood ketone bodies are mainly produced as metabolites in the oxidation process of fatty acids in the liver, but they also serve as an indicator of whether carbohydrates are being used appropriately as an energy source. Ketone bodies are a general term for acetoacetic acid, 3-hydroxybutyric acid, and acetone. Most ketone bodies in blood are made up of acetoacetic acid and 3-hydroxybutyric acid. 3-hydroxybutyrate dehydrogenase (hereinafter also referred to as "HBDH") is an industrially useful enzyme used in the quantification of ketone bodies.

HBDH (E.C1.1.1.30) is known as 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). It is also known to exist in microbial enzymes such as Rhodospirillum rubrum (Non-Patent Document 1), Pseudomonas remiogneii (Non-Patent Document 2), Rhizobium meliotialca (Non-Patent Document 3), Ligenes faecalis (Patent Document 1), and Rhodobacter sphaeroides (Patent Document 2).

Liquid reagents and ketone sensors are mainly used to measure blood ketone bodies. Enzymes used in reagents and sensors are often distributed as dried products (hereafter also referred to as "dried preparations"). There are various methods for drying enzymes. For example, there is the spray drying method, in which a solution containing the enzyme is sprayed and dried by applying hot air, and the freeze-drying (lyophilization) method, in which a solution containing the enzyme is frozen and then dried under reduced pressure.

Japanese Patent Application Publication No. 8-7085 Japanese Patent Application Publication No. 11-318438

　In any method, when the enzyme is dried, problems such as loss of activity due to denaturation and generation of turbidity when redissolved can occur. In such cases, a stabilizer is often added to avoid drying the enzyme protein alone. For HBDH, a technique has been known in the past whereby it is freeze-dried in the presence of bovine serum albumin. However, from the perspective of safety regarding bovine transmissible spongiform encephalopathy, a composition that does not contain bovine-derived raw materials, even among animal-derived raw materials, is preferable.

The inventors have investigated the composition of the dry preparation, particularly from the perspective of application to a ketone sensor. In a ketone sensor, the hygroscopicity of the dry preparation, which is composed of HBDH and a stabilizer, leads to deterioration of the sensor performance. Specifically, amino acids such as serine and glutamine have hydrophilic groups such as amino and carboxyl groups, and it is believed that these hydrophilic groups are hygroscopic because they adsorb water. In addition, it is known that salts such as calcium chloride are deliquescent.

On the other hand, in the case of a ketone sensor, the enzyme reaction begins when the enzyme immobilized on the surface of the sensor chip is dissolved by a very small amount of blood liquid, so the sensor chip must be moderately hydrophilic so that the enzyme can be dissolved even in the small amount of water in the sample.

One of the objectives of the present invention is to provide a dried preparation of 3-hydroxybutyrate dehydrogenase suitable for use in ketone sensors, etc.

The present invention includes the following aspects:

Item 1.
A dry preparation of 3-hydroxybutyrate dehydrogenase comprising 3-hydroxybutyrate dehydrogenase, a hydrophilic polymer, and a sugar (one or more types of sugars).
Item 2.
Item 2. A dry preparation of 3-hydroxybutyrate dehydrogenase according to Item 1, wherein the saccharide is a disaccharide and/or a sugar alcohol.
Item 3.
Item 3. A dry preparation of 3-hydroxybutyrate dehydrogenase according to Item 1 or 2, wherein the content of the hydrophilic polymer is 1 to 30% of the total protein content, or the content of the hydrophilic polymer is 1 to 30% of the protein content.
Item 4.
Item 4. The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of Items 1 to 3, wherein the concentration of the sugar (or each of the sugars when there are two or more kinds) is 1 to 60% of the total protein concentration, or the content of the sugar is 1 to 60% of the protein.
Item 5.
Item 5. A 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 total protein concentration, and the concentration of the saccharide (or each of the saccharides when there are two or more kinds) is 1 to 60% of the total protein concentration, or the content of the hydrophilic polymer is 1 to 30% of the protein, and the content of the saccharide is 1 to 60% of the protein.
Item 6.
Item 6. A 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 (or each of the saccharides when there are two or more kinds) is 1 to 40% of the total protein concentration, or the content of the hydrophilic polymer is 1 to 20% of the protein, and the content of the saccharide is 1 to 40% of the protein.
Item 7.
Item 7. A dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of Items 1 to 6, wherein the concentration of the hydrophilic polymer is 1 to 10% of the total protein concentration, and the concentration of the saccharide (or saccharides, if there are two or more kinds) is 1 to 20% of the total protein concentration, or the content of the hydrophilic polymer is 1 to 10% of the protein, and the content of the saccharide is 1 to 20% of the protein.
Item 8.
Item 8. The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of Items 1 to 7, wherein the hydrophilic polymer is a nonionic polymer.
Item 9.
Item 9. The dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of Items 1 to 8, wherein the hydrophilic polymer is polyvinylpyrrolidone.
Item 10.
Item 10. A dry preparation of 3-hydroxybutyrate dehydrogenase according to any one of Items 1 to 9, wherein the saccharide comprises a disaccharide and a sugar alcohol.
Item 11.
Item 11. A 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.

The present invention can provide a 3-hydroxybutyrate dehydrogenase dry preparation suitable for ketone sensors, etc. For example, by optimizing the type and amount of stabilizer added for the target protein, it is possible to produce a 3-hydroxybutyrate dehydrogenase dry preparation that has excellent powder shape, solubility of the dry preparation, and clarity of the protein solution while maintaining the function of the target protein in the powder process.

The present invention will be described in detail below. The HBDH used in the present invention is an enzyme that acts on 3-hydroxybutyric acid in the presence of nicotinamide adenine dinucleotide (NAD) and reversibly catalyzes the reaction of oxidizing 3-hydroxybutyric acid to produce acetoacetic acid and reduced NAD. The origin of the HBDH used in the present invention is not particularly limited and may be any, and may be produced by genetic recombination.

In the present invention, a dry preparation refers to a preparation obtained by a process of drying the composition containing the above-mentioned HBDH using a drying method that is commonly used by those skilled in the art, such as freeze-drying or air-drying. The drying method is not particularly limited, but freeze-drying is particularly preferred from the viewpoint of minimizing loss of enzyme activity.

The 3-hydroxybutyrate dehydrogenase dry preparation of the present invention is characterized by the coexistence of a hydrophilic polymer and a sugar (one or more types of sugar).

Hydrophilic polymers are polymers that are water-soluble, for example by containing polar or charged functional groups, and that are capable of interacting with or dissolving in water or other polar substances. Specific examples include nonionic polymers such as polyvinylpyrrolidone, polyethylene glycol, polyethyleneimine, and polyvinyl alcohol. Polyvinylpyrrolidone 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, for example, HPLC.

Examples of sugars include monosaccharides, disaccharides, polysaccharides, and sugar alcohols. Of these, disaccharides and/or sugar alcohols are preferred. Examples of disaccharides include sucrose, lactose, maltose, and melibiose. On the other hand, examples of sugar alcohols include mannitol, erythritol, lactitol, maltitol, sorbitol, and xylitol. Either one of the disaccharides or the sugar alcohol may be used, or both may be used in combination. In particular, it is preferred to use sucrose and mannitol in combination.

The purpose of adding these stabilizers is to suppress the loss of enzyme activity of HBDH during the freeze-drying process and to improve the powder shape and solubility of the resulting dried preparation and the clarity of the enzyme solution, so the amount added can be set appropriately within a range that can achieve this purpose. Therefore, although not particularly limited, the content concentration of the hydrophilic polymer is, for example, 1 to 35% of the content concentration of HBDH or, in the case of containing proteins other than HBDH, the content concentration of the total protein (concentration when the absorbance at 280 nm of 1 is 1 mg/mL), preferably 1 to 30%, more preferably 1 to 25%, even more preferably 1 to 20%, even more preferably 1 to 15%, and particularly preferably 1 to 10%. The concentration of the saccharide (or each of the saccharides when there are two or more kinds) is, for example, 1 to 65% of the concentration of HBDH or, when a protein other than HBDH is contained, the concentration of the total protein (concentration when absorbance at 280 nm of 1 is taken as 1 mg/mL), preferably 1 to 60%, more preferably 1 to 50%, even more preferably 1 to 40%, still more preferably 1 to 30%, and particularly preferably 1 to 20%. In this specification, "concentration" can be read as "content", "solid content", etc. In one embodiment, when polyvinylpyrrolidone, sucrose, and mannitol are used, the polyvinylpyrrolidone is 1-30%, the sucrose is 1-60%, and the mannitol is 1-60% relative to the solid content of HBDH, or the total protein solid content when a protein other than HBDH is contained, more preferably 1-20% polyvinylpyrrolidone, 1-40% sucrose, and 1-40% mannitol, and even more preferably 1-10% polyvinylpyrrolidone, 1-20% sucrose, and 1-20% mannitol. The concentration of HBDH in the dry preparation is 50% or more relative to the total protein concentration, and is preferably 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more. In one embodiment, the protein contained in the dry preparation may be only HBDH.

The 3-hydroxybutyrate dehydrogenase dry preparation of the present invention may contain any other component as necessary in addition to the above, and the composition is not particularly limited. An example of the optional component is a buffering agent. As the buffering agent, a buffering agent having a buffering capacity in the range of pH 4 to 9 may be appropriately added, and examples thereof include boric acid, Tris-HCl, potassium phosphate, and other buffering agents, and Good's buffers such as ACES, BES, Bis-Tris, CHES, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TES, and Tricine. Also included are buffering agents based on dicarboxylic acids, such as phthalic acid, maleic acid, and glutaric acid. Only one of these buffering agents may be used, or two or more may be used. Furthermore, the composition may be a composite composition of one or more buffering agents that includes other than the above.

If necessary, the preparation may contain a chelating agent such as EDTA and/or a surfactant such as polyoxyethylene (10) octylphenyl ether (TritonX-100) or polyoxyethylene sorbitan monolaurate (Tween20). The concentration of these additives is not particularly limited as long as they have a buffering capacity, but the preferred upper limit is 20 mM or less, and more preferably 10 mM or less. The preferred lower limit is 1 mM or more. The content of the buffering agent in the 3-hydroxybutyrate dehydrogenase dry preparation of the present invention is not particularly limited, but is preferably 0.1% (mass ratio) or more, and particularly preferably in the range of 0.5 to 2% (mass ratio).

The enzyme solution to be subjected to the drying step is preferably adjusted so that its protein concentration A280 (A280=1 is 1 mg/mL) is 25±8, more preferably 25±4, and even more preferably 25±2. When the concentration of the enzyme to be subjected to the drying step is within the above range, the recovery rate does not decrease during the drying step, and the obtained dried product often has a shape that is easy to handle. Also, drying does not take much time.

One preferred embodiment of the present invention is a 3-hydroxybutyrate dehydrogenase dry preparation that contains polyvinylpyrrolidone as well as sucrose and mannitol. The 3-hydroxybutyrate dehydrogenase dry preparation can be produced by the method described above, and can be in the form of, for example, a dry powder or a freeze-dried preparation.

The present invention can reduce the hygroscopicity of a dried preparation of 3-hydroxybutyrate dehydrogenase. In this invention, low hygroscopicity means that when the dried preparation is stored at 25°C and 70% humidity for 6 hours and then the powder is mixed with a spatula or the like, the powder does not turn into a clay-like substance or adhere to the spatula.

The present invention can improve the solubility of a dried preparation of 3-hydroxybutyrate dehydrogenase. High solubility in the present invention means that when the dried preparation is dissolved in, for example, a PBS buffer to an enzyme concentration of about 1 KU/mL, the protein dissolves quickly, rather than remaining insoluble due to aggregation or the like.

The present invention can improve the clarity of a dried 3-hydroxybutyrate dehydrogenase preparation. In the present invention, high clarity means that when the dried preparation is dissolved in, for example, a PBS buffer to an enzyme concentration of about 1 KU/mL, no suspended matter or turbidity is generated in the solution.

The present invention can improve the stability of a dried preparation of 3-hydroxybutyrate dehydrogenase. In the present invention, high stability means that after the dried preparation is stored at 37°C for one week, the percentage of HBDH activity that is maintained is increased or at least maintained compared to the case in which no stabilizer is added.

Specifically, whether or not the stability has been improved can be evaluated as follows.
In the activity measurement method described below in the section on measuring HBDH activity, the HBDH activity value (a) per dry mass after drying and the HBDH activity value (b) per dry mass after storage at a constant temperature for a constant period are measured, and the relative value (b) ((b)/(a)×100) of the measured value (b) with the measured value (a) taken as 100 is calculated. This calculated relative value is the residual activity rate. Then, by comparing with and without the addition of the compound, if the residual activity rate increases with the addition of the compound, it is determined that stability has been improved.

Method for measuring HBDH activity
Reagent: 100 mM Tris-HCl buffer (pH 8.5; 25°C)
158 mM 3-hydroxybutyric acid solution 27.9 mM NAD ⁺ solution 2.3 mL of the above Tris-HCl buffer, 0.5 mL of 3-hydroxybutyric acid solution, and 0.2 mL of NAD ⁺ solution were mixed to prepare a reaction reagent.

Measurement principle D-3-hydroxybutyric acid + NAD ⁺ → acetoacetic acid + NADH + H ⁺
The increase in NADH caused by the above reaction is evaluated by measuring absorbance at 340 nm.

Definition of Enzyme Activity The amount of enzyme that catalyzes the production of 1 μmol of NADH per minute under the reaction conditions described below is defined as 1 U (U=μmol/min).

Measurement Method Specifically, HBDH activity can be measured as follows.
3 mL of reaction solution consisting of 0.1 M Tris-HCl (pH 8.5), 25 mM sodium DL-3-hydroxybutyrate, and 1.8 mM NAD ⁺ was placed in 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 to this and gently mixed, after which the absorbance at 340 nm was recorded for 2-3 minutes using a spectrophotometer controlled at 37°C. The absorbance change per minute (ΔOD) was determined from the part showing a linear increase in absorbance, and 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) so that the HBDH activity was 0.15 to 0.5 U/mL. A blind test can be performed by replacing the sample with the enzyme dilution buffer.

Here, 3.1 is the volume of the reaction solution (mL), 6.22 is the millimolar extinction 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).

The present invention will be explained in more detail below with reference to examples, but the present invention is not particularly limited to the following examples.

Cultivation of HBDH-producing E. coli Chemically competent cells of E. coli JM109 (DE3) strain were transformed by heat shock method after adding plasmid pET-24b(+)-HBDH containing HBDH gene. After recovery culture in SOC medium, the cells were inoculated on LB agar medium containing 0.5% glucose and 100 mg/L kanamycin sulfate, and cultured overnight at 37°C to obtain colonies of transformants. Several colonies were scraped off and inoculated into 50 mL of LB medium containing 0.5% glucose and 100 mg/L kanamycin sulfate, and seed culture was performed by shaking at 30°C for 16 hours. Next, 70 mL of seed culture liquid was added to 7 L of medium (composition shown in Table 1) placed in a 10 L jar fermenter, and main culture was performed at 30°C. When OD660 reached 7 (approximately 8 hours after the start of culture), IPTG was added to a final concentration of 0.4 mM, and culture was continued for an additional 16 hours. At the end of culture, a sample was taken, and the HBDH activity of the cell lysate was analyzed.

The thus obtained bacterial cells were collected by centrifugation, suspended in 50 mM phosphate buffer solution (pH 7.5), fed to a French press (manufactured by Niro Soavi) at a flow rate of 160 mL/min, disrupted at 700-800 bar , treated to remove nucleic acids, and centrifuged to obtain a supernatant. Ammonium sulfate (manufactured by Sumitomo Chemical Co., Ltd.) was gradually added to the supernatant to a saturation of 0.6, and the mixture was stirred at room temperature for 30 minutes to precipitate the target protein. The precipitate collected by centrifugation was redissolved in 50 mM phosphate buffer solution (pH 7.5). Then, gel filtration using a Sephadex G-25 column, anion exchange chromatography using a DEAE-Sepharose column (elution conditions for both were a 0 to 1 M sodium chloride gradient to extract peak fractions), and hydrophobic chromatography using a Phenyl-Sepharose column (elution conditions for both were a 25% saturation to 0% ammonium sulfate gradient to extract peak fractions) were performed, and ammonium sulfate was removed by gel filtration using a G-25 Sepharose column to obtain an HBDH solution.

Preparation of dry preparation A solution containing various additives was prepared in the HBDH solution obtained in this way. The concentration of the HBDH solution was adjusted to A280 (absorbance at 280 nm) = 25. The concentration of the various stabilizers added was calculated as a ratio to the enzyme concentration (A280 = 1 is 1 mg / mL). Polyvinylpyrrolidone, sucrose, and mannitol with a molecular weight of 24,500 were added at 30% (mass ratio), 60% and 60% of the enzyme concentration. Specifically, since the enzyme concentration A280 = 25, various stabilizers were added to polyvinylpyrrolidone, sucrose, and mannitol so that the final concentrations were 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 each of these was accurately divided into 2 mL portions and dispensed into vials. In addition, a control was prepared without adding any stabilizer. This was freeze-dried in vacuum to completely evaporate the water, and then pulverized with a spatula into powder. Several dried preparations with different stabilizer concentrations were prepared in the same manner as above (each level is shown in Table 2), and used in the following tests.

FDR Yield: The total HBDH activity before freeze-drying was measured (the activity value at this time was designated as (a)). Subsequently, the total HBDH activity after freeze-drying was measured (the activity value at this time was designated as (b) and shown as the powder titer in Table 3). The residual activity rate was calculated by taking the measured value (a) as 100% and calculating the relative value ((b)/(a)×100) of the measured value (b), and this relative value was designated as the residual activity rate (FDR yield).

Powder shape test: Approximately 10 mg of the pulverized powder preparation was accurately weighed and placed in a medicine packaging paper, and evaluated according to the following criteria.
++: No solids of 1 mm or more are present +: 1 to 10 solids of 1 mm or more are present -: 10 or more solids of 1 mm or more are present

Solubility test: Approximately 10 mg of the powder formulation was accurately weighed and placed in a beaker, and PBS buffer was added to give a concentration of approximately 0.25 KU/mL. The time until complete dissolution was then measured and evaluated according to the following criteria.
++: Time required for complete dissolution is 5 seconds or less +: Time required for complete dissolution is more than 5 seconds but less than 10 seconds -: Time required for complete dissolution is more than 10 seconds

Clarity test: Approximately 10 mg of the powder formulation was accurately weighed and placed in a beaker, and PBS buffer was added to a concentration of approximately 0.25 KU/mL. After gentle stirring at room temperature for approximately 1 hour, OD660 was measured using a spectrophotometer and judged according to the following criteria.
++: OD660 is 10 or less +: OD660 is more than 10 and less than 70 -: OD660 is more than 70

Hygroscopicity test: About 10 mg of the powder formulation was accurately weighed and placed in a Spitz roll, and stored at 70% humidity, 25° C. for 7 hours. The mixture was then mixed with a spatula and evaluated according to the following criteria.
++: The shape remains the same as before moisture absorption +: The shape is not the same as before moisture absorption, but it does not become clay-like and does not adhere to the spatula -: The shape becomes clay-like or adheres to the spatula

Stability test: HBDH activity (a) per weight of the dried product after drying and HBDH activity (b) per weight of the dried product after storage at 37°C for one week were measured, and the relative value of the measured value (b) to the measured value (a) taken as 100 ((b)/(a) x 100) was calculated. This calculated relative value was taken as the residual activity rate. Then, a comparison was made between the presence and absence of the compound, and if the residual activity rate increased due to the addition of the compound, it was determined that stability had been improved.

The results of HBDH activity (powder titer) and FDR yield after freeze-drying of the powder formulation are shown in Table 3. The powder titer was over 300 U/mg at all levels, a value that is within the practical range and does not pose a problem. Regarding the FDR yield, the residual activity rate was improved at all levels by adding polyvinylpyrrolidone, sucrose, and mannitol, compared to when nothing was added.

The results of the powder shape test are shown in Table 4. With regard to powder shape, no solids of 1 mm or more were observed in the case of no addition and in level 4. Although there was more solid matter in the other levels compared to the case of no addition, it was determined that this was not a problem in practical use.

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 amount of time as the one without any addition.

The results of the clarity test are shown in Table 6. In terms of clarity, level 4 had the smallest OD660 value, and no suspended matter or aggregates were generated. Levels 2 and 3 had smaller OD660 values than those with no additions.

The results of the moisture absorption test are shown in Table 7. In terms of moisture absorption, level 4 had the same shape as before moisture absorption. The one with no additives was adsorbed to the spatula and changed to a clay-like shape. Levels 2 and 3 were not the same as before moisture absorption, but they did not become clay-like and did not adsorb to the spatula.

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

The dried 3-hydroxybutyrate dehydrogenase preparation of the present invention is particularly useful as a reagent or sensor for measuring ketone bodies, and is therefore expected to be widely applicable in the fields of clinical testing, diagnostic medicine, and the life science industry.

Claims

A dried preparation of 3-hydroxybutyrate dehydrogenase comprising 3-hydroxybutyrate dehydrogenase, a hydrophilic polymer, and a sugar.
The dried 3-hydroxybutyrate dehydrogenase preparation according to claim 1, wherein the sugar is a disaccharide and/or a sugar alcohol.
The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein the hydrophilic polymer content is 1 to 30% of the total protein content.
The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein the concentration of the sugar (or each of the sugars when there are two or more sugars) is 1 to 60% of the total protein concentration.
The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein the hydrophilic polymer concentration is 1-30% of the total protein concentration, and the sugar concentration (each of two or more sugars) is 1-60% of the total protein concentration.
The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein the hydrophilic polymer concentration is 1-20% of the total protein concentration, and the sugar concentration (each of two or more sugars) is 1-40% of the total protein concentration.
The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein the hydrophilic polymer concentration is 1-10% of the total protein concentration, and the sugar concentration (each of two or more sugars) is 1-20% of the total protein concentration.
The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein the hydrophilic polymer is a non-ionic polymer.
The 3-hydroxybutyrate dehydrogenase dry preparation according to claim 1, wherein the hydrophilic polymer is polyvinylpyrrolidone.
The dried preparation of 3-hydroxybutyrate dehydrogenase according to claim 1, wherein the sugars consist of 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.