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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/0103—3-Hydroxybutyrate dehydrogenase (1.1.1.30)

Definitions

• 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.
• spray drying method in which a solution containing the enzyme is sprayed and dried by applying hot air
• freeze-drying (lyophilization) method in which a solution containing the enzyme is frozen and then dried under reduced pressure.
• HBDH a technique has been known in the past whereby it is freeze-dried in the presence of bovine serum albumin.
• 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.
• a ketone sensor the hygroscopicity of the dry preparation, which is composed of HBDH and a stabilizer, leads to deterioration of the sensor performance.
• 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.
• salts such as calcium chloride are deliquescent.
• 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. 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.
• Item 4. Item 4.
• 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 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.
• 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 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.
• a 3-hydroxybutyrate dehydrogenase dry preparation suitable for ketone sensors, etc.
• 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 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.
• NAD nicotinamide adenine dinucleotide
• 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.
• 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.
• 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 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 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%.
• concentration can be read as “content", "solid content”, etc.
• the polyvinylpyrrolidone 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.
• 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.
• 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.
• 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.
• 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).
• 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).
• 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.
• 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.
• 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.
• 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.
• the stability has been improved can be evaluated as follows.
• 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.
• 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 ⁇ L 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 ( ⁇ OD) was determined from the part showing a linear increase in absorbance, and HBDH activity was calculated based on formula (1).
• ⁇ OD absorbance change per minute
• 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.
• enzyme dilution buffer 0.1 M Tris-HCl (pH 8.5) containing 0.1% BSA.
• a blind test can be performed by replacing the sample with the enzyme dilution buffer.
• 3.1 is the volume of the reaction solution (mL)
• 6.22 is the millimolar extinction coefficient of NADH at 340 nm (cm 2 / ⁇ mol)
• 1.0 is the optical path length of the absorbance cell (cm)
• 0.1 is the volume of the enzyme solution (mL).
• E. coli JM109 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.
• 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.
• 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.
• 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).
• 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
• 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.
• 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.
• 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 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.

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• 2024
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