JP2007129965A - Method for improving thermal stability of composition containing soluble coenzyme-bound type glucose dehydrogenase (gdh) - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for improving the thermal stability of a composition containing a soluble coenzyme-bound type glucose dehydrogenase (GDH). <P>SOLUTION: A dicarboxylic acid or a salt compound is added to the composition containing a soluble coenzyme-bound type glucose dehydrogenase (GDH). Thereby, the thermal stability can be improved, and the enhancement in the measurement accuracy of a glucose-measuring reagent can be expected. And, the composition is held at acidic side pH, whereby the thermal stability is obtained at such a high level that the composition can thermally be dried, and the preservation stability and measurement accuracy of the glucose-measuring reagent can be enhanced. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for improving the thermal stability of a composition comprising a soluble coenzyme-bound glucose dehydrogenase such as glucose dehydrogenase using a flavin compound as a coenzyme (herein, glucose dehydrogenase is also referred to as GDH) and the method. The present invention relates to a composition using

  Blood glucose self-measurement is important for diabetics to grasp their normal blood glucose level and use it for treatment. An enzyme using glucose as a substrate is used for a sensor used for blood glucose self-measurement. An example of such an enzyme is glucose oxidase (EC 1.1.3.4). Glucose oxidase has been used as an enzyme for blood glucose sensors for a long time because it has the advantage of high specificity for glucose and excellent thermal stability, and its first announcement dates back to about 40 years ago. . In a blood glucose sensor using glucose oxidase, measurement is performed by passing electrons generated in the process of oxidizing glucose and converting it to D-glucono-δ-lactone to the electrode through a mediator. There is a problem that dissolved oxygen affects the measured value because the protons generated in the above are easily passed to oxygen.

In order to avoid such problems, for example, NAD (P) -dependent glucose dehydrogenase (EC 1.1.1.47) or pyrroloquinoline quinone (herein, pyrroloquinoline quinone is also referred to as PQQ) -dependent glucose dehydrogenase. (EC 1.1.5.2 (formerly EC 1.1.9.17)) is used as an enzyme for blood glucose sensors. These are superior in that they are not affected by dissolved oxygen, but the former NAD (P) -dependent glucose dehydrogenase (NAD (P) -dependent glucose dehydrogenase is also referred to as NADGDH in this document) has poor stability. There is a complication that requires the addition of a coenzyme. On the other hand, the latter PQQ-dependent glucose dehydrogenase (in this document, PQQ-dependent glucose dehydrogenase is also referred to as PQQGDH) has poor substrate specificity and acts on saccharides other than glucose such as maltose and lactose, so that the accuracy of the measured value is improved. There is a drawback of losing.

Patent Document 1 discloses an Aspergillus-derived flavin-binding glucose dehydrogenase (herein, flavin-binding glucose dehydrogenase is also referred to as FADGDH). This enzyme is superior in that it has excellent substrate specificity and is not affected by dissolved oxygen. About thermal stability, it is said that the activity remaining rate is about 89% after treatment at 50 ° C. for 15 minutes, and the stability is also excellent. However, in view of the fact that heat treatment may be required in the sensor chip manufacturing process, it cannot be said that the stability is sufficient.
WO 2004/058958

  An object of the present invention is to provide a composition that can be used as a reagent for measuring blood glucose level, which is advantageous in practical use, by overcoming the drawbacks related to the thermal stability of known enzymes for blood glucose sensor as described above. .

The present inventors have obtained a large number of multiple mutants that have improved the substrate specificity of the enzyme in previous PQQGDH studies, but some of them have reduced thermal stability compared to wild-type PQQGDH. Mutants were seen. Therefore, in order to solve this problem, intensive research was conducted on the cause of the problem. By adding a certain compound to the enzyme presented in this patent, the three-dimensional structure of PQQGDH can be stably maintained and the stability can be improved. all right.
The present inventors have further studied and found that FADGDH and NADGDH have the same effect and completed the present invention.
To date, there is Patent Document 2 as a report on measures for improving the stability of PQQGDH, in which studies using PQQGDH modification means at the gene level have been reported, but stability without enzyme modification is reported. The possibility of increasing the value was not even mentioned.
WO02 / 072839

  In addition, as a result of intensive studies, the inventors of the present invention have maintained the acidic pH at the pH of the composition containing GDH containing a flavin compound, so that the composition can be heated and dried at a high level of heat stability. It was found that sex can be obtained.

  The heat-dryable level is a state where the remaining activity after treatment at 50 ° C. for 15 minutes is 10% or more, preferably 45% or more, and more preferably 70% or more. In this state, the remaining activity of can be obtained.

  Furthermore, the present inventors searched for a simpler measure for improving the thermal stability from a viewpoint different from the past measures, and as a result of further earnest research, as a result, the pH on the acidic side was reduced with the composition containing GDH. Furthermore, it has been clarified that the stability of the whole composition can be improved by coexisting one or more kinds of dicarboxylic acids and salt compounds, and the present invention has been finally completed.

That is, the present invention has the following configuration.
[Claim 1]
A composition comprising a soluble coenzyme-linked glucose dehydrogenase (GDH) comprising the enzyme and a sugar alcohol, a carboxyl group-containing compound, an alkali metal-containing compound, an alkaline earth metal compound, an ammonium salt, a sulfate, and a protein A method for improving the thermal stability of GDH, comprising the step of coexisting any one or more compounds selected from those in the absence of the compound.
[Section 2]
Item 2. The method for improving stability according to Item 1, wherein the coenzyme is pyrroloquinone quinoline, a flavin compound, or nicotinamide adenine dinucleotide (NAD).
[Section 3]
Item 3. The composition having improved thermal stability according to Item 1 or 2, wherein the final concentration of each compound to be present is 0.1% or more.
[Claim 4]
The compound to be added is mannitol, inositol, arabitol, adonitol, galactitol, valine, histidine, phenylalanine, leucine, inositol, calcium glycerate, succinic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, fumaric acid, malonic acid, pimelin It is any one or more selected from the group consisting of acid, 3-3′dimethylglutaric acid, lysine, phthalic acid, maleic acid, glutaric acid, ammonium sulfate, sodium sulfate, sodium chloride, and bovine serum albumin (BSA). Item 4. The method for improving thermal stability according to Item 1 to 3.
[Section 5]
Item 5. A composition comprising a soluble coenzyme-linked glucose dehydrogenase having improved thermal stability by the method according to any one of Items 1 to 4.
[Claim 6]
A method for measuring a glucose concentration using the composition according to Item 5.
[Claim 7]
A glucose sensor comprising the composition of Item 6.
[Section 8]
A composition comprising a soluble coenzyme-linked glucose dehydrogenase (GDH) comprising the enzyme and a sugar alcohol, a carboxyl group-containing compound, an alkali metal-containing compound, an alkaline earth metal compound, an ammonium salt, a sulfate, and a protein The manufacturing method of the composition which improved the thermal stability of GDH compared with the case where this compound is not coexisted including the process of coexisting any one or more compounds chosen from more.
[Claim 9]
In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), the step of maintaining the pH of the composition on the acidic side of pH 7 or lower, compared to the case where the composition is adjusted to pH 7.3 or higher A method for improving the thermal stability of the composition.
[Section 10]
In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), the step of maintaining the pH of the composition at pH 3.1 to 7.0, compared with the case where the composition is adjusted to pH 7.4 And improving the thermal stability of the composition.
[Section 11]
Item 11. The method for improving the thermal stability of the composition according to Item 9 or 10, wherein the soluble coenzyme-linked glucose dehydrogenase (GDH) is glucose dehydrogenase (GDH) having a flavin compound as a coenzyme.
[Claim 12]
Item 12. The method for improving thermal stability according to Item 11, wherein glucose dehydrogenase (GDH) using a flavin compound as a coenzyme is derived from a filamentous fungus.
[Claim 13]
Item 13. A composition having improved thermal stability by the method according to any one of Items 9 to 12.
[Section 14]
In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), 10% or more of GDH activity remains even when treated at 50 ° C. for 15 minutes compared to the composition stored at 4 ° C. Item 14. A GDH-containing composition according to Item 13.
[Section 15]
In a composition containing glucose dehydrogenase (GDH) containing a flavin compound as a coenzyme, 45% or more of GDH activity remains even when treated at 50 ° C. for 15 minutes compared to the composition stored at 4 ° C. Item 14. A GDH-containing composition according to Item 13.
[Section 16]
Item 14. The composition according to Item 13, comprising at least one dicarboxylic acid or salt compound.
[Section 17]
Item 14. The composition according to Item 13, comprising at least one compound selected from sodium chloride, sodium sulfate, trisodium citrate, ammonium sulfate, succinic acid, malonic acid, glutaric acid, phthalic acid, and maleic acid.
[Section 18]
Item 14. A method for measuring glucose concentration using the composition according to Item 13.
[Section 19]
Item 14. A glucose sensor comprising the composition according to item 13.
[Section 20]
In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), the step of maintaining the pH of the composition on the acidic side of pH 7 or lower, compared to the case where the composition is adjusted to pH 7.3 or higher A method for producing a composition having improved thermal stability.
[Claim 21]
In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), the step of maintaining the pH of the composition at pH 3.1 to 7.0, compared with the case where the composition is adjusted to pH 7.4 And a method for producing a composition having improved thermal stability.

  The improvement of the thermal stability according to the present invention reduces the heat inactivation of the enzyme during preparation of the glucose measurement reagent, glucose assay kit, and glucose sensor, thereby enabling the use amount of the enzyme to be reduced and the measurement accuracy to be improved. In addition, it is possible to provide a blood sugar level measuring reagent using GDH having excellent storage stability.

GDH is an enzyme that catalyzes the following reaction.
D-glucose + electron transfer material (oxidized type)
→ D-glucono-δ-lactone + electron transfer material (reduced form)
It is an enzyme that catalyzes a reaction in which D-glucose is oxidized to produce D-glucono-1,5-lactone, and the origin and structure are not particularly limited.

The GDH that can be applied to the method of the present invention is not particularly limited as long as it is a soluble coenzyme-linked glucose dehydrogenase (GDH).
As the coenzyme, for example, pyrroloquinone quinoline or flavin compound or nicotinamide adenine dinucleotide (NAD) can be used.

GDH (PQQGDH) taking pyrroloquinone quinoline as a coenzyme that can be applied to the method of the present invention is not particularly limited. For example, Acinetobacter calcoaceticus LMD79.41 From (A.M. Cleton-Jansen et al., J. Bacteriol., 170, 2121 (1988) and Mol. Gen. Genet., 217, 430 (1989)), from Escherichia coli (AM Cleton-Jansen et al., J. Bacteriol., 172, 6308 (1990)), derived from Gluconobacter oxydans. (Mol.Gen.Genet., 229,206 (1991)), and, such as those derived from microorganisms such as patents Acinetobacter baumannii that have been reported in the literature 1 (Acinetobacter baumanni) NCIMB11517 can be exemplified.
However, it is difficult to modify a membrane enzyme present in Escherichia coli to make it soluble, and it is preferable to select soluble PQQGDH such as Acinetobacter calcoaceticus or Acinetobacter baumannii as the origin. .
In addition, Acinetobacter baumannii (Acinetobacter baumannii) NCIMB11517 strain was formerly classified into Acinetobacter calcaceticus.

  As these PQQGDH, for example, commercially available products such as GLD-321 manufactured by Toyobo Co., Ltd. can be used. Alternatively, it can be easily manufactured by those skilled in the art using known techniques in the technical field.

  The GDH that takes FAD as a coenzyme that can be applied to the method of the present invention (FAD-binding GDH) is not particularly limited, but for example, Penicillium of a filamentous fungus belonging to the category of eukaryotes ( Examples include those derived from microorganisms such as the genus Penicillium and the genus Aspergillus. These microbial strains can be easily obtained by requesting distribution from each strain storage organization. For example, the Penicillium genus Penicillium Lirashino Echinulatam is registered in the Product Evaluation Technology Foundation / Biological Resources Division under the deposit number NBRC6231.

  GDH that takes NAD as a coenzyme that can be applied to the method of the present invention is not particularly limited, but a commercial product (GLD-311) sold by Toyobo is obtained and used. Can do. Alternatively, it can be prepared by various known methods.

As long as GDH that can be applied to the method of the present invention has glucose dehydrogenase activity, some of the other amino acid residues may be deleted or substituted with those exemplified above. An amino acid residue may be added.
Such modifications can be easily carried out by those skilled in the art using known techniques in the art. For example, various methods for substituting or inserting a base sequence of a gene encoding a protein in order to introduce a site-specific mutation into the protein are described in Sambrook et al., Molecular Cloning; A Laboratory Manual 2nd edition (1989). Cold Spring Harbor Laboratory Press, New York.

  For example, the above-mentioned natural microorganism producing GDH or the gene encoding natural GDH as it is or after being mutated, is a vector for expression (many things are known in the art. For example, plasmids ), And cultivates the transformant transformed into an appropriate host (many are known in the art. For example, E. coli), and recovers the cell from the culture by centrifugation. After that, the bacterial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, a chelating agent such as EDTA or a surfactant is solubilized, and a water-soluble fraction containing GDH. You can get minutes. Alternatively, the expressed GDH can be secreted directly into the culture medium by using an appropriate host vector system.

  The GDH-containing solution obtained as described above is precipitated by, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. You just have to let them know. Heat treatment and isoelectric point treatment are also effective purification means. Further, purified GDH can be obtained by performing gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

  Before and after the above step, in order to improve the ratio of holo-type GDH to the total GDH enzyme protein, a heat treatment of preferably 25 to 50 ° C., more preferably 30 to 45 ° C. may be performed.

The concentration of GDH in the present invention is not particularly limited. Although an appropriate range varies depending on the characteristics of the enzyme used, any concentration may be used as long as a person skilled in the art can determine that glucose can be measured with sufficient reliability in practice.
For example, the concentration of PQQGDH in the present invention is not particularly limited, but is preferably 0.1 to 100 U / mL, more preferably 1 to 50 U / mL, and still more preferably 2 to 10 U / mL in the solution. is there. A similar concentration is desirable in powders or lyophilized products, but for the purpose of preparing a powder preparation, the concentration can be 100 U / mL or more.
For example, the NADGDH concentration in the present invention is not particularly limited, but is preferably 10 to 1000 U / mL, more preferably 20 to 500 U / mL, and still more preferably 50 to 150 U / mL in the solution. A similar concentration is desirable in powders or lyophilized products, but for the purpose of preparing a powder preparation, the concentration can be 1000 U / mL or more.
For example, the concentration of FADGDH in the present invention is not particularly limited, but in the solution, it is preferably 0.01 to 100 U / mL, more preferably 0.1 to 50 U / mL, and still more preferably 0.2 to 10 U / mL. A similar concentration is desirable in powders or lyophilized products, but for the purpose of preparing a powder preparation, the concentration can be 100 U / mL or more.

  The medium for culturing the microorganism is not particularly limited as long as the microorganism grows and can produce the GDH shown in the present invention. More preferably, the carbon source, inorganic nitrogen source, and One containing an organic nitrogen source is preferable, and a liquid medium suitable for aeration stirring is more preferable. In the case of a liquid medium, for example, glucose, dextran, soluble starch, and sucrose are used as the carbon source, and examples of the nitrogen source are ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, defatted soybeans, potato An extract etc. are illustrated. Moreover, other nutrients (for example, inorganic salts such as calcium chloride, sodium dihydrogen phosphate, magnesium chloride, vitamins, etc.) may be contained as desired.

  The culture method follows a conventional method known in the art. For example, spore of microorganisms or growing cells are inoculated in a liquid medium containing the above nutrients, and the cells are allowed to grow by standing or stirring with aeration. Preferably, the cells are cultured by stirring with aeration. The pH of the culture solution is preferably 5-9, more preferably 6-8. The temperature is usually 14 to 42 ° C, more preferably 20 to 40 ° C. Usually, the culture is continued for 14 to 144 hours. Preferably, the culture is terminated when the expression level of GDH is maximized under each culture condition. As a measure to identify such a time point, the change is monitored by sampling the culture solution and measuring the GDH activity in the culture solution, and the time point when the increase in GDH activity with time is stopped is regarded as a peak. What is necessary is just to stop culture | cultivation.

  As a method for extracting GDH from the culture solution, when collecting GDH accumulated in the microbial cells, only the microbial cells are collected by an operation such as centrifugation or filtration, and the microbial cells are collected in a solvent, preferably water. Alternatively, resuspend in buffer. The resuspended cells can be crushed by a known method to extract GDH in the cells into a solvent. As a crushing method, a lytic enzyme can be used, or a physical crushing method may be used. The lytic enzyme is not particularly limited as long as it has the ability to digest fungal cell walls. Examples of applicable enzymes include “Lyticase” manufactured by Sigma. Examples of the physical crushing method include ultrasonic crushing, glass bead crushing, and French press crushing. The residue after the crushing treatment can be removed by centrifugation or filtration to obtain a GDH crude extraction solution.

  In addition, the culture method in the present invention may be solid culture. Preferably, the eukaryotic microorganism having the ability to produce the GDH of the present invention is grown on bran such as wheat while appropriately controlling temperature, humidity and the like. At this time, the culture may be performed by standing or may be mixed by stirring the culture. GDH extraction is performed by adding a solvent, preferably water or a buffer, to the culture to dissolve GDH, and removing solids such as bacterial cells and bran by centrifugation or filtration.

  Purification of GDH can be performed by appropriately combining various separation techniques that are usually used depending on the fraction in which GDH activity is present. From the GDH extract, for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denaturing PAGE, SDS-PAGE, ion exchange chromatography, hydroxyapatite chromatography, affinity chromatography, reverse phase high performance liquid chromatography A known separation method such as isoelectric focusing can be selected appropriately.

One form of the method for improving the thermal stability of GDH of the present invention is a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), in which (1) the enzyme, (2) sugar alcohol, carboxyl And a step of coexisting one or more compounds selected from the group consisting of a group-containing compound, an alkali metal-containing compound, an alkaline earth metal compound, an ammonium salt, a sulfate, and a protein.
Preferred compounds to be added include mannitol, inositol, arabitol, adonitol, galactitol, valine, histidine, phenylalanine, leucine, inositol, calcium glycerate, succinic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, fumaric acid, Any one or more selected from the group consisting of malonic acid, pimelic acid, 3-3′dimethylglutaric acid, lysine, phthalic acid, maleic acid, glutaric acid, ammonium sulfate, sodium sulfate, sodium chloride, bovine serum albumin (BSA) Can be mentioned.
The concentration of each compound to be coexistent is not particularly limited, but in the solution, it is preferably 0.001 to 30% by weight, more preferably 0.01 to 5%, and still more preferably 0.00. 01 to 1%. The same concentration is desirable in the powder or lyophilized product, but in the powder or lyophilized product, the effectiveness tends to be exhibited by addition of a compound at a lower concentration than in the solution.
In addition, the density | concentration of the compound described in the Example is a final density | concentration when preserve | saving together with a GDH enzyme. Preferred combinations include those in salt compounds with similar properties and combinations of carboxylic acid-containing compounds. For example, a combination of a salt compound and a carboxylic acid-containing compound can complement each other's effects. Those that do are preferred.
In order to further improve the thermal stability in the above form, the pH of the composition can be maintained on the acidic side of pH 7 or lower. Alternatively, dicarboxylic acids such as succinic acid, malonic acid, glutaric acid, phthalic acid and maleic acid, and salt compounds such as sodium chloride, sodium sulfate, trisodium citrate and ammonium sulfate can be contained.

Another embodiment of the method for improving the thermal stability of GDH of the present invention is a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), wherein the pH of the composition is maintained on the acidic side of pH 7 or lower. And a step of improving the thermal stability of the composition as compared with the case where the composition is adjusted to pH 7.3 or higher.
Alternatively, still another embodiment of the method for improving the thermal stability of GDH of the present invention is a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), wherein the pH of the composition is adjusted to pH 3.1 to 3.1. This is a method for improving the thermal stability of the composition as compared with the case where the composition is adjusted to pH 7.4, including the step of maintaining the composition at 7.0.
The pH is preferably 3.1 to 7.0, more preferably 4.0 to 6.5, and even more preferably 4.0 to 6.0.
The composition described above retains 10% or more of GDH activity even when treated at 50 ° C. for 15 minutes, compared to the composition stored at 4 ° C. Alternatively, in a composition containing glucose dehydrogenase (GDH) having a flavin compound as a coenzyme, 45% or more of GDH activity remains even when treated at 50 ° C. for 15 minutes compared to the composition stored at 4 ° C. To do.
In the said form, it is preferable to contain 1 or more types of dicarboxylic acid and a salt compound in a composition. Examples of the dicarboxylic acid include succinic acid, malonic acid, glutaric acid, phthalic acid, maleic acid, and examples of the salt compound include sodium chloride, sodium sulfate, trisodium citrate, and ammonium sulfate. In this invention, 1 or more types of compounds can be contained among these.
The concentration of each compound to be coexisted is not particularly limited, but in the solution, it is preferably 1 mM to 10M, more preferably 5 mM to 5M, and further preferably 20 mM to 1M. When preparing a powder or lyophilized product, a dried preparation having the same effect can be obtained by applying a drying treatment with a composition containing a compound concentration comparable to that in the solution.
In addition, the density | concentration of the compound described in the Example is a final density | concentration when preserve | saving together with a GDH enzyme. Preferred combinations include those in salt compounds with similar properties and combinations of carboxylic acid-containing compounds. For example, a combination of a salt compound and a carboxylic acid-containing compound can complement each other's effects. Those that do are preferred.
In order to further improve the thermal stability in the above form, sugar alcohol, carboxyl group-containing compound, alkali metal-containing compound, alkaline earth metal compound, ammonium salt, sulfate, protein such as mannitol, inositol, arabitol, adonitol, Lactitol, valine, histidine, phenylalanine, leucine, inositol, calcium glycerate, succinic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, fumaric acid, malonic acid, pimelic acid, 3-3'dimethylglutaric acid, lysine, phthalate Compounds such as acid, maleic acid, glutaric acid, ammonium sulfate, sodium sulfate, sodium chloride, bovine serum albumin (BSA) can be contained.

  The composition containing GDH of the present invention can be provided in a liquid state, but can be pulverized by freeze drying, vacuum drying, spray drying or the like. At this time, GDH dissolved in a buffer solution or the like can be used, and sugars / sugar alcohols other than the above compounds used in the present invention, amino acids, proteins, peptides, etc. are added as excipients or stabilizers. be able to. Further, it can be further granulated after pulverization. Examples of such substances include trehalose, sucrose, sorbitol, erythritol, amino acids typified by sugar, sugar alcohols, glutamic acid, arginine and the like typified by glycerol, bovine serum albumin, egg white albumin, and various chaperones. Representative proteins and peptides can be mentioned.

The composition of the buffer used for GDH extraction, purification, pulverization, and stability test described above is not particularly limited, but any buffer having a buffering ability in the range of pH 5 to 8 is preferable. For example, boric acid, Buffers such as Tris-HCl and potassium phosphate, and good buffers such as BES, Bicine, Bis-Tris, CHES, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TES, Tricine Can be mentioned.
Of these, only one type may be applied, or two or more types may be used. Furthermore, one or more composite compositions including those other than the above may be used.
In addition, the concentration of these additives is not particularly limited as long as it has a buffer capacity, but the preferable upper limit is 100 mM or less, more preferably 50 mM or less. A preferred lower limit is 5 mM or more.
The content of the buffer in the powder or lyophilized product is not particularly limited, but is preferably 0.1% (weight ratio) or more, particularly preferably 0.1 to 30% (weight ratio). Used in range.
These can use various commercially available reagents.

  The various compounds shown above may be added at the time of measurement, or may be contained in advance when a glucose measuring reagent, a glucose assay kit, or a glucose sensor described later is prepared. Moreover, it can also add to a process liquid in each manufacturing process, such as extraction, refinement | purification, and pulverization of GDH. In this case, the liquid state, the dry state, etc. are not limited, and it is sufficient to function at the time of measurement.

  The improvement in thermostability as used in the present invention means that the residual ratio (%) of the maintained GDH enzyme increases after heat treatment of the composition containing the GDH enzyme at a certain temperature for a certain time. . In the present invention, a sample stored at 4 ° C. in which the activity is almost completely maintained is assumed to be 100%, and this is compared with the activity value of the GDH solution after heat treatment at a constant temperature for a certain time to calculate the residual ratio of the enzyme. is doing. When this residual ratio was increased as compared with that without addition of the compound, it was judged that the thermal stability of GDH was improved.

Specifically, whether or not the stability is improved was determined as follows.
In the activity measurement method described in the method for measuring GDH enzyme activity described below, the GDH activity value (a) of a solution stored at 4 ° C. and the GDH activity value (b) after heat treatment at a constant temperature for a certain time are measured and measured. The relative value ((b) / (a) × 100) with respect to the value (a) of 100 was determined. This relative value was defined as the residual rate (%). And the presence or absence of the addition of the compound was compared, and when the residual ratio increased by the addition, it was determined that the thermal stability was improved.

The effect of the present invention becomes more remarkable in a system including a mediator. The mediator applicable to the method of the present invention is not particularly limited, but a combination of phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCPIP), a combination of PMS and nitroblue tetrazolium (NBT), or DCPIP alone And ferricyanide ions (such as potassium ferricyanide as a compound) alone, ferrocene alone and the like. Of these, ferricyanide ions (such as potassium ferricyanide) are preferable.
Since each of these mediators has various differences in sensitivity, it is not necessary to uniformly define the addition concentration, but it is generally desirable to add 1 mM or more.
These mediators may be added at the time of measurement, or may be contained in advance when a glucose measuring reagent, a glucose assay kit, or a glucose sensor described later is prepared. In this case, the liquid state, the dry state, and the like are not limited, and it is only necessary to dissociate at the time of the reaction during the measurement so as to be in an ion state.

In the present invention, various components can coexist if necessary. For example, you may add surfactant, a stabilizer, an excipient | filler, etc.
For example, one or more amino acids selected from the group consisting of glutamic acid, glutamine, and lysine may be used. Further, bovine serum albumin (BSA) and ovalbumin (OVA) may be added.

In the case of PQQGDH, for example, PQQGDH can be further stabilized by adding calcium ions or salts thereof, amino acids such as glutamic acid, glutamine, and lysine, and serum albumin.
For example, PQQGDH can be stabilized by containing calcium ions or calcium salts. Examples of calcium salts include calcium chloride, calcium salts of inorganic acids such as calcium acetate and calcium citrate, and organic acids. In the aqueous composition, the content of calcium ions is preferably 1 × 10 ⁻⁴ to 1 × 10 ⁻² M.
The stabilizing effect of PQQGDH by containing calcium ions or calcium salts is further improved by containing an amino acid selected from the group consisting of glutamic acid, glutamine and lysine. One or more amino acids selected from the group consisting of glutamic acid, glutamine and lysine may be used. It may further contain ovalbumin (OVA).
Or (1) one or more compounds selected from the group consisting of aspartic acid, glutamic acid, α-ketoglutaric acid, malic acid, α-ketogluconic acid, α-cyclodextrin, and salts thereof; and (2) By coexisting albumin, PQQGDH can be stabilized.

In the present invention, glucose can be measured by the following various methods.
The glucose measurement reagent, glucose assay kit, and glucose sensor of the present invention can take various forms such as liquid (aqueous solution, suspension, etc.), powdered by vacuum drying or spray drying, freeze drying, and the like. The drying method is not particularly limited and may be performed according to a conventional method. The composition containing the enzyme of the present invention is not limited to a lyophilized product, and may be in a solution state in which the dried product is redissolved.
In the present invention, glucose can be measured by the following various methods.

Glucose Measuring Reagent The glucose measuring reagent of the present invention typically contains GDH, a buffer, a mediator, and other necessary reagents for measurement, a glucose standard solution for preparing a calibration curve, and usage guidelines. The kit of the invention can be provided, for example, as a lyophilized reagent or as a solution in a suitable storage solution. Preferably, the GDH of the present invention is provided in a holified form, but may be provided in the form of an apoenzyme and hololated at the time of use.

Glucose Assay Kit The present invention also features a glucose assay kit comprising GDH according to the present invention. The glucose assay kit of the present invention comprises GDH according to the present invention in an amount sufficient for at least one assay. Typically, the kit includes the GDH of the present invention, plus buffers necessary for the assay, mediators, glucose standard solution for creating a calibration curve, and directions for use. The GDH according to the present invention can be provided in various forms, for example as a lyophilized reagent or as a solution in a suitable storage solution. Preferably, the GDH of the present invention is provided in a holified form, but may be provided in the form of an apoenzyme and hololated at the time of use.

Glucose sensor The present invention also features a glucose sensor using GDH according to the present invention. As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on this electrode. As immobilization methods, there are a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc. It may be fixed or adsorbed and fixed on the electrode, or a combination of these may be used. Preferably, the GDH of the present invention is immobilized on the electrode in a holo form, but it may be immobilized in the form of an apoenzyme and the coenzyme supplied as a separate layer or in solution. Typically, the GDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, and then treated with a reagent having an amine group to block glutaraldehyde.

  The glucose concentration can be measured as follows. Put the buffer in a thermostatic cell and add a mediator to maintain a constant temperature. An electrode on which the GDH of the present invention is immobilized is used as a working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

Hereinafter, the present invention will be described in more detail based on examples.
Example 1 Construction of PQQ-Dependent Glucose Dehydrogenase Gene Expression Plasmid Wild-type PQQ-dependent glucose dehydrogenase expression plasmid pNPG5 was derived from the Acinetobacter baumannii17 strain NCIMB115 at the multiple cloning site of the vector pBluescript SK (-). A structural gene encoding a PQQ-dependent glucose dehydrogenase is inserted. The base sequence is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of PQQ-dependent glucose dehydrogenase deduced from the base sequence is shown in SEQ ID NO: 1 in the sequence listing.
5 μg of DNA of pNPG5 was cleaved with restriction enzymes BamHI and XhoI (manufactured by Toyobo), and the structural gene portion of mutant PQQ-dependent glucose dehydrogenase was isolated. The isolated DNA was reacted with pTM33 (1 μg) cleaved with BamHI and XhoI with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA. Ligated DNA was transformed with Escherichia coli DH5α competent cells. The resulting expression plasmid was named pNPG6.

Example 2 Production of Pseudomonas Bacteria Transformant Pseudomonas putida TE3493 (Microtechnical Research No. 12298) was cultured in LBG medium (LB medium + 0.3% glycerol) at 30 ° C. for 16 hours and centrifuged (12 The bacterial cells were collected by 2,000 rpm for 10 minutes, and 8 ml of 5 mM K-phosphate buffer (pH 7.0) containing 300 mM sucrose ice-cooled was added to the bacterial cells to suspend the bacterial cells. The bacterial cells were collected again by centrifugation (12,000 rpm, 10 minutes), and 0.4 ml of 5 mM K-phosphate buffer (pH 7.0) containing 300 mM sucrose ice-cooled was added to the bacterial cells. Suspended.
0.5 μg of the expression plasmid pNPG6 obtained in Example 1 was added to the suspension and transformed by electroporation. A target transformant was obtained from a colony grown on an LB agar medium containing 100 μg / ml streptomycin.

Example 3 Preparation of PQQ-Dependent GDH Standard 500 ml of terrific broth was dispensed into a 2 L Sakaguchi flask, autoclaved at 121 ° C. for 20 minutes, allowed to cool, and then separately sterile filtered streptomycin to 100 μg / ml. Added to. This medium was inoculated with 5 ml of Pseudomonas putida TE3493 (pNPG6) cultured in PY medium containing 100 μg / ml streptomycin in advance at 30 ° C. for 24 hours, and cultured at 30 ° C. for 40 hours with aeration and stirring. The PQQ-dependent glucose dehydrogenase activity at the end of the culture was about 30 U / ml per 1 ml of the culture solution in the activity measurement.
The cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.0), disrupted by sonication, and further centrifuged to obtain a supernatant as a crude enzyme solution. . The obtained crude enzyme solution was separated and purified by HiTrap-SP (Amersham-Pharmacia) ion exchange column chromatography. Next, after dialyzing with 10 mM PIPES-NaOH buffer (pH 6.5), calcium chloride was added to a final concentration of 1 mM. Finally, it was separated and purified by HiTrap-DEAE (Amersham-Pharmacia) ion exchange column chromatography to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band by SDS-PAGE.
The purified enzyme thus obtained was used as a PQQ-dependent GLD evaluation sample.

Example 4 Preparation of NAD-Dependent GDH Standard A NAD-dependent GDH standard was obtained by using a commercial product (GLD-311) sold by Toyobo.

Example 5: Preparation of FAD-dependent GDH preparation Aspergillus terreus subspecies and Penicillium lilacinoechinulatum NBRC6231 (purchased from the National Institute of Technology and Evaluation Technology) were used as FAD-dependent GDH-producing bacteria. It was reconstituted by inoculating agar medium (Difco) and incubating at 25 ° C. The restored mycelium on the plate was collected together with the agar and suspended in filter sterilized water. Prepare 6 L of production medium (1% malt extract, 1.5% soybean peptide, 0.1% MgSO ₄ .7 hydrate, 2% glucose, pH 6.5) in two 10 L jar fermenters, After the autoclave sterilization at 120 ° C. for 15 minutes, the above mycelial suspensions were added and the culture was started. The culture conditions were a temperature of 30 ° C., an aeration rate of 2 L / min and a stirring rate of 380 rpm. The culture was stopped 64 hours after the start of the culture, and the cells of each strain were collected on the filter paper by suction filtration using a Nutsche filter. Concentrate 5 L of the culture solution to 1/10 volume using a hollow fiber module for ultrafiltration with a molecular weight of 10,000 cut, and add and dissolve ammonium sulfate in the concentrate so that the final concentration is 60% saturation (456 g / L). did. Subsequently, after centrifuging at 8000 rpm for 15 minutes in a Hitachi high-speed cooling centrifuge, the residue was precipitated, the supernatant was adsorbed on an Octyl-Sepharose column, and gradient elution was performed with an ammonium sulfate concentration of 0.6 to 0.0 saturation. Minutes were collected. The obtained GDH solution was subjected to gel filtration with a G-25 Sepharose column to recover the protein fraction, and desalted to add 0.6 ammonium sulfate equivalent to the desalted solution and dissolved. This was adsorbed on a Phenyl-Sepharose column and eluted with a gradient at an ammonium sulfate concentration of 0.6 to 0.0 to collect a fraction having GDH activity. Further, the obtained GDH solution was subjected to gel filtration with a G-25 Sepharose column to collect a protein fraction, and the obtained purified enzyme was used as a FAD-dependent GLD evaluation preparation.

  The mediator used in the composition for measuring glucose, the glucose assay kit, the glucose sensor, or the glucose measuring method of the present invention is not particularly limited, but preferably 2,6-dichlorophenol-indophenol (abbreviated as DCPIP), ferrocene. Alternatively, derivatives thereof (for example, potassium ferricyanide, phenazine methosulfate, etc.) may be used. These mediators can be obtained commercially.

Test Example 1: Method for measuring PQQ-dependent GDH activity In the present invention, the activity of PQQ-dependent GDH is measured under the following conditions.
Measurement principle D-glucose + PMS + PQQGDH → D-glucono-1,5-lactone + PMS (red)
PMS (red) + DCPIP → PMS + DCPIP (red)
The presence of DCPIP (red) formed by the reduction of 2,6-dichlorophenol-indophenol (DCPIP) with phenazine methosulfate (PMS) (red) was measured spectrophotometrically at 600 nm. In the examination of substrate specificity, the D-glucose part was changed to another saccharide, and the specificity for each substrate was measured.
Unit Definition One unit refers to the amount of PQQGDH enzyme that forms 1.0 mmol of DCPIP (red) per minute under the conditions described below.
Methods Reagent A. D- glucose solution: 1.0 M (1.8 g D- glucose (molecular weight 180.16) / 10ml H ₂ O)
B. PIPES-NaOH buffer, pH 6.5: 50 mM (1.51 g of PIPES (molecular weight 302.36) suspended in 60 mL of water is dissolved in 5N NaOH and 2.2 ml of 10% Triton X-100 is added. The pH was adjusted to 6.5 ± 0.05 at 25 ° C. using 5N NaOH, and water was added to make 100 ml.)
C. PMS solution: 24 mM (73.52 mg phenazine methosulfate (molecular weight 817.65) / 10 ml H ₂ O)
D. DCPIP solution: 2.0 mM (6.5 mg of nitrotetrazolium blue (molecular weight 817.65) / 10 ml H ₂ O)
E. Enzyme dilution: 50 mM PIPES-NaOH buffer (pH 6.5) containing 1 mM CaCl ₂ , 0.1% Triton X-100, 0.1% BSA

Procedure 1. Prepare the following reaction mixture in a light-proof bottle and store on ice (prepared at the time of use)
4.5 ml D-glucose solution (A)
21.9 ml PIPES-NaOH buffer (pH 6.5) (B)
2.0ml PMS solution (C)
1.0 ml DCPIP solution (D)

The concentration of the assay mixture in the reaction solution is as follows.
PIPES buffer 36 mM
D-glucose 148 mM
PMS 1.58mM
DCPIP 0.066 mM

2. 3.0 ml of the reaction mixture was placed in a test tube (made of plastic) and preheated at 37 ° C. for 5 minutes.
3. 0.1 ml enzyme solution was added and mixed by gentle inversion.
4). The decrease in absorbance for water at 600 nm was recorded on a spectrophotometer while maintaining at 37 ° C. for 4-5 minutes, and the ΔOD per minute from the initial linear portion of the curve was calculated (OD test).
At the same time, the same method was repeated except that the enzyme diluent (E) was added instead of the enzyme solution, and a blank (ΔOD blank) was measured.
Immediately before the assay, the enzyme powder was dissolved in ice-cooled enzyme diluent (E) and diluted to 0.05-0.10 U / ml with the same buffer (use of a plastic tube for adhesion of the enzyme) Is preferred).
For the purpose of evaluating substrate specificity, the above activity measurement procedure was performed using another type of sugar solution as a substrate instead of a glucose solution.
Calculate the calculated activity using the following formula:
U / ml = {ΔOD / min (ΔOD test−ΔOD blank) × Vt × df} / (16.8 × 1.0 × Vs)
U / mg = (U / ml) × 1 / C
Vt: Total volume (3.1 ml)
Vs: Sample volume (0.1 ml)
16.8: Millimolecular extinction coefficient of DCPIP under the above measurement conditions (cm ² / micromol)
1.0: Optical path length (cm)
df: Dilution factor C: Enzyme concentration in solution (c mg / ml)

Test Example 2: Method for Measuring NAD-Dependent GDH Activity In the present invention, NAD-dependent GDH activity is measured under the following conditions. As the NAD-dependent GDH enzyme preparation, glucose dehydrogenase (GLD311) manufactured by Toyobo was used.
Measuring principle D-glucose + NAD ⁺ → D-glucono-1,5-lactone + NADH + H ⁺
The amount of NADH produced was measured by the change in absorbance at 340 nm.
Unit Definition One unit refers to the amount of NADGDH enzyme that forms 1.0 micromole of NADH per minute under the conditions described below.
Methods Reagent A. D-glucose solution: 1.5 M (2.7 g D-glucose (molecular weight 180.16) / 10 ml H ₂ O)
B. Tris-HCl buffer, pH 8.0: 100 mM (1.21 g of tris (hydroxymethyl) aminomethane (molecular weight 121.14) suspended in 90 mL of water, 5N HCl at 25 ° C., pH 8. Adjusted to 0 ± 0.05 and added water to make 100 ml.)
C. NAD solution: 8% (80 mg NAD (molecular weight 717.48) / 1 ml H ₂ O)
D. Enzyme dilution: potassium phosphate buffer (pH 7.2)

Procedure 1. Prepare the following reaction mixture in a light-proof bottle and store on ice (prepared at the time of use)
0.9 ml D-glucose solution (A)
7.8 ml Tris-HCl buffer (pH 8.0) (B)
0.3ml NAD solution (C)

The concentration of the assay mixture in the reaction solution is as follows.
D-glucose 148 mM
Tris-HCl buffer 77 mM
NAD 0.26%

2. 3.0 ml of the reaction mixture was placed in a test tube (made of plastic) and preheated at 37 ° C. for 5 minutes.
3. 0.05 ml enzyme solution was added and mixed by gentle inversion.
4). The change in absorbance for water at 340 nm was recorded for 4-5 minutes on a spectrophotometer while maintaining at 37 ° C., and the ΔOD per minute from the initial linear portion of the curve was calculated (OD test).
At the same time, the same method was repeated except that the enzyme diluent (D) was added instead of the enzyme solution, and a blank (ΔOD blank) was measured.
Immediately before the assay, the enzyme powder was dissolved in an ice-cooled enzyme diluent (D) and diluted to 0.10-0.70 U / ml with the same buffer (use of a plastic tube for adhesion of the enzyme) Is preferred).
For the purpose of evaluating substrate specificity, the above activity measurement procedure was performed using another type of sugar solution as a substrate instead of the glucose solution.

Calculate the calculated activity using the following formula:
U / ml = {ΔOD / min (ΔOD test−ΔOD blank) × Vt × df} / (6.22 × 1.0 × Vs)
U / mg = (U / ml) × 1 / C
Vt: Total volume (3.05 ml)
Vs: Sample volume (0.05 ml)
6.22: mmol molecular absorption coefficient of NADH (cm ² / micromol)
1.0: Optical path length (cm)
df: Dilution factor C: Enzyme concentration in solution (c mg / ml)

Test Example 3: Method for measuring FAD-dependent GDH activity In the present invention, the activity of FAD-dependent GDH is measured under the following conditions.
<Reagent>
50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100)
14 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1M D-glucose solution 15.8 ml of the above PIPES buffer, 0.2 ml of DCPIP solution, and 4 ml of D-glucose solution are mixed to obtain a reaction reagent.

<Measurement conditions>
Prewarm 2.9 ml of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 ml of GDH solution and mixing gently, the absorbance change at 600 nm was recorded for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control, and the absorbance change per minute (ΔOD _TEST) ). In the blind test, a change in absorbance per minute (ΔOD _BLANK ) is similarly measured by adding a solvent that dissolves GDH to the reagent mixture instead of the GDH solution. From these values, the GDH activity is determined according to the following formula. Here, 1 unit (U) in GDH activity is defined as the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of 200 mM D-glucose.

Activity (U / ml) =
{− (ΔOD _TEST −ΔOD _BLANK ) × 3.0 × dilution ratio} / {16.3 × 0.1 × 1.0}

In the formula, 3.0 is the amount of the reaction reagent + enzyme solution (ml), 16.3 is the molar molecular extinction coefficient (cm ² / micromol) under the conditions for this activity measurement, and 0.1 is the enzyme solution solution. Amount (ml), 1.0 indicates the optical path length (cm) of the cell.

  GDH using three different types of coenzymes is examining improvement in thermal stability under different conditions. For example, in Examples 6-8 below, in PQQGDH, an enzyme solution prepared to 5 U / ml with a pH 6.5 buffer solution was heat-treated at 50 ° C. for 16 hours, and then the remaining PQQGDH activity was compared and heat stable. The improvement of sex is confirmed. Similarly, in NADGDH, after the enzyme solution prepared to 85 U / ml with the buffer solution of pH 7.2 was heat-processed at 50 degreeC for 1 hour, the remaining NADGDH activity was compared and the improvement in thermal stability was confirmed. Similarly, in FADGDH, an enzyme solution prepared to 5 U / ml with a buffer solution of pH 7.2 is heat-treated at 50 ° C. or 55 ° C. for 15 to 30 minutes, and then the remaining FADGDH activity is compared to determine the thermal stability. Improved.

  Moreover, in the following Examples 9 and 10, after actually measuring the pH of the enzyme solution composition (0.4 to 5.1 U / ml) prepared with various 50 mM buffers, 50 ° C. for 15 minutes, or Heat treatment was performed at 50 ° C. for 30 minutes, or 55 ° C., 15 minutes, or 55 ° C. for 30 minutes, and GDH activity was measured to calculate the residual activity rate (%). The improvement in thermal stability was confirmed by comparing the activity residual ratio.

Example 6: Confirmation of thermal stability using a glucose measurement system 1
The examination was performed according to the method for measuring PQQGDH activity in Test Example 1 described above. In addition, in order to measure the enzyme activity of PQQGDH including the apo type, the activity was also measured in a reaction mixture to which PQQ having a final concentration of 860 nM was added.
First, enzyme dilution solution (50 mM PIPES-NaOH buffer (pH 6.5) containing 1 mM CaCl ₂ , 0.1% Triton X-100, 0.1% BSA) so that PQQGDH is about 5.0 U / ml. 50 ml of the product dissolved in was prepared. To 0.33 ml of this enzyme solution, 0.1 ml of 10-fold concentrations of various compounds listed in Tables 1 and 2 were added, and the base buffers listed in Tables 1 and 2 were added to make the total volume 1.0 ml. Two were prepared. In addition, two controls were prepared by adding 0.1 ml of distilled water instead of various compounds. Of the two, one was stored at 4 ° C. for 16 hours, and the other was treated at 50 ° C. for 16 hours. After the treatment, each sample was diluted 10-fold with an enzyme diluent, and then PQQGDH activity was measured. The enzyme activity of each sample stored at 4 ° C. for 16 hours was defined as 100, and the activity values after treatment at 50 ° C. for 16 hours were compared and calculated as a relative value (%).
In all the compounds shown in Tables 1 and 2 in the PQQGDH composition, an improvement in thermal stability was observed by coexisting them. In the case of the one based on potassium phosphate buffer, when succinic acid, pimelic acid and dimethylglutaric acid are added, the thermal stability of holo-type PQQGLD is lowered, but this is because PQQ is dropped from the enzyme. It seems to be caused by this. The thermal stability including the apo type has been improved, and it seems that these compounds are effective in maintaining the three-dimensional structure of the enzyme itself.
Table 1 shows the residual rate (%) of PQQGDH activity after treatment of PQQGDH composition containing PPIES buffer (pH 6.5) in the presence of various compounds at 50 ° C. for 16 hours.
Table 2 shows the residual rate of PQQGDH activity after treatment of PQQGDH composition in the presence of various compounds based on phthalate buffer (pH 7.0) and potassium phosphate buffer (pH 7.0) at 50 ° C. for 16 hours (% ).

Example 7: Confirmation of thermal stability using glucose measurement system 2
The examination was performed according to the method for measuring NADGDH activity in Test Example 2 described above.
First, 50 ml of NADGLD (Toyobo GLD-311) dissolved in an enzyme diluent so as to be about 250 U / ml was prepared. To 0.33 ml of this enzyme solution, 0.1 ml of various compounds having a 10-fold concentration shown in Table 3 was added, and potassium phosphate buffer (pH 7.2) was added to make the total volume 1.0 ml. I prepared a book. In addition, two controls were prepared by adding 0.1 ml of distilled water instead of various compounds. Of the two, one was stored at 4 ° C and the other was treated at 50 ° C for 1 hour. After the treatment, each sample was diluted 500-fold with an enzyme diluent, and then NADGDH activity was measured. In each case, the enzyme activity of those stored at 4 ° C. was defined as 100, and the activity value after treatment at 50 ° C. for 1 hour was compared to calculate the activity remaining rate (%).
As a result, all dicarboxylic acids examined showed an effect, and the highest thermal stability improvement effect was observed when succinic acid or maleic acid was added.
Table 3 shows the residual ratio (%) of NADGDH activity after treatment for 1 hour at 50 ° C. of the NADGDH composition in which various compounds coexist.

Example 8: Confirmation of thermal stability using glucose measurement system 3
The examination was performed according to the method for measuring FADGDH activity in Test Example 3 described above.
First, 50 ml of FADGLD obtained in Example 5 dissolved in an enzyme diluent (50 mM potassium phosphate buffer (pH 7.2)) so as to be about 10 U / ml was prepared. Two solutions were prepared by adding 0.5 ml of 1% and 0.5% BSA to 0.5 ml of this enzyme solution to a total volume of 1.0 ml. In addition, succinic acid, malonic acid, phthalic acid, maleic acid, glutaric acid, sodium chloride, sodium sulfate, trisodium citrate, and ammonium sulfate having double concentrations shown in Tables 5 and 6 were prepared. Two bottles were prepared by adding 5 ml to a total volume of 1.0 ml. For control, two were prepared by adding 0.1 ml of distilled water instead of various compounds.
Of the two, one was stored at 4 ° C and the other was treated at 50 ° C for 30 minutes. After the treatment, each sample was diluted 21 times with an enzyme diluent, and then FADGDH activity was measured. In each case, the enzyme activity of those stored at 4 ° C. was defined as 100, and the activity value after treatment at 50 ° C. for 1 hour was compared to calculate the activity remaining rate (%).
As a result of these studies, it has been clarified that the thermal stability of FAD-GLD is increased by adding a proteinaceous stabilizer (BSA) (Table 4). In addition, the addition of various dicarboxylic acid compounds or various salt compounds is effective in improving the thermal stability of FAD-GLD. Among the dicarboxylic acid compounds, succinic acid and malonic acid are the largest, and among the salt compounds, sodium sulfate is the largest. The effect was seen (Tables 5 and 6). In the case of succinic acid, malonic acid, and sodium sulfate, it is considered that the effect of improving the stability can be seen even with the addition of several moles. Moreover, since a sufficient effect is seen even with a simple salt compound such as sodium chloride, it has been found for the first time in FAD-GLD that the thermal stabilization is achieved simply by increasing the ionic strength.
Table 4 shows the residual ratio (%) of FADGDH activity after treatment for 30 minutes at 50 ° C. of the FADGDH composition in the presence of a proteinaceous stabilizer.
Table 5 shows the residual ratio (%) of the FADGDH activity after treatment for 30 minutes at 50 ° C. of the FADGDH composition containing the dicarboxylic acid compound.
Table 6 shows the residual ratio (%) of FADGDH activity after treatment for 15 minutes at 50 ° C. of the FADGDH composition in the presence of a salt compound.

Example 9: Examination of the effect of improving the thermal stability of the GDH composition with the acidic pH maintained was carried out in accordance with the method for measuring FADGDH activity in Test Example 3 described above.
First, 10 ml prepared by dissolving the two types of FADGDH obtained in Example 5 with an enzyme diluent (50 mM potassium phosphate buffer (pH 6.5)) so as to be about 10 U / ml each was prepared. To 0.2 ml of this enzyme solution, 1. Add 50 mM potassium phosphate buffer (pH 4.3), 50 mM potassium phosphate buffer (pH 5.6), or 50 mM potassium phosphate buffer (pH 7.0). 8 ml was added to bring the total volume to 2.0 ml. When the pH of each enzyme solution was measured, it varied to around pH 5.6, 6.0, and 7.2. Two aliquots of 1 ml were prepared, one was stored at 4 ° C., and the other was heat-treated at 50 ° C. After the treatment, each sample was diluted 2-fold with an enzyme diluent, and then FADGDH activity was measured. The percentage of remaining GDH activity (%) was calculated by comparing the activity values after treatment at 50 ° C. for 15 minutes or 30 minutes with the enzyme activity of those stored at 4 ° C. as 100%.
As a result of these studies, it has been clarified that the thermal stability of the FADGLD-containing composition increases when the pH of the GDH-containing composition is maintained on the acidic side (Table 7).
Furthermore, in order to examine the pH conditions in detail, as in the previous method, the enzyme solution was diluted 10-fold with various buffers shown in Table 8, and after confirming the measured pH, heat treatment was performed at 50 ° C. for 15 minutes, and GDH was performed. Activity was measured and the residual ratio (%) was calculated.
As a result, in the composition containing FADGLD derived from any of Aspergillus terreus subspecies and Penicillium lilacinoechinulatum NBRC6231, the thermal stability is improved in the range of pH 3.14 to 6.97 as compared to the vicinity of pH 7.4. (Table 8, FIG. 1 and FIG. 2).
When preparing a sensor chip for a glucose sensor, which is a general purpose use of the GDH composition, there is a step of heating and drying, and maintaining the FADGDH-containing composition on the acidic side improves the stability of the composition itself. It is very important for improving the stability and measurement accuracy of the sensor chip product used.

Example 10: Examination and examination of the effect of improving the thermal stability of the composition obtained by allowing one or more salt compounds or dicarboxylic acids to coexist in the GDH composition having an acidic pH maintained is the test example described above. This was carried out according to the method for measuring FADGDH activity of No. 3.
First, various compound solutions were prepared by dissolving sodium sulfate, trisodium citrate, and ammonium sulfate in a 50 mM potassium phosphate buffer solution (pH 6.5) so as to be 1 M each. Further, 20 ml of the two types of FADGDH obtained in Example 5 dissolved in an enzyme diluent (50 mM potassium phosphate buffer (pH 6.5)) so as to be about 10 U / ml was prepared. Eight kinds of enzyme solution samples (2 ml) in which two kinds of FADGDH were mixed 1: 1 were prepared for each of the four kinds of compound solutions. In addition, as a control without adding the compound solution, a control enzyme solution sample prepared in the vicinity of pH 6.25 and 6.65 with 50 mM potassium phosphate buffer was also prepared. The measured pH of each enzyme solution sample was measured, and it was confirmed that a close pH was maintained. Two pieces of enzyme solution sample dispensed by 1 ml were prepared, one was stored at 4 ° C., and the other was heat-treated at 55 ° C. for 30 minutes. After the treatment, each sample was diluted 10-fold with an enzyme diluent, and then FADGDH activity was measured. The percentage of remaining GDH activity (%) was calculated by comparing the activity values after treatment at 55 ° C. for 30 minutes, assuming that the enzyme activity of those stored at 4 ° C. was 100%.
As a result of these studies, the pH of the GDH-containing composition is maintained on the acidic side, and the pH is maintained on the acidic side by adding salt compounds such as sodium sulfate, trisodium citrate, and ammonium sulfate. It became clear that thermal stability more than a sample was obtained (Table 9).
Next, an equal amount of 2M sodium chloride, 1M sodium sulfate, or 1M tricitrate is added to an enzyme solution sample (5 U / ml) prepared by adjusting the above two types of FADGDH with succinate buffer to pH 5.3. Sodium or 1M ammonium sulfate was added to prepare 8 types of enzyme solution samples (2 ml). As a control without adding the compound solution, a control enzyme solution sample diluted twice with 50 mM sodium succinate buffer (pH 5.3) was also prepared. The actually measured pH of each enzyme solution sample was measured, and it was confirmed that the pH close to the control was maintained. Two pieces of enzyme solution sample dispensed by 1 ml were prepared, one was stored at 4 ° C., and the other was heat-treated at 55 ° C. for 30 minutes. After the treatment, each sample was diluted 5-fold with an enzyme diluent, and then FADGDH activity was measured. The percentage of remaining GDH activity (%) was calculated by comparing the activity values after treatment at 55 ° C. for 30 minutes, with the enzyme activity of each stored at 4 ° C. being 100%.
As a result of these studies, it was confirmed that the thermal stability can be improved by adding salt compounds such as sodium chloride, sodium sulfate, trisodium citrate, and ammonium sulfate even at a low pH of about 5. It was speculated that the addition effect was recognized even in a compound having less characteristics such as sodium chloride, and that the thermal stability of the FADGDH composition can be improved by coexisting them in general in general salt compounds. (Table 10).
In addition, when a succinic acid buffer was used, there was a tendency for thermal stability to be improved as compared to the case of using a potassium phosphate buffer as a base. Like the compound, it was speculated that there was an effect of improving the thermal stability. Therefore, it was investigated whether the thermal stability fluctuates by maintaining the pH of the GDH-containing composition on the acidic side and adding a dicarboxylic acid compound.
First, 100 mM sodium acetate (pH 5.0) and 100 mM potassium phosphate buffer (pH 5.6) were prepared as base buffers. Further, 20 ml of the two types of FADGDH obtained in Example 5 dissolved in an enzyme diluent (50 mM potassium phosphate buffer (pH 6.5)) so as to be about 10 U / ml was prepared. Next, 0.4M succinic acid (adjusted to pH 7.0 with NaOH), 0.4M malonic acid (adjusted to pH 7.0 with NaOH), and 0.4M glutaric acid (adjusted to pH 7.0 with NaOH) were prepared. .
After mixing 0.8 ml of base buffer and 0.2 ml of enzyme solution (10 U / ml), 1 ml of various dicarboxylic acid compounds were added, and the pH of each was measured. In addition, as a control in which the compound solution was not added, distilled water was added instead of the dicarboxylic acid compound to prepare a sample adjusted to the vicinity of the measured pH value of each sample. Two pieces of the enzyme solution sample dispensed by 1 ml were prepared, one was stored at 4 ° C., and the other was heat-treated at 55 ° C. After the treatment, each sample was diluted 2-fold with an enzyme diluent, and then FADGDH activity was measured. The percentage of remaining GDH activity (%) was calculated by comparing the activity values after treatment at 55 ° C. for 15 minutes or 30 minutes with the enzyme activity of those stored at 4 ° C. as 100%.
As a result of these studies, it was confirmed that the thermal stability can be improved by adding dicarboxylic acid compounds such as succinic acid, malonic acid, and glutaric acid at a pH of about 5.5 or 6.0. From the above results, it is speculated that the thermal stability of the FADGDH composition can be improved by coexisting them widely in all dicarboxylic acid compounds (Table 11).

Example 11: Examination of confirmation of storage stability using a glucose measurement system was performed according to the method for measuring PQQGDH activity in Test Example 1 described above. In addition, in order to measure the enzyme activity of PQQGDH including the apo type, the activity was also measured in a reaction mixture to which PQQ having a final concentration of 860 nM was added.
First, 10 ml of PQQGDH dissolved in an enzyme diluent (1 mM CaCl ₂ , 50 mM PIPES-NaOH buffer (pH 6.5)) so as to be about 10 U / ml was prepared. Three compounds having a total volume of 0.6 ml were prepared by adding 0.06 ml of 10-fold concentrations of various compounds listed in Table 12 to 0.54 ml of this enzyme solution. In addition, three controls were prepared by adding 0.06 ml of distilled water instead of various compounds. After the prepared vial was freeze-dried (FDR) to completely evaporate the water, the control vial was immediately subjected to activity measurement. On the other hand, the sample vial was treated at 25 ° C. and 70% humidity for 6 hours and then stored at 37 ° C., and the residual activity after 1 week or 2 weeks was measured. The activity remaining rate was calculated from the activity value of each sample with the activity value immediately after FDR as 100%. It was judged that the storage stability was improved as the activity remaining rate was higher.
By adding succinic acid, ammonium chloride, malonic acid, etc., the effect of suppressing the decrease in the holoformation rate was observed, and the powder stability was also improved. When the difference in the shape of the powder depending on whether or not a compound was added was compared, it was easily expected that the added powder had a compacted powder body and increased moisture absorption resistance. In this study, we examined a limited number of carboxylic acid-containing compounds due to the relationship of materials, but it seems that a wide variety of compounds exhibit the same effect. Moreover, since it can be estimated that the change in powder shape leads to an increase in storage stability, it can be similarly analogized that the same effect can be obtained with NAD-GLD and FAD-GLD.

The improvement in thermal stability according to the present invention can improve the storage stability and measurement accuracy of the glucose measurement reagent, glucose assay kit, and glucose sensor.

PH dependence of thermal stability of FADGDH derived from Aspergillus terreus subspecies PH dependence of thermal stability of FADGDH derived from Penicillium lilacinoechinulatum NBRC6231

Claims (21)

  A composition comprising a soluble coenzyme-linked glucose dehydrogenase (GDH) comprising the enzyme and a sugar alcohol, a carboxyl group-containing compound, an alkali metal-containing compound, an alkaline earth metal compound, an ammonium salt, a sulfate, and a protein A method for improving the thermal stability of GDH, comprising the step of coexisting any one or more compounds selected from those in the absence of the compound.   The method for improving stability according to claim 1, wherein the coenzyme is pyrroloquinone quinoline or a flavin compound or nicotinamide adenine dinucleotide (NAD).   The composition having improved thermal stability according to claim 1 or 2, wherein the final concentration of each compound to be coexisted is 0.1% or more.   The compound to be added is mannitol, inositol, arabitol, adonitol, galactitol, valine, histidine, phenylalanine, leucine, inositol, calcium glycerate, succinic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, fumaric acid, malonic acid, pimelin It is any one or more selected from the group consisting of acid, 3-3′dimethylglutaric acid, lysine, phthalic acid, maleic acid, glutaric acid, ammonium sulfate, sodium sulfate, sodium chloride, and bovine serum albumin (BSA). The method for improving thermal stability according to claim 1, wherein the thermal stability is improved.   A composition comprising a soluble coenzyme-linked glucose dehydrogenase having improved thermal stability by the method according to claim 1.   A method for measuring glucose concentration using the composition of claim 5.   A glucose sensor comprising the composition of claim 6.   A composition comprising a soluble coenzyme-linked glucose dehydrogenase (GDH) comprising the enzyme and a sugar alcohol, a carboxyl group-containing compound, an alkali metal-containing compound, an alkaline earth metal compound, an ammonium salt, a sulfate, and a protein The manufacturing method of the composition which improved the thermal stability of GDH compared with the case where this compound is not coexisted including the process of coexisting any one or more compounds chosen from more.   In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), the step of maintaining the pH of the composition on the acidic side of pH 7 or lower, compared to the case where the composition is adjusted to pH 7.3 or higher A method for improving the thermal stability of the composition.   In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), the step of maintaining the pH of the composition at pH 3.1 to 7.0, compared with the case where the composition is adjusted to pH 7.4 And improving the thermal stability of the composition.   The method for improving the thermal stability of the composition according to claim 9 or 10, wherein the soluble coenzyme-linked glucose dehydrogenase (GDH) is glucose dehydrogenase (GDH) having a flavin compound as a coenzyme.   The method for improving thermal stability according to claim 11, wherein glucose dehydrogenase (GDH) having a flavin compound as a coenzyme is derived from a filamentous fungus.   A composition having improved thermal stability by the method according to claim 9.   In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), 10% or more of GDH activity remains even when treated at 50 ° C. for 15 minutes compared to the composition stored at 4 ° C. The GDH containing composition of Claim 13 characterized by these.   In a composition containing glucose dehydrogenase (GDH) containing a flavin compound as a coenzyme, 45% or more of GDH activity remains even when treated at 50 ° C. for 15 minutes compared to the composition stored at 4 ° C. The GDH containing composition of Claim 13 characterized by these.   The composition of Claim 13 containing 1 or more types of dicarboxylic acid and a salt compound.   The composition according to claim 13, comprising at least one compound selected from sodium chloride, sodium sulfate, trisodium citrate, ammonium sulfate, succinic acid, malonic acid, glutaric acid, phthalic acid, and maleic acid.   A method for measuring glucose concentration using the composition according to claim 13.   A glucose sensor comprising the composition according to claim 13.   In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), the step of maintaining the pH of the composition on the acidic side of pH 7 or lower, compared to the case where the composition is adjusted to pH 7.3 or higher A method for producing a composition having improved thermal stability.   In a composition containing soluble coenzyme-linked glucose dehydrogenase (GDH), the step of maintaining the pH of the composition at pH 3.1 to 7.0, compared with the case where the composition is adjusted to pH 7.4 And a method for producing a composition having improved thermal stability.