WO2006068170A9

WO2006068170A9 - Method for improvement of heat stability of glucose dehydrogenase

Info

Publication number: WO2006068170A9
Application number: PCT/JP2005/023468
Authority: WO
Inventors: Tadanobu Matsumura; Masao Kitabayashi
Original assignee: Toyo Boseki; Tadanobu Matsumura; Masao Kitabayashi
Priority date: 2004-12-22
Filing date: 2005-12-21
Publication date: 2006-10-19
Also published as: WO2006068170A1

Abstract

[PROBLEMS] To provide a method for improving stability of a composition comprising coenzyme-binding soluble glucose dehydrogenase. [MEANS FOR SOLVING PROBLEMS] A method for improving heat stability of coenzyme-binding soluble glucose dehydrogenase in a composition comprising the enzyme, the method comprising treating the composition with heat; a process for producing a composition comprising the enzyme having improved heat stability; a composition produced by the method; a glucose assay kit and a glucose sensor comprising the composition; and a method for the determination of a glucose concentration by using the composition.

Description

Specification

Method for improving the thermal stability of glucose dehydrogenase

Technical field

[0001] The present invention relates to a method for improving the stability of a composition comprising a soluble coenzyme-bound type of gnolecose dehydrogenase (hereinafter referred to as GDH). In addition, the present invention relates to a glucose measurement method using GDH with improved stability and a genolecose sensor.

The present invention also relates to a method for improving the holoformation rate or stability in a dry state of a composition containing soluble coenzyme-linked gnolecose dehydrogenase (hereinafter, dalcose dehydrogenase is also referred to as GDH). In addition, the present invention relates to a glucose measuring method using a GDH with improved holo-formation rate or stability, and a genolecose sensor.

The PQQGDH of the present invention is useful for the determination of glucose in clinical examinations and food analysis.

Background art

[0002] Blood glucose self-measurement is important for diabetics to grasp their normal blood glucose level and use it for treatment. For sensors used for blood glucose self-measurement, an enzyme using gnolecose as a substrate is used. An example of such an enzyme is gnorecoxidase (EC 1. 1. 3. 4). Glucose oxidase has been used for a long time as an enzyme for blood glucose sensors because of its high specificity to glucose and excellent heat stability, and its first announcement was made about 40 years ago. Go back. 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 dalcono δ rataton 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.

[0003] In order to avoid such a problem, for example, NAD (Ρ) -dependent gnolecose dehydrogenase (EC 1. 1. 1. 47) or pyroguchi quinoline quinone (in this book, pyroguchi quinoline quinone is used). P QQ is also described.) Dependent glucose dehydrogenase (EC1. 1. 5. 2 (BEC1. 1. 99. 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 has poor stability and requires the addition of a coenzyme. On the other hand, the latter PQQ-dependent glucose dehydrogenase (PQQ-dependent glucose dehydrogenase is

Also described as QQGDH. ) Has the disadvantage that it affects the accuracy of measured values because it also acts on sugars other than glucose, such as maltose and ratatoose, which have poor substrate specificity.

[0004] Patent Document 1 discloses a flavin-binding glucose dehydrogenase derived from the genus Aspergillus. This enzyme is superior in that it has excellent substrate specificity and is not affected by dissolved oxygen. With regard to thermal stability, the activity remaining rate is about 89% after 15 minutes of treatment at 50 ° C, and it is said that the stability is also excellent. However, considering the fact that heat treatment may be required in the process of manufacturing the sensor chip, it is by no means sufficient stability.

Patent Literature l: WO 2004/058958

Brief Description of Drawings

[0005] [Fig. 1] Fig. 1 shows the residual rate (%) of PQQGDH activity after treatment for 16 hours at 50 ° C in a PQQGDH composition based on PIPES buffer (pH 6.5) in the presence of various compounds. Show.

[Figure 2] Figure 2 shows the results 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. P Indicates the residual rate (%) of QQGDH activity.

FIG. 3 shows the residual rate (%) of NADGDH activity after 1 hour treatment of NADGDH composition coexisting with various compounds at 50 ° C.

[FIG. 4] FIG. 4 shows the residual rate (%) of FADGDH activity after treatment for 30 minutes at 50 ° C. with a FADGDH composition in the presence of a proteinaceous stabilizer.

FIG. 5 shows the residual ratio (%) of FADGDH activity after treatment of a FADGDH composition coexisting with a dicarboxylic acid compound at 50 ° C. for 30 minutes.

FIG. 6 shows the FADGDH activity remaining rate (%) after a 15 minute treatment at 50 ° C. with a FADGDH composition in the presence of a salt compound. [Fig. 7] Fig. 7 shows the optimum temperature for PQQ-dependent GDH.

[Fig. 8] Fig. 8 shows the thermal stability of PQQ-dependent GDH.

[FIG. 9] FIG. 9 shows the results of studying the thermal stability effect of heating treatment. 4 after heating

Residual activity was calculated with the activity value of Sampu Nore stored at ° C as 100%.

[FIG. 10] FIG. 10 shows the results of the examination of the heat treatment conditions (temperature X time). The residual activity was calculated with the activity value of the sample stored at 4 ° C after the heating treatment as 100%.

[FIG. 11] FIG. 11 shows the results of an examination of the effect of re-cooking treatment at 50 ° C for 16 hours after the heating treatment. The residual activity after heat treatment at 55 ° C for 1 hour was calculated with the activity value of the sampnore stored at 4 ° C as 100%.

[Fig. 12] Percentage of expression level of holo-type PQQGDH calculated by dividing holo-type PQQGDH activity (U / ml) expressed per unit liquid volume by total PQQGDH activity (U / ml).

[Fig. 13] Percentage of expression level of holo-type PQQGDH calculated by dividing holo-type PQQGDH activity (U / ml) expressed per unit volume by total PQQGDH activity (U / ml).

Disclosure of the invention

Problems to be solved by the invention

[0006] An object of the present invention is to provide a reagent for measuring a blood glucose level that overcomes the above-mentioned drawbacks related to the stability of known enzymes for blood glucose sensors and is more practically advantageous.

Means for solving the problem

[0007] In the previous PQQGDH studies, the present inventors have obtained a large number of multiple mutants that have improved the substrate specificity of the enzyme, and some of them have storage stability compared to wild-type PQQGDH. A mutant with reduced was observed. Therefore, in order to solve this problem, intensive research was conducted on the cause of the problem, and by applying the heating treatment of the enzyme presented in this patent, the three-dimensional structure of PQ QGDH was stably maintained and the stability was improved. I found that it can be improved. In addition, it was found that by adding certain compounds to the enzyme presented in this patent, the three-dimensional structure of PQQGDH is stably maintained and the stability can be improved.

To date, Patent Document 2 has reported on measures to improve the stability of PQQGDH, among which there are reports of studies using PQQGDH modification means at the gene level. The possibility of increasing means It was not touched.

Patent Document 2: WO02 / 072839

[0008] As a result of further diligent research, the present inventors have sought to find a simpler method for improving stability from a viewpoint different from that of the past policy. It has been clarified that the stability of DH itself can be increased, and that the GDH holo-type ratio or storage stability can be increased by improving the GDH composition, and finally the present invention has been completed.

That is, the present invention has the following configuration.

[Section 1

A method for improving the thermal stability of a composition comprising a soluble coenzyme-linked glucose dehydrogenase, comprising a step of heating the composition.

[Section 2]

Item 2. The method for improving thermal stability according to Item 1, wherein the coenzyme is pyrroloquinone quinoline or a flavin compound.

[Section 3

2. The method for improving the thermal stability according to claim 1, wherein the heating treatment temperature is not higher than a temperature at which a large heat inactivation of the enzyme occurs.

[Section 4

A composition containing a soluble coenzyme-linked glucose dehydrogenase comprising a soluble coenzyme-linked glucose dehydrogenase having improved thermal stability, comprising a step of heating the composition. How to manufacture.

[Section 5]

A composition containing a soluble coenzyme-bound glucose dehydrogenase produced by the method according to Item 4, which contains a soluble coenzyme-bound glucose dehydrogenase with improved thermal stability. Composition.

[Section 6

A glucose assembly kit comprising the composition according to Item 5.

[Section 7] Item 6. A glucose sensor comprising the composition according to Item 5.

[Section 8

6. A method for measuring glucose concentration using the composition according to item 5.

The invention's effect

[0010] The improvement in thermal stability according to the present invention reduces the heat inactivation of the enzyme during preparation of the glucose measurement reagent, the gnolecose assay kit, and the glucose sensor, thereby reducing the amount of the enzyme used and the measurement accuracy. Enable improvement. It also makes it possible to provide a blood glucose measurement reagent using GDH, which has excellent storage stability.

The improvement of the holo-type ratio or storage stability according to the present invention also improves the storage stability of glucose measuring reagents, glucose assembly kits, and glucose sensors, thereby reducing the amount of the enzyme used and improving the measurement accuracy. To. It also makes it possible to provide a blood glucose level measurement reagent using GDH, which has excellent storage stability.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] GDH is an enzyme that catalyzes the following reaction.

D—glucose + electron mediator (oxidized)

→ D—Dalcono δ —Rataton + electron transfer material (reduced form)

It is an enzyme that catalyzes the reaction when D-gnolecose is oxidized to produce D-gnolecono-1,5-latataton, and there is no particular limitation on the origin and structure.

[0012] GDH that can be applied to the method of the present invention is not particularly limited as long as it is a soluble coenzyme-bound dalcos dehydrogenase (GDH).

As the coenzyme, for example, pyrroloquinone quinoline or flavin compound or nicotinamide adenine dinucleotide (NAD) can be used.

[0013] The GD H (PQQGDH) taking pyrroloquinone quinoline as a coenzyme that can be applied to the method of the present invention is not particularly limited, but for example, acinetopacter • force no coreaceticus (Acinetobacter calcoaceticus) from LMD79.41 (A. M. Cleton—Jansen et al., J. Bacteriol., 170, 2121 (1988) and Mol. Gen. Genet., 217, 430 (1989)), Escherichia E. coli) (A. M. Cleton—Jansen et al., J. Bacteriol., 172, 6308 (1990)), Ku, Norecono Oxidans (Gluconobacter oxydans) (Mol. Gen. Genet., 229, 206 (1991)), and those derived from microorganisms such as Acinetobacter baumanni NCIMB11517 reported in Patent Document 1 Can be illustrated.

However, it is difficult to modify the membrane enzyme present in Escherichia coli to make it soluble, and the origin is to select soluble PQQGDH such as Acinetobacter's Calcoaceticus or Acinetopacter 'Baumannii Is preferred.

In addition, Acinetobacter baumannii (CIcinetobacter baumannii) NCIMB1151 7 or more, ij, Acinetobacter calcoaceticus (this was classified).

[0014] PQQGDH that can be applied to the method of the present invention is not limited to those exemplified above, as long as it has glucose dehydrogenase activity. Other amino acid residues 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; (1989) As described in Cold Spring Harbor Laboratory Press, New York.

[0015] 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 produced by those skilled in the art using known techniques in the technical field.

[0016] For example, the above-mentioned natural microorganisms that produce PQQGDH, or the gene encoding natural PQQGDH as it is or after mutation, expression vectors (many are known in the art) Cultivated transformants transformed into suitable plasmids (many are known in the art, eg, E. coli), and culture fluid force centrifugation After harvesting the cells, etc., destroy the cells by mechanical methods or enzymatic methods such as lysozyme, and if necessary, solubilize by adding a chelating agent such as EDT A or a surfactant. A water-soluble fraction containing PQQGDH can be obtained. Or expressed P by using an appropriate host vector system. QQGDH can be secreted directly into the culture medium.

[0017] The PQQGDH-containing solution obtained as described above is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment such as ammonium sulfate or sodium sulfate, or hydrophilic organic solvents such as methanol, ethanol, acetone and the like. Precipitate by the fractional precipitation method. Heat treatment and isoelectric point treatment are also effective purification means. In addition, purified PQQGDH can be obtained by performing gel filtration using an adsorbent or gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified enzyme preparation should preferably be purified to such an extent that it shows a single band on electrophoresis (SDS-PAGE).

[0018] Before and after the above steps, 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. .

[0019] The concentration of PQQGDH in the present invention is not particularly limited.

[0020] GDH taking FAD as a coenzyme (FAD-dependent GDH) that can be applied to the method of the present invention is not particularly limited. For example, filamentous fungi belonging to the category of eukaryotes And those derived from microorganisms such as Penicillium and Aspergillus. These microbial strains can be easily obtained by requesting distribution from each strain storage organization. For example, Penicillium Penicillium, Lirashino Echinulatam, is registered with the Product Evaluation Technology Foundation's “Biological Resources Department” under the deposit number NBRC6092.

[0021] 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 medium is a carbon source or inorganic necessary for the growth of the microorganism. A liquid medium containing a nitrogen source and Z or an organic nitrogen source is more preferable, and a liquid medium suitable for aeration stirring is preferred. In the case of liquid medium, for example, glucose, dextran, soluble starch, sucrose, etc. as the carbon source. As the nitrogen source, for example, ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, defatted soybeans, A potato extract etc. are illustrated. If desired, other nutrients (for example, inorganic salts such as calcium chloride, sodium dihydrogen phosphate, magnesium chloride, Vitamins and the like).

[0022] The culture method follows a 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 aeration stirring, but preferably cultured by aeration stirring. 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, but the culture is preferably 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 medium and measuring the GDH activity in the culture liquid, and the point when the increase in GDH activity over time is eliminated is regarded as the peak. To stop the culture.

[0023] As a method of extracting GDH from the above 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 as a solvent, Preferably resuspended in water or buffer. The resuspended cells can be disrupted 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 can be used. The lytic enzyme is not particularly limited as long as it has the ability to digest the fungal cell wall. Examples of applicable enzymes include “Lyticase” manufactured by Sigma. Examples of the physical crushing method include ultrasonic crushing, glass bead crushing, and French press. After the crushing treatment, the residue can be removed by centrifugation or filtration to obtain a GDH crude solution.

[0024] Further, the culture method in the present invention may be solid culture. Preferably, the eukaryotic microorganism having the ability to produce GDH of the present invention is grown on wheat bran or the like 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 solid matter such as bacterial cell bran by centrifugation or filtration.

[0025] 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 above, 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, isoelectric A known separation method such as point electrophoresis can be selected appropriately. Various stabilizers and the like can also be added to the extracted GDH or the purified GDH solution. Examples of such substances include sugars and sugar alcohols such as mannitol, trehalose, sucrose, sonorebitonore, erythritole, glyceronole, etc., amino acids such as bovine serum albumin, egg white albumin, Proteins such as chaperones and peptides.

[0026] The GDH of the present invention can also be pulverized by force S, lyophilization, vacuum drying, spray drying or the like which can be provided in a liquid state. In this case, GDH dissolved in a buffer solution or the like can be used, and it is preferable to add sugar / sugar alcohols, amino acids, proteins, peptides, etc. as excipients or stabilizers. It can also be granulated after pulverization.

[0027] The composition of the buffer used for the GDH extraction, purification, pulverization, and stability test described above is not particularly limited, but preferably has a buffer capacity in the range of pH 5-8. For example, buffers such as boric acid, Tris hydrochloric acid, potassium phosphate, BES, Bicine, Bis-Tris, CHES, EPPS, HEPES, HEPPSO, MES, MOPS, M〇PS〇, PIPES, P〇 Good buffering agents such as PS0, TAPS, TAPS0, TES, Tricine are listed.

Of these, only one type may be applied, or two or more types may be used. Further, one or more composite compositions including those other than the above may be used.

The concentration of these additives is not particularly limited as long as it has a buffering capacity, but the preferable upper limit is 10 mM or less, more preferably 50 mM or less. A preferred lower limit is 5 mM or more.

The content of the buffering agent in the lyophilized product is not particularly limited, but is preferably 0.1% (weight ratio) or more, particularly preferably 0.:! To 30% (weight ratio). Used in the range of.

These can use various commercially available reagents. [0028] These buffers 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, etc. are not limited, and it is sufficient to function during measurement.

[0029] The improvement in stability as used in the present invention means that the residual rate (%) of the maintained GDH enzyme increases after heat treatment of the composition containing the GDH enzyme at a certain temperature for a certain period of time. Means. For example, in the present invention, when the activity is almost completely maintained, the sampnore stored at 4 ° C is regarded as 100%, and the activity value of the GDH solution after heat treatment at 55 ° C for 1 hour is compared with that of the enzyme. Survival rate is calculated. If this residual rate was increased compared to that without heating treatment, it was judged that the thermal stability of GDH was improved.

Judgment as to whether or not stability was improved was performed as follows.

In the activity measurement method described in the method for measuring GDH enzyme activity described below, the PQQGDH activity value (a) after 4 hours at 16 ° C and the GDH activity value (b) after heat treatment were measured. The relative value ((b) / (a) X 100) was calculated when a) was 100. This relative value was defined as the residual rate (%). Then, the presence or absence of the heating treatment was compared, and it was determined that the thermal stability was improved when the residual rate increased due to the execution.

It should be noted that the “heat treatment” in the above description refers to a treatment for confirming the stability of the enzyme and is different from the “warming treatment” of the present invention. The same applies to the explanations other than in this paragraph.

[0030] The heating treatment requires that at least 50% or more of the activity be maintained before and after the heating treatment, preferably 80% or more of the activity is maintained, and more preferably 90% or more. It is desirable that the activity of is maintained.

In this patent, the temperature at which large heat inactivation of the enzyme occurs is the absolute value of the slope of the approximate expression obtained from three or more consecutive data when the residual activity of the enzyme is examined at several different processing temperatures. This means the point where the temperature changes more than twice (processing temperature). For example, in Fig. 1, the absolute expression of the approximate expression at 0, 30, and 40 ° C is 0.23, and the inclination of the approximate expression at 40, 50, and 60 ° C is 0.233. The absolute value is 3.63. Therefore, under this heat treatment condition, 40 ° C is a temperature at which large thermal deactivation occurs. Alternatively, the temperature at which large heat inactivation of the enzyme in this patent occurs is the temperature at which the residual activity of the enzyme is 80% or less when heat-treated at several different temperatures.

In the present invention, among the temperatures obtained by the above two methods, the lower temperature is referred to as “temperature at which large heat inactivation of the enzyme occurs”. If it can only be determined by one method, the temperature determined by that method is defined as “the temperature at which a large heat inactivation of the enzyme occurs”.

[0031] The temperature at which this large heat inactivation occurs varies depending on the type of enzyme, and even for the same enzyme, it varies depending on the enzyme concentration used in the study. For example, in the PQQGD H variant used in the study of this patent, when the enzyme concentration was examined at 5 U / ml, the temperature at which large heat inactivation occurred was 40 ° C (Fig. 1), When examined at 20-30 U / ml, it was 55 ° C. Therefore, in the examination of the enzyme concentration of 20-30 U / ml, considering the safety side below this temperature, the heating treatment is examined at 50 ° C. or less. In addition, as a result of examining the heat treatment conditions, below the temperature at which large heat deactivation occurs, no large heat deactivation was observed even if the treatment time was extended, and 90% or more of the activity was maintained. It seemed to be most effective to treat for a long time at a temperature (about 5 ° C) slightly lower than the temperature at which large heat deactivation occurs.

[0032] The improvement of the holo-type ratio of the present invention is thought to lead to an increase in stability. The improvement of the holo-type ratio in the present invention means that the residual rate (%) of GDH enzyme is maintained after the composition containing GDH enzyme is heat-treated at a certain temperature for a certain time. Means. In the present invention, 100 sampnole stored at 4 ° C, in which the activity is almost completely maintained, is 100. / ₀ and to, by comparing the activity value of GDH solution after heat treatment certain time and this at a constant temperature, and calculates the residual rate of that enzyme. When this residual ratio was increased compared to that without the compound, it was judged that the holo ratio of GDH was improved.

[0033] The improvement in stability as used in the present invention means that the residual rate (%) of the maintained GDH enzyme increases after heat treatment of the composition containing the GDH enzyme at a certain temperature for a fixed time. Means. In the present invention, 100 sampnole stored at 4 ° C, in which the activity is almost completely maintained. / _0, by comparing the activity value of GDH solution after heat treatment predetermined time which is constant temperature, and calculates the residual rate of the enzyme. When this residual ratio was increased as compared with that without the compound, it was judged that the storage stability of GDH was improved.

[0034] Specifically, whether the holo-type ratio or stability is improved is determined as follows. went.

The GDH activity value (a) of the solution stored at 4 ° C and the GDH activity value after heat treatment at a constant temperature for a certain time (b) ) Was measured, and the relative value ((b) / (a) X 100) with respect to the measured value (a) as 100 was determined. This relative value was defined as the residual rate (%). Then, by comparing the presence or absence of the additive of the compound, it was determined that the stability was improved when the residual ratio increased by the addition.

[0035] Each GDH using different coenzymes is considering improvement of the holo-type ratio or stability under different conditions. For example, in PQQGDH, the enzyme solution prepared to 5 U / ml with a pH 6.5 buffer solution is heat-treated at 50 ° C for 16 hours, and then the remaining PQQGDH activity is compared to improve the holo ratio or stability. I have confirmed. At the same time, after NADGDH was heat-treated at 50 ° C for 1 hour with a pH 8.0 buffer solution at 85 U / ml, the remaining N ADGDH activity was compared to improve the holo ratio or stability. I have confirmed. In the same FADGDH, the enzyme solution prepared at 5 U / ml in a buffer solution of ρΗ6.5 was heat-treated at 50 ° C for 15-30 minutes, and then the remaining FADGDH activity was compared to determine the holo ratio or stability. The improvement is confirmed.

[0036] The determination as to whether the holo-type ratio is improved can also be made as follows.

The GDH activity value (a) measured by adding a sufficient amount of coenzyme to the activity measurement method described in the measurement method for GDH enzyme activity described below, and measured without adding any coenzyme. The GDH activity value (b) was measured, and the relative value ((b) Z (a) X 100) with respect to the measured value (a) as 100 was determined. This relative value was defined as a holo-formation rate (%). Then, by comparing the presence or absence of the addition of the compound, it was determined that the stability was improved when the holoformation rate was increased by the additive.

[0037] 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-dichlorophenol indophenol (DCPIP), PMS and nitro blue tetra Combinations with Zorium (NBT), DCPIP alone, ferricyanide ions (such as ferricyanium potassium as a compound) alone, and feucene alone. Of these, ferricyanide ions (such as potassium ferricyanide as the compound) are preferable. Each of these mediators has various differences in sensitivity, so it is not necessary to uniformly define the concentration of addition, but generally an additive of ImM or higher is desirable.

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 gnolecose sensor described later is prepared. In this case, the liquid state, the dry state, etc. may be used, and it may be dissociated during the reaction at the time of measurement so as to be in an ionic state.

[0038] In the present invention, various components can be allowed to coexist if necessary. For example, surfactants, stabilizers, excipients and the like may be added.

For example, it is possible to further stabilize PQQGDH by adding calcium ions or salts thereof, amino acids such as glutamic acid, gnoretamine, and lysine, and serum albumin.

For example, PQQGDH can be stabilized by containing calcium ions or calcium salts. Examples of the calcium salt include calcium chloride, calcium acetate, calcium salt of inorganic acid such as calcium citrate, and calcium salt of organic acid. In the aqueous composition, the calcium ion content is preferably 1 X 10-4-1 X 10-2M.

The stabilizing effect of PQQGDH by containing calcium ions or calcium salts can be further improved by containing an amino acid selected from the group consisting of gnoretamic acid, gnoletamine and lysine. The amino acid selected from the group consisting of glutamic acid, glutamine and lysine may be one type or two or more types. Furthermore, egg white albumin (OVA) may be added.

Or (1) one or more compounds selected from the group consisting of aspartic acid, glutamic acid, monoketoglutaric acid, malic acid, monoketognoreconic acid, _a- cyclodextrin and their salts, and (2 ) PQQGDH can be stabilized by coexisting albumin.

[0039] By the way, PQQGDH as an enzyme is a glucose dehydrogenase having pyroguchi quinoline quinone (PQQ) as a coenzyme. Since it catalyzes the reaction that oxidizes glucose to produce darconoratone, it can be used for blood glucose measurement. Blood glucose level is diabetes It is an extremely important index for clinical diagnosis as an important marker of disease. Currently, blood glucose levels are measured mainly using a biosensor that uses glucose oxidase, but the reaction is affected by the dissolved oxygen concentration, which may cause errors in the measured values. was there. PQQ-dependent glucose dehydrogenase is attracting attention as a new enzyme that replaces this glucose oxidase.

[0040] Our group found that Acinetobacter baumannii NCIMB11517 strain PQQ-dependent gnolecose dehydrogenase was produced, and constructed a gene cloning and expression system (see Japanese Patent Application Laid-Open No. 11 243949) . However, further improvement in the productivity of PQQGDH has been desired for industrial production. The present invention has been made against the background of the problems of the prior art, and also relates to the improvement of PQQGDH as an issue.

[0041] The inventors of the present invention have made extensive studies in order to solve the above-mentioned problems. As a result, the aqueous solution containing the PQQ-dependent dalcoyl dehydrogenase is subjected to heat treatment, so that the holo in the total GDH enzyme protein during production can be obtained. The proportion of type PQQGDH could be improved and the present invention was finally completed.

[0042] Improvement in productivity as a holo-type PQQGDH according to the present invention results in a reduction in manufacturing cost. Furthermore, the increase in the proportion of holo-type PQQGDH eliminates the need to add a PQQ to form holo to obtain active PQQGDH, resulting in a reduction in manufacturing costs. These make it possible to manufacture PQQGDH inexpensively. Furthermore, it is also possible to provide a dull course assembly kit and a gnore course sensor at a low price.

[0043] Further, since the activity value per unit protein weight of PQQGDH is improved by increasing the proportion of holo-type PQQGDH, it is possible to reduce the amount of protein added to the glucose assembly kit and glucose sensor.

[0044] PQQGDH used in the present invention is an enzyme that catalyzes the reaction when P-Q quinoline quinone is coordinated as a coenzyme to oxidize D-darcose to produce D-darcono 1,5 ratatones. (ECl. 1. 5. 2 (formerly ECl. 1. 99. 17)), and the origin and structure are not particularly limited.

At this time, cloning of PQQGDH is Seticus (Acinetobacter calcoaceticus) LMD79. 41 (AM Cleton—Jans en et al., J. Bacteriol., 170, 2121 (1988) and Mol. Gen. Genet., 217, 430 (1989)), Escherichia coli ) (AM Cleton—Jansen et al., J. Bacteriol., 172, 6308 (1990)), Gnoreconactor oxydans (Mol. Gen. Genet., 229, 206 (1991)) And Acinetobacter baumanni NCIMB11517 reported in Patent Document 1. However, it is difficult to modify the membrane enzyme present in Escherichia coli etc. to make it soluble, and as a source, select soluble PQQGDH such as Acinetopacter 'Calcoaceticus or Acinetobacter baumannii I prefer that.

Acinetobacter baumannii NCIMB11517 strain was categorized as ij, Acinetobacter calcoaceticus.

[0045] The amino acid sequence of PQQGDH derived from the genus Acinetobacter is preferably the AQ / DH of PQQu ^ DH derived from Acinetob acter calcoaceticus or AcinetoDacter baumanmi. Among them, SEQ ID NO: 1 is preferable. The wild-type PQ QGDH protein represented by SEQ ID NO: 1 and its base sequence represented by SEQ ID NO: 2 originated from Acinetobacter baumannii strain NCIMB 11517, and Japanese Patent Application Laid-Open No. 11-243949 Is disclosed. In the above and SEQ ID NO: 1, the amino acid notation is numbered with 1 for aspartic acid from which the signal sequence has been removed.

[0046] For example, when the amino acid sequence of SEQ ID NO: 1 is compared with the amino acid sequence of the enzyme derived from Acine tobacter calcoaceticus LMD79.41, the difference is slight and the homology is 92.3. % (Including the signal sequence), which are very similar, it is easy to recognize which amino acid residue of an enzyme of other origin corresponds to a residue in SEQ ID NO: 1. These PQQGDHs are also preferred as PQQGDHs used in the present invention.

[0047] As long as PQQGDH used in the present invention has glucose dehydrogenase activity, a part of another amino acid residue may be deleted or substituted. Residues may be added.

[0048] The produced DNA having the genetic information of the modified protein is transferred into a host microorganism in a state of being linked to a plasmid, and becomes a transformant that produces the modified protein. As the plasmid at this time, for example, pBluescript, pUC18 and the like can be used when Escherichia coli is used as a host microorganism. Examples of host microorganisms that can be used include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, and Escherichia coli DH5. As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism belongs to the genus Escherichia, a method of transferring the recombinant DNA in the presence of calcium ions can be employed. Further, an elect port position method may be used. Furthermore, a commercially available combi- tive cell (for example, combitent high JM109; manufactured by Toyobo) may be used.

[0049] Such a gene can be extracted from these strains or chemically synthesized. Furthermore, a DNA fragment containing the PQQGDH gene can be obtained by using the PCR method.

[0050] In the present invention, methods for obtaining a gene encoding PQQGDH include the following methods. For example, after isolating and purifying the chromosome of Acinetobacter calcoaceticus NCIMB11517, DNA was cleaved using sonication, restriction enzyme treatment, etc., linear expression vector and blunt ends or attachment of both DNAs A recombinant vector is constructed by closing and ligating DNA ends with DNA ligase. Recombinant vector containing a gene encoding GDH having PQQ as a prosthetic group after transferring the recombinant vector into a replicable host microorganism and screening using the expression of the vector marker and enzyme activity as an indicator To obtain microorganisms that retain

[0051] In the next step, the microorganism carrying the recombinant vector is cultured, the recombinant vector is isolated and purified from the cells of the cultured microorganism, and a gene encoding GDH is obtained from the expression vector. Can be collected. For example, the chromosomal DNA of the gene donor Acinetopacter 'calcoaceticus NCIMB11517 is specifically collected as follows.

[0052] The gene-donating microorganism is centrifuged, for example:! ~ For 3 days with stirring. The lysate containing GDH gene having PQQ as a prosthetic group can be prepared by collecting the bacterium and then lysing it. As a method for lysis, for example, treatment is performed with a lytic enzyme such as lysozyme, and a protease or other enzyme or a surfactant such as sodium lauryl sulfate (SDS) is used in combination as necessary. Furthermore, it may be combined with a physical crushing method such as freeze-thawing or French press treatment.

[0053] In order to separate and purify DNA from the lysate obtained as described above, a method such as deproteinization by phenol treatment or protease treatment, ribonuclease treatment, alcohol precipitation treatment or the like is appropriately performed according to a conventional method. It can be done by combining.

[0054] A method for cleaving DNA separated and purified from a microorganism can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. Type II restriction enzymes that act on specific nucleotide sequences are suitable.

[0055] As a vector for cloning, a vector constructed for gene recombination from a phage or plasmid capable of autonomously growing in a host microorganism is suitable. Examples of the fage include Lambda gtlO and Lambda gtl l when Escherichia coli is used as a host microorganism. Examples of plasmids include pBR322, pUC19, and pBluescript when Escherichia coli is used as a host microorganism.

[0056] During cloning, a vector fragment can be obtained by cleaving the above-described vector with the restriction enzyme used for cleaving the microbial DNA that is the gene donor encoding GDH described above. It is not necessary to use the same restriction enzyme as that used to cleave the microbial DNA. The microbial DNA fragment and the vector DNA fragment may be combined by any known DNA ligase method. For example, after annealing the attachment end of the microbial DNA fragment and the attachment end of the vector fragment, an appropriate DNA ligase may be used. Use to create a recombinant vector of microbial and vector DNA fragments. If necessary, after annealing, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.

[0057] The host microorganism used for cloning is not particularly limited as long as the recombinant vector is stable, can autonomously proliferate, and can express a foreign gene. In general, Escherichia coli W3110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH5, etc. can be used.

[0058] As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method using calcium treatment, an electo-baudation method, or the like can be used.

[0059] The microorganism, which is a transformant obtained as described above, can stably produce a large amount of GDH by being cultured in a nutrient medium. The selection of whether or not the target recombinant vector is transferred to the host microorganism may be performed by searching for a microorganism that simultaneously expresses GDH activity by adding PQQ and the drug resistance marker of the vector holding the target DNA. For example, a microorganism that grows on a selective medium based on a drug resistance marker and produces GDH may be selected.

[0060] The base sequence of the GDH gene having PQQ obtained by the above method as a prosthetic group was decoded by the dideoxy method described in Science, 214, 1205 (1981). The amino acid sequence of GDH was estimated from the base sequence determined as described above.

[0061] As described above, transfer from a recombinant vector carrying a GDH gene having PQQ selected once as a prosthetic group to a recombinant vector capable of replicating in a microorganism capable of producing PQQ is as follows. It can be easily carried out by collecting DNA, which is a GDH gene, from a recombinant vector carrying the GDH gene by restriction enzymes or PCR and linking it to other vector fragments. In addition, the transformation of microorganisms capable of producing PQQ with these vectors can be carried out by using the calcium treatment, the electoral cell method, the electopore method, or the like.

[0062] Examples of microorganisms capable of producing PQQ include methanol-utilizing bacteria such as Methylobacterium, acetic acid bacteria belonging to the genus Acetobacter and Gluconobacter, Flavobacterium (Flavobacterium) And bacteria of the genus Genus, Syudomonas, and Acinetopacter. Among them, a host-vector system that can use a genus Pseudomonas and a bacterium belonging to the genus Acinetopacter has been established and is preferable because it is easy to use. [0063] In the genus Pseudomonas, Pseudomonas' Elginosa, Pseudomonas' fluorescens, Pseudomonas' Putida and the like can be used. For bacteria belonging to the genus Acinetopacter, Acinetobacter 1 'Calcoaceticus, Acinetobacter 1' Baumannii and the like can be used.

[0064] As a recombinant vector that can be replicated in the above microorganism, a vector derived from RSF1010 or a vector having a similar replicon can be used for bacteria belonging to the genus Pseudomonas. For example, ρΚΤ240, ρΜΜΒ24, etc. (MM Bagdasarian et al., Gene, 26, 273 (1983)), pCN40, pCN60 etc. (CC Nieto et al., Gene, 87, 145 (1990)) and pTS 1137 (gene recombination practical technology) Technology, Vol. 4, p73-85, 1983, published by Silence Forum, Inc.). Moreover, pME290 etc. (Y. Itoh et al., Gene, 36, 27 (1985)), pNIll, pNI20C (N. Itoh et al., J. Biochem., 110, 614 (1991)) can also be used.

[0065] In bacteria belonging to the genus Acinetopacter, pWM43 etc. (W. Minas et al., Appl. Environ. Mic robiol., 59, 2807 (1993)), pKT230, pWH1266 etc. (Μ. Hunger et al., Gene, 87, 45 (1990) ) Can be used as a vector.

[0066] Microorganisms, which are transformants thus obtained, can be produced as a whole P QQGDH protein by cultivating them in a nutrient medium, 力 the ability to produce QQGDH protein S, and adding an organic solvent to the medium. Both productivity improvement and productivity improvement as holo-type PQQGDH protein are possible. For the culture form of the host microorganism, which is a transformant, the culture conditions should be selected in consideration of the nutritional physiological properties of the host. Industrially, aeration and agitation culture is advantageous.

[0067] In the present invention, the holo-type PQQGDH refers to the PQQGDH enzyme itself in which the coenzyme PQQ is bound to the GDH protein and the state thereof, and the enzyme having the GDH activity without the addition of PQQ and the state thereof. It is. On the other hand, all PQQGDH means (1) holo-type PQQGDH, (2) apo-type PQQGDH in which coenzyme PQQ is not bound to GDH protein, and (3) the binding state of PQQGDH that is further bound by PQQ This refers to the total GDH enzyme protein combined with PQ QGDH, which does not have GDH activity because it is complete, and its state.

[0068] In the claims of the present invention, "PQQ-dependent gnolecose dehydrogenase" or “PQQGDH” is a single protein, but as an enzyme co-enzyme complex, it is a mixture containing at least one of the above (1) to (3) when viewed from the binding state of PQ Q. means.

[0069] As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used.

Any carbon compound that can be assimilated may be used as the carbon source. For example, gnolecose, sucrose, ratatoses, maltose, ratatoses, molasses, pyruvic acid and the like are used. The nitrogen source may be any available nitrogen compound. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc and other salts, specific amino acids, specific vitamins and the like are used as necessary.

[0070] The culture temperature is a force that can be appropriately changed within the range in which the bacteria grow and produce PQQGDH. In the case of the microorganism having the PQQ production ability as described above, preferably 20 to 42 ° C, more preferably 30 to 37 ° C. About C. The culture time is slightly different depending on the conditions. It is usually about 6 to 48 hours, preferably about 16 to 36 hours if the culture is completed at an appropriate time in consideration of the time when the maximum yield of PQQGDH is reached. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce PQQGDH, but is preferably in the range of about ρΗ6.0 to 9.0, more preferably in the range of ρΗ6.5 to 8.0.

[0071] A culture solution containing cells that produce PQQGDH in the culture can be collected and used as is, but generally, if PQQGDH is present in the culture solution, it is filtered and centrifuged according to conventional methods. It is used after separating the PQQGDH-containing solution and microbial cells by the above. When PQQGDH is present in the microbial cells, the microbial cells are collected from the obtained culture by means of filtration or centrifugation, and then the microbial cells are collected by a mechanical method or an enzymatic method such as lysozyme. Destroy it, and if necessary, add a chelating agent such as EDTA and a surfactant to solubilize GDH and separate and collect it as an aqueous solution.

[0072] The PQQGDH-containing solution obtained as described above is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment such as ammonium sulfate or sodium sulfate, or hydrophilic organic solvents such as methanol, ethanol, acetone, etc. It may be precipitated by the fractional precipitation method. Heat treatment and isoelectric point treatment are also effective purification means. Also, adsorbent or gel Purified PQQGDH can be obtained by gel filtration using a filter medium, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0073] For example, gel filtration using Sephadex gel (Farmasia Biotech), DEAE Sepharose CL-6B (Farmasia Biotech), Octyl Sepharose CL-6B (Farmasia Biotech), etc. The purified enzyme preparation can be obtained by separation and purification by column chromatography. The purified enzyme preparation is preferably purified to such an extent that it shows a single band on electrophoresis (SDS-PAGE).

[0074] The heat treatment described above plays a role of improving the ratio of holo-type PQQGDH to the total GDH enzyme protein. This treatment that improves PQQGDH activity without adding PQQ from the outside is extremely useful for industrial applications. This phenomenon is presumed that GDH enzyme protein strength S, which is inactive due to imperfect binding state of GQ enzyme protein, but that binds to PQQ, is confirmed by heat treatment. Yong's change is considered to have improved the binding state with PQQ and became active. Thus, it was surprising and surprising that there may be GDH enzyme proteins that bind to PQQ but are inactive due to imperfect binding. The heat treatment conditions are preferably 25 ° C to 50 ° C, more preferably 30 ° C to 45 ° C.

[0075] This heat treatment is not only useful for production using a host producing PQQ as described above. It is also useful when preparing a holo-type PQQGDH by holizing apo-type GDH protein. For example, after cloning the GDH gene, the apo-type GDH protein is expressed directly using Escherichia coli DH5, purified in the same manner as PQQGDH, and then the purified apo-type GDH protein is holoed to prepare the holo-type PQQGDH And so on. Because normally, when apo-type GDH protein is made into a holo, the power to obtain a holo-type PQQGDH by adding a PQQ solution to the apo-type GDH protein solution and mixing it, PQQ as described above in this state It is very likely that there are many GDH enzyme proteins that are inactive due to imperfect binding. Even in such a case, holoformation by heat treatment is extremely useful, The effect is high.

[0076] The purified enzyme obtained as described above can be pulverized and distributed, for example, by freeze drying, vacuum drying, spray drying, or the like. In this case, the purified enzyme can be dissolved in phosphate buffer, Tris-HCl buffer or GOOD buffer. Preferred are GOOD buffers, with PIPES, MES or MOPS buffers being particularly preferred. Furthermore, PQQGDH can be further stabilized by adding calcium ions or salts thereof, amino acids such as gnoretamine, gnoretamine, and lysine, and serum albumin.

[0077] The PQQGDH protein can take various forms such as liquid (aqueous solution, suspension, etc.), powder, and lyophilized. The freeze-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 lyophilized product is redissolved. Further, when measuring the genole course, various forms such as a glucose assembly kit and a glucose sensor can be taken. The purified modified protein thus obtained can be stabilized by the following method.

[0078] The purified modified protein includes (1) one or more selected from the group consisting of aspartic acid, glutamic acid, α-ketoglutanolic acid, malic acid, ketogluconic acid, α-cyclodextrin, and salts thereof By coexisting the above compound and (2) albumin, the modified protein can be further stabilized.

[0079] In the lyophilized composition, the PQQGDH content varies depending on the origin of the enzyme, but is usually suitably used in the range of about 5 to 50% (weight ratio). In terms of enzyme activity, it is preferably used in the range of 100 to 2000 U / mg.

[0080] Salts of aspartic acid, gnoretamic acid, α-ketoglutaric acid, malic acid, and hyketogluconic acid include salts such as sodium, potassium, ammonium, calcium, and magnesium, but are not particularly limited. . The addition amount of the above compound, its salt and dicyclodextrin is preferably 1 to 90% (weight ratio). These substances can be used alone or in combination.

[0081] The buffer solution to be contained is not particularly limited, but Tris buffer solution, phosphate buffer Examples include impulse solution, borate buffer solution, and GOOD buffer solution. The pH of the buffer solution is adjusted in accordance with the intended use within a range of about 5.0.90. The content of the buffer in the lyophilized product is not particularly limited, but is preferably 0.1% (weight ratio) or more, particularly preferably 0. 30% (weight ratio). Used in range.

[0082] Examples of albumin that can be used include bovine serum albumin (BSA) and ovalbumin (OVA). BSA is particularly preferable. The albumin content is preferably in the range of 180% (weight ratio), more preferably 570% (weight ratio).

[0083] Further stabilizers and the like may be added to the composition in a range that does not particularly adversely affect the reaction of PQQGDH. The blending method of the stabilizer of the present invention is not particularly limited. For example, a method in which a stabilizer is added to a buffer solution containing PQQGDH, a method in which PQQGDH is added to a buffer solution containing a stabilizer, or a method in which PQQGDH and a stabilizer are simultaneously added to a buffer solution are included.

[0084] A stabilizing effect can also be obtained by adding calcium ions. That is, the modified protein can be stabilized by containing calcium ions or calcium salts. Examples of the calcium salt include calcium chloride, calcium acetate, calcium salt of inorganic acid such as calcium citrate, and calcium salt of organic acid. Further, in an aqueous composition, the content of calcium ions is preferably ^{1 X 10 1 X 10_ 2 M} .

[0085] The stabilizing effect 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.

[0086] The amino acid selected from the group consisting of gnoretamic acid, glutamine and lysine may be one kind or two or more kinds. In the aqueous composition, the content of an amino acid selected from the group consisting of gnoretamic acid, gnoletamine and lysine is preferably 0.01 to 0.2% by weight.

[0087] Further, serum albumin may be added. When serum albumin is added to the aqueous composition, the content is preferably 0.05 to 0.5% by weight.

[0088] As the buffering agent, a normal one is used, and the buffer having a pH of 5 to 10 is usually used. I like it. Specifically, Tris-HCl, boric acid, and the power for which Good's buffer is used Any buffer that does not form an insoluble salt with calcium can be used.

[0089] If necessary, other components such as a surfactant, a stabilizer, and an excipient may be added to the aqueous composition.

[0090] In addition, as exemplified above, the present invention includes a step of heating the composition in a composition containing a soluble coenzyme-bound type of gnolecose dehydrogenase.

This is a method for producing a composition containing soluble coenzyme-linked gnolecose dehydrogenase with improved thermostability.

[0091] Further, the present invention provides a composition comprising a soluble coenzyme-bound glucose dehydrogenase produced by the method described above, wherein a soluble coenzyme-bound glucose dehydrogenase having improved thermal stability is obtained. It is a composition to contain.

[0092] In the present invention, gnole course can be measured by the following various methods.

The reagent for measuring gnolecose, the gnolecose assembly kit, and the gnolecose sensor of the present invention are various forms such as liquid (aqueous solution, suspension, etc.), powdered by vacuum drying or spray drying, freeze drying, etc. Can be taken. 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, but may be a solution in which the dried product is redissolved.

In the present invention, gnole course can be measured by the following various methods.

[0093] Glucose measuring reagent

The reagent for measuring gnolecose of the present invention typically includes reagents necessary for measurement such as GDH, buffer solution, mediator, glucose standard solution for preparing a calibration curve, and usage guidelines. The kit of the present 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 the form of a holo, but it may be provided in the form of an apoenzyme and holo- ed at the time of use.

[0094] Glucose Atsy Kit

The invention also features a glucose assembly kit comprising GDH according to the invention. The glucose assay kit of the present invention is a GDH according to the present invention is converted into at least one assay. Contains enough. Typically, the kit contains the GDH of the present invention plus the buffers necessary for assembly, mediators, glucose standard solutions for creating calibration curves, and usage guidelines. 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 the form of a holo, but it can also be provided in the form of an apoenzyme and hololated at the time of use.

[0095] Glucose sensor

The invention also features a glucose sensor using GDH according to the invention. As an 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. Immobilization methods include 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, an oxidation-reduction polymer, or the like in the polymer together with a mediator. These can be adsorbed and fixed on the electrode, or a combination of these may be used. Preferably, the GDH of the present invention may be immobilized in the form of apoenzyme in the form of a holoform and immobilized on the electrode, and the coenzyme may be supplied as a separate layer or in solution. Typically, the GDH of the present invention is immobilized on a carbon electrode using dartalaldehyde, and then treated with a reagent having an amine group to block dartalaldehyde.

[0096] The glucose concentration can be measured as follows. Put the buffer in the thermostatic cell and add the mediator to maintain a constant temperature. As the working electrode, an electrode on which the GDH of the present invention is immobilized is used, and a counter electrode (for example, platinum electrode) and a reference electrode (for example, Ag / Ag C1 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. It is possible to calculate the glucose concentration in the sample according to the calibration curve prepared with the standard concentration glucose solution.

Note that the present invention has the following configuration from another viewpoint.

[1] A method for improving the stability of the enzyme, comprising a step of subjecting the composition to a heating treatment, wherein the composition comprises a soluble coenzyme-linked glucose dehydrogenase.

[2] The stability according to [1], wherein the coenzyme is pyrroloquinone quinoline or a flavin compound. How to improve.

[3] The method for improving stability according to [1], [2], wherein the heating treatment temperature is not higher than a temperature at which a temperature at which a large thermal inactivation of the enzyme occurs.

[4] A soluble complement with improved thermal stability produced by a method comprising a step of subjecting a composition comprising a soluble coenzyme-linked glucose dehydrogenase to a heating treatment. Composition comprising enzyme-linked glucose dehydrogenase

[5] A method for measuring glucose concentration using the composition according to [4].

[6] A glucose sensor comprising the composition according to [5].

[7] Soluble coenzyme-bound glucose dehydrogenase with improved stability, comprising a step of subjecting the composition to soluble coenzyme-bound glucose dehydrogenase to a heating treatment. A method for producing a composition comprising:

Furthermore, the present invention has the following configuration from another viewpoint.

[1] A method comprising a composition containing a soluble coenzyme-bound glucose dehydrogenase, wherein a compound having a carbocinole group or an amino group is added to the composition to improve the mouth ratio of the enzyme.

[2] The method for improving the holo-type ratio according to [1], wherein the coenzyme is pyrroloquinone quinoline or a flavin compound.

[3] The method for improving the holo-type ratio according to [1] or [2], wherein the compound to be added is succinic acid, malonic acid, or salt ammonium.

[4] A composition comprising a soluble coenzyme-bound dalcoose dehydrogenase having an improved holo-type ratio by the method of [1] to [3].

[5] Improve the powder stability of the enzyme by adding a compound having a carbocinole group or an amino group to the composition containing a soluble coenzyme-bound glucose dehydrogenase. Method.

[6] The method for improving stability according to [4], wherein the coenzyme is pyrroloquinone quinoline or a flavin compound.

[7] The method for improving stability according to [4] or [5], wherein the compound to be added is succinic acid, malonic acid, or salt ammonium. [8] A composition comprising a soluble coenzyme-bound darcos dehydrogenase improved in thermal stability by the method of [5] to [7].

[9] A method for measuring glucose concentration using the composition according to [4] or [8].

[10] A glucose sensor comprising the composition according to [ ⁹ ].

[0099] Further, another aspect of the present invention is configured as follows.

[1] A method for improving the ratio of holo-type PQQGDH to total GDH enzyme protein, characterized by heat-treating an aqueous solution containing PQQ-dependent glucose dehydrogenase

[2] A method for producing a PQQ-dependent glucose dehydrogenase, comprising heat-treating an aqueous solution containing the PQQ-dependent glucose dehydrogenase

[3] The aqueous solution containing PQQ-dependent glucose dehydrogenase is heated, and the PQQ-dependent gnolecose dehydrogenase having an improved ratio of holo-type PQQGDH to the total GDH enzyme protein according to [2] How to manufacture

[4] An aqueous solution containing a PQQ-dependent glucose dehydrogenase is subjected to a heat treatment, and the ratio of the holo-type PQQ-dependent glucose dehydrogenase described in [2] is 90% or more. How to make

[5] PQQ-dependent glucose dehydrogenase produced by the method according to [2]

[6] Gnore course assembly kit containing PQQ-dependent glucose dehydrogenase produced by the method according to [2]

[7] A glucose sensor comprising a PQQ-dependent glucose dehydrogenase produced by the method according to [2].

[8] A method for measuring glucose containing a PQQ-dependent glucose dehydrogenase produced by the method according to [2].

Example

[0100] Hereinafter, the present invention will be described in more detail based on examples.

Example 1: Construction of an expression plasmid for the PQQ-dependent gnolecose dehydrogenase gene Wild-type PQQ-dependent glucose dehydrogenase expression plasmid PNPG5 is expressed in the multiple cloning site of the vector pBluescript SK (-). Acinetobacter baumannii) A structural gene encoding PQQ-dependent glucose dehydrogenase derived from NCIMB 1 1517 strain. 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.

PNPG5 DNA (5 μg) was cleaved with restriction enzymes BamHI and Xhol (Toyobo Co., Ltd.), and the structural gene part of mutant PQQ-dependent glucose dehydrogenase was isolated. The isolated DNA and pTM33 (1 μg) cleaved with BamHI and Xhol were reacted with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA. The ligated DNA was transformed using a competent cell of Escherichia coli DH5a. The resulting expression plasmid was named pNPG6.

[0101] Example 2: Preparation of transformants of Pseudomonas bacteria

Pseudomonas' Putida TE3493 (Microeken No. 12298) was cultured in LBG medium (LB medium + 0.3% glycerol) at 30 ° C for 16 hours, and the cells were collected by centrifugation (12,000 rpm, 10 minutes) Then, 8 ml of 5 mM K-phosphate buffer (PH7.0) containing 300 mM sucrose cooled on ice was added to the cells to suspend the cells. The cells are collected again by centrifugation (12,000 rpm, 10 minutes), and 0.4 ml of 5 mM K-phosphate buffer (ρΗ7.0) containing 300 mM sucrose ice-cooled is added to the cells. The body was suspended.

The suspension was transformed with the expression plasmid PNPG6 obtained in Example 1 by the electrovolution method. The desired transformant was obtained from a colony grown on LB agar medium containing 100 μg Zml of streptomycin.

[0102] Example 3: Preparation of PQQ-dependent GDH preparation

Dispense 500 ml of Terrific broth into a 2 L volumetric flask. C, autoclaved for 20 minutes, and after standing to cool, separately sterile filtered streptomycin was added to 100 μg / ml. This medium was inoculated with 5 ml of Pseudomonas' Putida TE3493 (pNPG6) culture medium previously cultured for 24 hours at 30 ° C in PY medium containing 100 μg / ml streptomycin and cultured at 30 ° C for 40 hours with aeration and agitation. . The PQQ-dependent glucose dehydrogenase activity at the end of the culture is about 30 U / ml per 1 ml of the culture solution, as measured by the above activity measurement. The cells are 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. It was. The obtained crude enzyme solution was separated and purified by HiTrap_SP (Amersham-Falmasia) ion exchange column chromatography. Next, after dialyzing with 10 mM PIPES_Na 0 H buffer (pH 6.5), calcium chloride was added to a final concentration of mM. Finally, it was separated and purified by HiTrap-DEAE (Amersham-Falmacia) ion exchange column chromatography to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band on SDS-PAGE.

The purified enzyme thus obtained was used as a PQQ-dependent GLD evaluation sample. 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—Dalcono 1, 5—Lataton + PMS (red)

PMS (red) + DCPIP → PMS + DCPIP (red)

The presence of DCPIP (red) formed by the reduction of 2,6-dichlorophenol-indophenol (DCPIP) by phenazine methosulfate (PMS) (red) was measured spectrophotometrically at 600 nm. In addition, in the examination of substrate specificity, the part of D-gnolecose was changed to other saccharides, 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.

Method

Reagent

A. D-glucose solution: 1. OM (l. 8g D-glucose (molecular weight 180. 16) / l0ml H20)

B. PIPES_NaOH buffer solution, pH 6.5: 50 mM (1.51 g PIPES (molecular weight 302. 36) suspended in 60 mL water was dissolved in 5 N NaOH, and 2 ml 10% Triton X— 1 Get 00 power Q. 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: 24mM (73.52mg phenazine methosulfate (molecular weight 817.65) / l0mlH2O)

D. DCPIP solution: 2.0mM (6.5mg of nitrotetrazolium blue (molecular weight 817.65) / l0mlH2O)

E. Enzyme Diluent: ImM CaC12, 0.1% Triton X—100, 0.1 mM BSA in 50 mM PIPES—NaOH buffer (ρΗ6 · 5) Procedure

1. Prepare the following reaction mixture in a light-proof bottle and store on ice (prepared at the time of use)

4. 5ml D-glucose solution (A)

21. 9ml PIPES—NaOH buffer (ρΗ6 · 5) (Β)

2. 0ml PMS solution (C)

1. 0ml DCPIP solution (D) The concentration of the above mixture in the reaction solution is as follows.

PIPES buffer 36 mM

D—glucose 148 mM

PMS 1. 58mM

DCPIP 0. 066mM

2. 3. 0 ml of the reaction mixture was placed in a test tube (plastic) and pre-warmed at 37 ° C for 5 minutes.

3. 0.1 ml of enzyme solution was added and mixed by gentle inversion.

4. Record the decrease in absorbance at 600 nm for water at 37 ° C for 4-5 minutes with a spectrophotometer and calculate AOD per minute from the initial linear portion of the curve (OD test) At the same time, the same method was repeated except that the enzyme dilution solution (E) was added instead of the enzyme solution, and a blank (ΔD blank) was measured.

The enzyme powder was dissolved in ice-cooled enzyme diluent (E) immediately before the assembly and diluted to 0.05-0.10 U / ml with the same buffer (use a plastic tube for the adhesion of the enzyme). Is preferred).

For the purpose of evaluating substrate specificity, the above activity measurement procedure was carried out using another type of sugar solution as a substrate instead of the glucose solution.

Calculation

Activity is calculated using the following formula:

U / ml = {A〇D / min (A〇D test one AOD blank) XVtXdf} / (16.8X1. OXVs)

Vt: Total volume (3. lml)

Vs: Sample volume (0 · lml)

16.8: Millimolecular extinction coefficient of DCPIP under the above measurement conditions (cm2 / micromol) 1.0: Optical path length (cm)

df: dilution factor

C: Enzyme concentration in solution (c mg / ml)

Test example 2: NAD-dependent GDH activity measurement method

In the present invention, NAD-dependent GDH activity is measured under the following conditions. In addition, Toyobo's glucose dehydrogenase (GLD311) was used as the NAD-dependent GDH enzyme preparation.

Measurement principle

The amount of D-gnolecose + NAD + → D-gnolecono_1,5-latataton + NADH + H + NADH was measured by the change in absorbance at 340 nm.

Unit definition

One unit is the amount of NADGDH enzyme that forms 1.0 micromolar NADH per minute under the conditions described below. Method

Reagent

A. D-glucose solution: 1.5M (2.7g D-glucose (molecular weight 180.16) / l0ml H20)

B. Tris_HCl buffer, pH 8.0: 100 mM (1.2 g of tris (hydroxymethyl) aminomethane (molecular weight 121.14) suspended in 90 mL of water, pH 5 at 25 ° C with 5N HC1. (Adjusted to 0 ± 0.05, freed from water to make 100ml.)

C. NAD solution: 8% (80mg NAD (molecular weight 717.48) / lmlH20)

D. Enzyme diluent: Reagent B was used as it was. Steps

0. 9ml D-glucose solution (A)

7. 8ml Tris—HC1 buffer (ρΗ8 · 0) (Β)

0. 3ml NAD solution (C) The concentration of the above mixture in the reaction solution is as follows.

D—glucose 148 mM

Tris _HC1 buffer 77 mM

NAD 0. 26%

2. 3. Oml of reaction mixture was placed in a test tube (plastic) and pre-warmed at 37 ° C for 5 minutes.

3. Add 0.05 ml enzyme solution and mix by gentle inversion.

4. Record the change in absorbance for water at 340 nm at 37 ° C for 4 to 5 minutes with a spectrophotometer, and calculate AOD per minute from the initial linear part of the curve (OD test) At the same time, the same method is repeated except that enzyme diluent (D) is added instead of enzyme solution. The blank (ΔOD blank) was measured.

The enzyme powder was dissolved in ice-cold enzyme diluent (D) immediately before the assembly and diluted to 0.10-0.70 U / ml with the same buffer (use a plastic tube for the adhesion of the enzyme). Is preferred).

For the purpose of evaluating substrate specificity, the above activity measurement procedure was carried out using another type of sugar solution as a substrate instead of the glucose solution. Calculation

Activity is calculated using the following formula:

U / ml = {A〇D / min (A〇D test one Δ〇D blank) X Vt X df} / (6.22 X 1. O X Vs)

Vt: Total volume (3.05ml)

Vs: Sample volume (0 · 05ml)

6. 22: NADH millimolar extinction coefficient (cm2 / micromol)

1. 0: Optical path length (cm)

df: dilution factor

C: Enzyme concentration in solution (c mg / ml)

Example 4: Preparation of FAD-dependent GDH preparation

Aspergillus terreus subspecies and Penicillium as FAD-dependent GDH producing bacteria

Using lilacinoechinulatum NBRC6231 (purchased from National Institute for Product Evaluation and Technology), each L dry specimen was inoculated into potato dextrose agar medium (manufactured by Difco) and incubated at 25 ° C. The mycelium on the restored plate was collected together with the agar and suspended in filter sterilized water. Production medium (1% malt extract, 1.5% soy peptide, 0.1% MgS04'7 hydrate, 2./.Dalcos, pH6.5) in two 10L jar mentors After the autoclave sterilization at 120 ° C. for 15 minutes, the above mycelial suspensions were respectively added to start the culture. The culture conditions were a temperature of 30 ° C, an aeration rate of 2 L / min, and a stirring rate of 3 80 i "pm. After 64 hours from the start of the culture, the culture was stopped and a Nutsche filter was used. The cells of each strain were collected on the filter paper by suction filtration. Concentrate 5 L of the culture solution to 1/10 volume with a hollow fiber module for ultrafiltration with a molecular weight of 10,000,000 cut, and add ammonium sulfate to the concentrate to a final concentration of 60% saturation (456 g / U). Next, after centrifuging at 8000 rpm for 5 minutes in a Hitachi high-speed cooling centrifuge to precipitate the residue, the supernatant was adsorbed on an Octyl_Sepharose column and eluted with a gradient of ammonium sulfate from 0.6 to 0.0 saturation. The fraction with GDH activity was collected, and the resulting GDH solution was subjected to gel filtration on a G-25 sepharose column and the protein fraction was collected for desalting. Ammonium sulfate equivalent to 6 saturation was added and dissolved, and this was adsorbed on a Phenyl- Sepharose column, and the fraction with GDH activity was recovered by gradient elution at an ammonium sulfate concentration of 0.6 to 0.0 saturation. Furthermore, the obtained GDH solution was added to G-25 The protein fraction by gel filtration was recovered by Sukaramu, using purified enzyme obtained as FA D dependent GLD evaluation standard.

[0106] 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 (abbreviation). DCPIP), ferrocene or their derivatives (eg potassium ferricyanide, phenazine methosulfate, etc.). These mediators are commercially available.

[0107] 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.

50 mM PIPES buffer pH 6.5 (including 0.1% TritonX_ 100)

14mM 2,6-dichlorophenol indophenol (DCPIP) solution

1M D_glucose solution

Mix the above PIPES buffer (15.8 ml), DCPIP solution (0.2 ml) and D-glucose solution (4 ml) to make the reaction reagent.

[0108] <Measurement conditions>

Pre-warm the reaction reagent 2. 9 ml at 37 ° C for 5 minutes. Add 0.1 ml of GDH solution and gently After mixing, record the change in absorbance at 600 nm for 5 minutes using a spectrophotometer controlled at 37 ° C with water as the control, and measure the change in absorbance per minute (A OD) from the straight line. Blinded

TEST

Add a solvent that dissolves GDH to the reagent mixture instead of the GDH solution, and measure the change in absorbance per minute (A OD). From these values, GDH according to the following formula

BLANK

Ask for activity. Here, 1 unit (G) 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-darcose.

Activity (U / ml) =

{-(Δ Οϋ-Δ Οϋ) Χ 3. O X dilution factor} / (16.3 X 0. 1 X 1. 0)

TEST BLANK In the formula, 3.0 is the volume of reaction reagent + enzyme solution (ml), 16.3 is the molar molecular extinction coefficient (cm2 / micromol) under the conditions for this activity measurement, 0.1 is the enzyme solution The amount of liquid (ml), 1.0 indicates the optical path length (cm) of the cell.

Example 5: Confirmation of holo ratio or storage stability using glucose measurement system 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 containing a final concentration of 860 nM PQQ.

First, dilute PQQGDH to about 5. OUZml with enzyme diluent (ImM CaC12, 0.1%

50 ml of a solution dissolved in 50 mM PIPES_NaOH buffer solution (pH 6.5) containing Triton X-100, 0.1% BSA was prepared. To 0.33 ml of this enzyme solution, add 0.1 ml of various compounds at 10-fold concentration shown in Figs. 1 and 2 and add the base buffer shown in Figs. 1 and 2 to make the total volume 1.0 ml. Two were prepared. In addition, two controls were prepared with addition of 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 (%). By making all the compounds shown in FIGS. 1 and 2 coexist in the PQQGDH composition, an improvement in the holo-type ratio or storage stability was recognized. For those based on potassium phosphate buffer, when succinic acid, pimelic acid, and dimethyldanoletalic acid are added, the holo-type ratio or storage stability of holo-type PQ QGLD is reduced. This is probably because PQ Q is missing. The holo-type ratio including the apo-type and the storage stability have been improved, and it seems that these compounds are effective in maintaining the three-dimensional structure of the enzyme itself.

FIG. 1 shows the residual rate (%) of PQQGDH activity after treatment of PQQ GDH composition coexisting with various compounds based on PIPES buffer (pH 6.5) at 50 ° C. for 16 hours. Figure 2 shows the residual rate of PQQG DH activity after treatment of PQQGDH composition in the presence of phthalate buffer (ρΗ7.0) and potassium phosphate buffer (ρΗ7.0) in the presence of various compounds at 50 ° C for 16 hours. (%).

Example 6: Confirmation of holo-type ratio or storage stability using glucose measurement system 1 The examination was performed according to the method for measuring GDH activity described above. First, NADGDH (Toyobo GLD-311) was diluted to 80-90 U / ml with enzyme dilution (ImM CaC12, 0.1%

50 ml of a solution dissolved in 50 mM potassium phosphate buffer (ρΗ6.5)) containing Triton X-100, 0.1% BSA was prepared. To 0.33 ml of this enzyme solution, 0.1 ml of 10-fold concentrated various compounds shown in FIG. 3 was added, and potassium phosphate buffer ( _PH 6.5) was added to make the total volume 1.0 ml. Two things were prepared. In addition, two controls prepared by adding 0.1 ml of distilled water instead of various compounds were prepared. 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 100-fold with an enzyme diluent, and then NADGDH activity was measured. The activity values after treatment at 50 ° C for 1 hour were calculated as the residual activity rate (%), with the enzyme activity of those stored at 4 ° C as 100, respectively.

In all the compounds shown in FIG. 3, the holo-type ratio or the storage stability was improved by adding the compounds to the NADGDH composition. Among them, the highest effect was observed when succinic acid or maleic acid was added.

Fig. 3 shows the residual ratio (%) of NA DGDH activity after 1 hour treatment of NADGDH composition coexisting with various compounds at 50 ° C. Example 7: Confirmation of holo ratio or storage stability using glucose measurement system 2 The study was conducted according to the NADGDH activity measurement method of Test Example 2 described above.

First, 50 ml of NADGLD dissolved in an enzyme dilution solution at about 250 U / ml was prepared. To 0.33 ml of this enzyme solution, add 0.1 ml of the 10-fold concentration of various compounds shown in Fig. 3 and remove the Tris_HCl buffer (pH 8.0) to make a total volume of 1. Oml. Prepared. In addition, two controls with 0.1 ml of distilled water added instead of various compounds were prepared as controls. Of the two, one was stored at 4 ° C and the other was treated at 50 ° C for 1 hour. After the treatment, each sampnore was diluted 500-fold with an enzyme diluent, and then FADGDH activity was measured. Each of those stored at 4 ° C was regarded as 100, and the activity value after 1 hour treatment at 50 ° C was compared to calculate the residual activity rate (%).

As a result, all the dicarboxylic acids studied showed an effect. In particular, when succinic acid or maleic acid was added, the highest holo-type ratio or storage stability was improved.

[0112] Example 8: Confirmation of holo ratio or storage 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, the enzyme dilution solution (ImM) was adjusted so that the FADGLD obtained in Example 4 was about 10 U / ml.

50 ml of CaC12, 0.1% Triton X—100, 0.1% BSA-containing 50 mM phosphate buffered saline (PH6.5) was prepared. Two 0.5 ml of 1% and 0.5% BSA were added to 0.5 ml of this enzyme solution to make a total volume of 1. Oml. In addition, the succinic acid, malonic acid, phthalic acid, maleic acid, daltaric acid, sodium chloride, sodium sulfate, trisodium citrate, and ammonium sulfate shown in FIGS. 5 and 6 were prepared in the same manner. Two bottles with a total volume of 1. Oml were prepared. Two types of controls were prepared, each containing 0.1 lml 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-fold 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 values after 1 hour treatment at 50 ° C. were compared to calculate the residual activity rate (%).

As a result of these studies, the addition of proteinaceous stabilizer (BSA) It became clear that the holo-type ratio or storage stability of _GLD increased (Fig. 4). In addition, the addition of various dicarboxylic acid compounds or various salt compounds has an effect of improving the FAD-GLD holo-type ratio or storage stability. Among dicarboxylic acid compounds, succinic acid and among malonic acid power compounds are sulfuric acid. Sodium had the greatest effect (Figures 5 and 6). In the case of succinic acid, malonic acid, and sodium sulfate, it seems that the stability improvement effect can be seen even with the addition of several moles. In addition, even simple salt compounds such as sodium chloride show sufficient effects, and FAD-GLD is the first to see that thermal stabilization is achieved simply by increasing the ionic strength. It was issued.

FIG. 4 shows the residual ratio (%) of FADGDH activity after treatment at 50 ° C. for 30 minutes with a FADGDH composition in the presence of a proteinaceous stabilizer.

FIG. 6 shows the residual rate (%) of the FAD GDH activity after treatment with a salt compound in the presence of a FADGDH composition at 50 ° C. for 15 minutes.

[0113] Example 7: Construction of PQQ-dependent Gnorecose Dehydrogenase Gene Expression Plasmid Wild-type PQQ-dependent glucose dehydrogenase expression plasmid PNPG5 was added to the multiple cloning site of the beta pBluescript SK (—). Acinetobacter baumannii) A structural gene encoding a PQQ-dependent darcos dehydrogenase derived from NCIMB11517 strain. 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.

[0114] Example 8: Production of mutant PQQ-dependent glucose dehydrogenase

QuickChangeTM Site -Directed Mutagenes is Kit (manufactured by STRATAGENE) based on a recombinant plasmid pN PG5 containing a wild-type PQQ-dependent glucose dehydrogenase gene and a synthetic oligonucleotide of about 40mer containing a triplet encoding the amino acid at the site of mutation introduction Mutation treatment operation according to the protocol using J, J168, Q168A, 169Y, L169P, A170L, E245D, M342I, N429D, 430P mutations introduced, mutant PQQ-dependent type with improved substrate specificity Glucose dehydro A recombinant plasmid (pNPG5_Q168A + 169Y + L169P + Al70L + E245D + M342I + N429D + 430P) encoding a genease was obtained. The nucleotide sequence of the obtained candidate strain was determined, and the 168th glutamine of the amino acid sequence described in SEQ ID NO: 1 was alanine, the 169th leucine was proline, the 170th alanine was leucine, and the 245th gnore. Mutant PQQ with thamic acid replaced with aspartic acid, 342th methionine replaced with isoleucine, 429th asparagine replaced with aspartic acid, 168th followed by tyrosine and 429th after proline It was confirmed that it encodes type glucose dehydrogenase.

E. coli competent cells (Escherichia coli JM 109; manufactured by Toyobo Co., Ltd.) were transformed with the recombinant plasmid to obtain transformants.

[0115] Example 9: Construction of an expression vector that can replicate in Pseudomonas bacteria

Recombinant plasmid obtained in Example 8 pNPG5-Q 168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P DNA 5 μg cleaved with restriction enzymes BamHI and Xhol (manufactured by Toyobo) and dependent on mutant PQQ The structural gene portion of type glucose dehydrogenase was isolated. The isolated DNA was reacted with pTM33 (1 ig) cleaved with BamHI and Xhol and 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA. The ligated DNA was transformed using a competent cell of Escherichia coli DH5a. The obtained expression plasmid was designated as pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P.

Example 10: Preparation of transformants of Pseudomonas bacteria

Pseudomonas.Putida TE3493 (Microeken No. 12298) was cultured in LBG medium (LB medium + 0.3% glycerol) at 30 ° C for 16 hours, and the cells were collected by centrifugation (12,000 rpm, 10 minutes) Then, 8 ml of 5 mM K_phosphate buffer solution (PH7.0) containing 300 mM sucrose cooled with ice was added to the cells to suspend the cells. The cells are collected again by centrifugation (12,000 rpm, 10 minutes), and 0.4 ml of 5 mM K-phosphate buffer (ρΗ7.0) containing 300 mM sucrose cooled on ice is added to the cells. The body was suspended.

Into the suspension, the expression plasmid pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P obtained in Example 9 was added to 0.5 / ig calecto, electopores. Transformation was performed by the Yong method. A target transformant was obtained from a colony grown on an LB agar medium containing 100 μg / ml streptomycin.

[0117] Example 11: Preparation of PQQ-dependent GDH preparation

Dispense 500 ml of Terrific broth into a 2 L volumetric flask. C, autoclaved for 20 minutes, and after standing to cool, separately sterile filtered streptomycin was added to 100 μg / ml. Pseudomonas putida TE3493 (pNPG6-Q 168 A + 169 Y + L169P + A170L + E245D + M342I + N429D + 430P) previously cultured in PY medium containing 100 μg / ml streptomycin at 30 ° C for 24 hours 5 ml of the culture solution was inoculated and cultured with aeration and stirring at 30 ° C for 40 hours. The PQQ-dependent glucose dehydrogenase activity at the end of the culture was about 30 U / ml per 1 ml of the culture solution after the measurement of the activity, and the cells were collected by centrifugation, and the 20 mM phosphate buffer solution ( After suspending in pH 7.0), the mixture was crushed by sonication and further centrifuged to obtain a supernatant as a crude enzyme solution. The resulting crude enzyme solution was separated and purified by HiTrap-SP (Amersham-Falmasia) ion exchange column chromatography. Then, after dialyzing with 10 mM PIPES-NaOH buffer (ρΗ6 · 5), calcium chloride was added so that the final concentration force was SlmM. Finally, it was separated and purified by HiTrap-DEAE (Amersham-Falmacia) ion exchange column chromatography to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band on SDS-PAGE.

The purified enzyme thus obtained was used as a PQQ-dependent GLD evaluation sample.

[0118] Example 12: Preparation of FAD-dependent GDH preparation

Using lilacinoechinulatum NBRC6231 (purchased from National Institute for Product Evaluation and Technology), each L dry specimen was inoculated into potato dextrose agar medium (manufactured by Difco) and incubated at 25 ° C. The mycelium on the restored plate was collected together with the agar and suspended in filter sterilized water. Production medium (1% malt extract, 1.5% soy peptide, 0.1% MgS04'7 hydrate, 2./.Dalcos, pH6.5) in two 10L jar mentors Prepare the above mycelial suspension after autoclaving at 120 ° C for 15 minutes. Each was charged and culture was started. The culture conditions were a temperature of 30 ° C., an aeration rate of 2 L / min, and a stirring rate of 3 80ι · ρπι. 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 with a hollow fiber module for ultrafiltration with a molecular weight of 10,000,000 cut, and add ammonium sulfate to the concentrate to a final concentration of 60% saturation (456 g / U). Next, after centrifuging at 8000 rpm for 5 minutes in a Hitachi high-speed cooling centrifuge to precipitate the residue, the supernatant was adsorbed on an Octyl_Sepharose column and eluted with a gradient of ammonium sulfate from 0.6 to 0.0 saturation. The fraction with GDH activity was collected, and the obtained GDH solution was subjected to gel filtration on a G-25 Sepharose column, and the protein fraction was collected for desalting to give a desalted solution of 0.6. Ammonium sulfate equivalent to saturation was added and dissolved, and this was adsorbed on a Phenyl-Sepharose column, and the fraction with GDH activity was recovered by gradient elution at an ammonium sulfate concentration of 0.6 to 0.0 saturation. Furthermore, the obtained GDH solution is added to G-25 Protein fractions were collected before gel filtration column was used purified enzyme obtained as FA D dependent GLD evaluation standard.

[0119] 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 (abbreviation). DCPIP), ferrocene or their derivatives (eg ferricyanium potassium, phenazine methosulfate, etc.). These mediators are commercially available.

[0120] Example 13: Optimal temperature and thermal stability of PQQ-dependent GDH

Examples 7 to: The optimum temperature and thermal stability were examined using the PQQ-dependent GDH obtained in 11. The optimum temperature was calculated as the relative activity (%) at each temperature with the temperature (40 ° C) showing maximum activity in 50 mM PIPES-NaOH (pH 6.5) buffer as 100% (Fig. 7). . The optimum temperature for the PQQ-dependent GDH used in the study was 40-45 ° C.

Thermal stability is 50, 40, 50, 60, 70 with 50 mM PIPES-NaOH (pH 6.5), ImU CaC12 composition and 5.0 U / ml PQQ-dependent GDH. Compare the GDH activity remaining after heat treatment at C for 30 minutes with the activity value of the sample stored at 4 ° C. Residual activity (%) was calculated (FIG. 8). Until the heat treatment temperature was 40 ° C, there was no significant decrease in activity, and there was a tendency to be largely deactivated by the 50 ° C treatment. Thermal stability is likely to vary depending on the solution composition and enzyme concentration. For example, the higher the enzyme concentration, the higher the temperature at which large heat inactivation is observed (it will be 50 ° C or higher). Example 14: Effect of heating treatment on the stability of PQQ-dependent GDH

Examples 7 to: Using the PQQ-dependent GDH obtained in 11, it was examined whether the stability of the holo-type enzyme was changed before and after the heating treatment. PQQ-dependent GDH solution in 20 mM PIPES buffer (ρΗ6 · 5), ImM

This was diluted with CaC12 to an activity of 20-30 U / ml, and this diluted solution was heated at 50 ° C. for 1 hour in a heat bath. After the heat treatment, the PQQ-dependent GDH activity (Test Example 1) before and after the heat treatment was compared after treatment under conditions where heat inactivation occurs (55 ° C, 1 hour). The residual activity after the heat treatment before the heat treatment was 31%, and the remaining activity after the heat treatment after the heat treatment was 42%. It was clarified that the stability of PQQ-dependent GDH was increased by heating treatment (Fig. 9). By carrying out the heating treatment, the holoformation rate of the enzyme increased, which seemed to lead to an increase in stability.

Figure 9 shows the results of studying the thermal stability effect of heating treatment. The residual activity was calculated with the activity value of the sample stored at 4 ° C after the heating treatment as 100%.

Next, the heat treatment conditions were extensively studied using the same operation method as in FIG. 9 (FIG. 10). Even when a treatment temperature of 35 ° C was applied for about 30 minutes, the effect of improving stability was observed although it was weak. However, at a treatment temperature of 35 ° C, a sufficient effect was not seen even if the treatment was performed for a long time. In addition, the stability of GDH, which does not impair the stability even when heated at 50 ° C for 16 hours, is stabilized for a long time at a slightly lower temperature than the enzyme denatures. Seemed to be most effective. In this experiment, the ability to use PQQ-dependent GDH mutants that had been destabilized due to amino acid mutations, even if wild-type PQQ-dependent GDH was used, so that the effect of heating treatment could be easily understood. It has been confirmed that the stability is improved by the caloric temperature treatment. In particular, mutants have reduced PQQ retention capacity due to structural distortion, and the structure of the holo-type enzyme can be determined by heating treatment. It is assumed that the stability of the holo-type enzyme itself is improved. In addition to the heating treatment, the same effect may be obtained by adding some energy to the enzyme.

Figure 10 shows the results of the examination of the heat treatment conditions (temperature X time). The residual activity was calculated by setting the activity value of the sample stored at 4 ° C after heating to 100%.

[0122] What is the thermal stability (heat treatment at 55 ° C for 1 hour) by applying the heat treatment (50 ° C, 16 hours) again after the heating treatment (50 ° C, 16 hours)? (Figure 11). When the heating treatment was insufficient, it was confirmed that the thermal stability was increased by heating again. From these studies, it became clear that the effect of heating treatment is proportional to temperature and time, but does not increase beyond a certain level.

Fig. 11 shows the results of studying the effects of rewarming after warming. The residual activity was calculated with the activity value of the sample stored at 4 ° C after the heating treatment as 100%.

[0123] The composition of the present invention is a composition comprising a soluble coenzyme-linked glucose dehydrogenase, which is produced by a method comprising a step of subjecting the composition to a heating treatment, and having improved heat stability. A composition comprising a coenzyme-linked glucose dehydrogenase, which is characterized by its production process.

In general, it is difficult to determine whether a composition has undergone a heating treatment step. In the composition of the present invention, from the result of FIG. 11 above, it is the second time at 50 ° C. for 16 hours. When heat treatment was performed, the value of (activity remaining after heat treatment for the first time at 55 ° C for 1 hour) ÷ (activity remaining after heat treatment for the second time at 55 ° C for 1 hour) exceeded 60% If this is the case, it can be estimated that the heat treatment has been performed.

Example 15: Construction of expression plasmid for PQQ-dependent gnolecose dehydrogenase gene Wild-type PQQ-dependent gnolecose dehydrogenase expression plasmid PNPG5 is a cinetopactor ^ ~ · at the multiple cloning site of the vector pBluescript SK (-). A structural gene encoding a PQQ-dependent glucose dehydrogenase derived from Acinetobacter baumannii NCIMB11517 strain was 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. [0125] Example 16: Construction of an expression vector capable of replicating in Pseudomonas bacteria DNA 5 µg of the recombinant plasmid pNPG5 obtained in Example 15 was cleaved with restriction enzymes BamHI and XHoI (Toyobo Co., Ltd.) to depend on PQQ. The structural gene portion of gnolecose dehydrogenase was isolated. The isolated DNA and pTS 1 137 (1 μg) cleaved with BamHI and XHoI were reacted with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA. The ligated DNA was transformed using Escherichia coli DH5a competent cells. The resulting expression plasmid was named PNPG6.

Example 17: Preparation of transformants of Pseudomonas bacteria

To this suspension, the expression plasmid pNPG6 obtained in Example 16 was transformed to 0. 0, and transformed by the electrovolution method. The desired transformant was obtained from a colony grown on LB agar medium containing 100 μg / ml streptomycin.

[0127] Test Example 4

Holo-type PQQGDH activity measurement method

Measurement principle

D—Glucose + PMS + PQQGDH → D—Dalcono 1,5—Lataton + PMS (red)

2PMS (red) + NTB → 2PMS + diformazan

The presence of diformazan formed by the reduction of nitrotetrazolium blue (NTB) with phenazine methosulfate (PMS) (red) was measured spectrophotometrically at 570 nm

One unit forms 0.5 mmol of diformazan per minute under the conditions described below Check the amount of PQQGDH enzyme.

(3) Method

Reagent

A. D—glucose solution: 0.5M (0.9 g D—glucose (molecular weight 180. 16) / l0ml H 2 O)

2

B. PIPES—NaOH buffer, pH 6.5: 50 mM (1.51 g of PIPES (molecular weight 302. 36) suspended in 60 mL of water, dissolved in 5N NaOH, 2. 2 ml of 10% Triton X Calorie free 100. Use 5N NaOH 25. Adjust the pH to 6.5 ± 0.05 with C and add 100 ml with water.)

C. PMS dissolved at night: 3.0 mM (9. 19 mg phenazine methosanolate (molecular weight 817. 65) / 10 ml H 2 O)

2

D. NTB solution: 6.6 mM (53. 96 mg of nitrotetrazolium blue (molecular weight 817. 65) / 10 ml H 2 O)

2

酵素 Enzyme Diluent: ImM CaCl, 0.1% Triton X—100, 0.1% BSA included

2

50mM PIPES—NaOH buffer (ρΗ6 · 5)

Steps

Prepare the following reaction mixture in a light-proof bottle and store on ice (prepared at the time of use)

1. 8ml D-glucose solution (A)

24. 6ml PIPES _Na 0H buffer (pH 6.5) (B)

2. 0ml PMS solution (C)

1. 0ml NTB solution (D) The concentration of the above mixture in the reaction solution is as follows.

PIPES buffer 42 mM

D_Gnore Course 30mM

PMS 0.20mM

NTB 0.22mM

3. Put 0ml reaction mixture into a test tube (plastic) and preheat at 37 ° C for 5 minutes 0.1 ml of enzyme solution was added and gently inverted to mix.

Record the increase in absorbance for water at 570 nm at 37 ° C for 4-5 minutes with a spectrophotometer, and calculate AOD per minute from the initial linear part of the curve (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 (ΔD blank) was measured.

The enzyme powder was dissolved in ice-cold enzyme diluent (E) immediately before the assembly and diluted to 0.1 -0.8 U / ml with the same buffer (use a plastic tube for the adhesion of the enzyme). Is preferred).

Calculation

Activity is calculated using the following formula:

U / ml = {A〇D / min (A〇D test Δ OD blank) X Vt X df} / (20 · 1 X 1. O X Vs)

Vt: Total volume (3. lml)

Vs: Sample volume (1.0 ml)

20. 1: 1/2 mmol molecular extinction coefficient of diformazan

1. 0: Optical path length (cm)

df: dilution factor

C: Enzyme concentration in solution (c mg / ml)

[0129] Test Example 5

Activity measurement method for all PQQGDH

In Test Example 4, instead of enzyme diluent (E), enzyme diluent (F): ImM CaCl,

2

The measurement was carried out in the same manner after the enzyme was diluted with 50 mM PIPES NaOH buffer (pH 6.5) containing 0.1% Triton X—100, 0.1% BSA and 測定 μΜ PQQ.

Example 18 Expression of PQQGDH and recovery of crude enzyme solution

Dispense 25 ml of GDH production medium (1.5% glycerol, 4% yeast extract, 1.25% K2HP04, 0.23% KH2P04, pH 6.8) into a 500 ml Sakaguchi flask. C, 20 minutes A one-clave was performed and the medium was sterilized. After standing to cool, separately sterile filtered streptomycin was added to 100 μg / ml, and ethanol was further added to 1% (V / V). The transformant obtained in Example 17 was cultured in this medium at 33 ° C for 24 hours, and the cells were collected by centrifugation (12000 rpm, 5 minutes). The cells are suspended in 20 mM phosphate buffer (pH 7.0), crushed by sonication, and further centrifuged to obtain a supernatant as a crude enzyme solution. According to Test Examples 4 and 5, PQQGDH Activity was measured.

The ratio of the expression level of holo PQQGDH was calculated by dividing the holo PQQGD H activity (U / ml) expressed per unit volume by the total PQQGDH activity (U / ml). Figure 12 shows the measurement results.

Example 19 Purification of PQQGDH and confirmation of improvement in proportion of holo-type PQQGDH by heat treatment HiTrap— SP (Amersham-Falmacia) ion buffered with 20 mM phosphate buffer (pH 7.0) The exchange column chromatography was charged and the GDH protein was adsorbed on the resin. After washing the resin with 20 mM phosphate buffer (pH 7.0), which is twice the amount of column resin, use 20 mM phosphate buffer (pH 7.0) containing twice the same amount of 0.3 M NaCl. GDH protein was eluted from the resin, and the eluted fraction was collected. Next, after dialyzing in 10 mM PIPES-NaOH buffer (pH 6.5) and desalting, calcium chloride was added to a final concentration of SlmM.

Hereinafter, the dialysate is divided into 2 minutes, (A) a method for obtaining a purified enzyme preparation after heat treatment, and (B) a method for obtaining a purified enzyme preparation without heat treatment, respectively. Carried out.

(A) Method for obtaining a purified enzyme preparation after heat treatment

Half of the dialysate was placed in warm water controlled at 35 ° C and heat-treated for 16 hours. After clarification by centrifuge separation, charge to Hi Trap—DEAE (Amersham-Falmacia) ion exchange column chromatography buffered with 10 mM PIPES _Na 0H buffer (pH 6.5), and collect the non-adsorbed fraction. As a result, a purified enzyme preparation was obtained.

(B) A method for obtaining a purified enzyme preparation without carrying out heat treatment

Charge remaining dialysate to HiTrap DEAE (Amersham-Falmacia) ion exchange column chromatography buffered with 10 mM PIPES—NaOH buffer (pH 6.5) The non-adsorbed fraction was collected to obtain a purified enzyme preparation.

(A) Each specimen obtained from (B) showed an almost single band on SDS-PAGE.

In the same manner as in Example 18, the ratio of holo-type PQQGDH in each enzyme solution was determined. The measurement results are shown in Fig. 13.

[0132] First, it was confirmed that the ratio of holo-type PQQGDH was significantly improved by heat treatment of the dialysate. Even if unbound PQQ is present in the GDH protein at the stage of the crude enzyme solution, PQQ is a small molecule, so free PQQ is removed by dialysis. Therefore, the result of the increase in the proportion of holo-type PQQGDH by heat treatment is that there is a GDH enzyme protein that is inactive due to imperfect binding state, although it is bound to GDH protein. It means that it was. In addition, it strongly suggests that the state of binding with PQQ has been improved by the change of the conformation due to heat treatment, and they have become active.

This change to the active form, in other words, the change in conformation, is because the ratio of holo-type PQQGDH does not change between the heat-treated solution (A) and the purified enzyme preparation (A) that has been further purified. 1 It turns out that it is permanent rather than transient.

Furthermore, as the results of the purified enzyme preparation (B) are clear, simply continuing the purification does not increase the proportion of holo-type PQQGDH, so the binding state is incomplete and the inactive form becomes inactive. Heat treatment is necessary to make the GDH enzyme protein active.

I understand.

The fact that there is a GDH enzyme protein in an incompletely bound state, and the fact that it can be converted into an active form simply by heat treatment was surprising and surprising. By carrying out the heat treatment, it becomes possible to effectively use such GDH enzyme protein, which accounts for more than 10% of the total, so it was judged as an industrially useful means.

Industrial applicability

[0133] According to the present invention, the stability of GDH itself is increased by heating the GDH composition. It is possible to provide a reagent with high storage stability by performing a heating process at the time of production of the glucose measuring reagent.

Further, the improvement of the holo-type ratio or the storage stability according to the present invention can improve the measurement accuracy with a glucose measuring reagent, a glucose assay kit and a glucose sensor. In addition, according to the present invention, productivity as a holo type PQQGDH can be improved, and PQQGDH can be manufactured at low cost. Furthermore, it is also possible to provide a gnolecose assembly kit and a glucose sensor at low cost. On the other hand, the activity value per unit protein weight of PQQGDH is improved by increasing the proportion of holo-type PQQG DH. PQQGDH is preferred for use because it allows a reduction in the amount of protein added to the glucose assembly kit and glucose sensor.

Claims

The scope of the claims

[1] In a composition containing a soluble coenzyme-linked glucose dehydrogenase

A method for improving the thermal stability of the enzyme, comprising a step of heating the composition.

[2] The method for improving thermostability according to claim 1, wherein the coenzyme is pyrroloquinone quinoline or a flavin compound.

[3] The heat treatment temperature is lower than the temperature at which a large heat inactivation of the enzyme occurs

The method for improving thermal stability according to claim 1.

[4] A composition containing a soluble coenzyme-linked glucose dehydrogenase is prepared.

A method for producing a composition containing a soluble coenzyme-linked glucose dehydrogenase having improved thermal stability, comprising a step of heating the composition.

[5] A composition containing a soluble coenzyme-linked gnolecose dehydrogenase produced by the method according to claim 4, wherein a soluble coenzyme-linked glucose dehydrogenase having improved thermal stability is used. Containing composition.

[6] A glucose assembly kit comprising the composition according to claim 5

[7] A glucose sensor comprising the composition according to claim 5.

[8] A method for measuring glucose concentration using the composition according to claim 5.