JP2007129966A - Method for improvement of heat stability of glucose dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for improving stability of a composition comprising a coenzyme-binding soluble glucose dehydrogenase (GDH). <P>SOLUTION: This method for improving heat stability of a coenzyme-binding soluble glucose dehydrogenase in a composition comprising the enzyme comprises treating the composition with heat. The improvement of the heat stability according to the present invention enables the reduction in the thermal deactivation of the enzyme on the production of a glucose-measuring reagent, a glucose assay kit, and a glucose sensor to improve the amount reduction and measurement accuracy of the enzyme, and also enables to provide a blood glucose level-measuring reagent using GDH excellent in preservation stability. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a method for improving the stability of a composition containing soluble coenzyme-linked glucose dehydrogenase (hereinafter, glucose dehydrogenase is also referred to as GDH). The present invention also relates to a glucose measurement method and glucose sensor using GDH with improved stability.

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

In order to avoid such a problem, for example, NAD (P) -dependent glucose dehydrogenase (EC 1.1.1.47) or PQQ-dependent glucose dehydrogenase (EC 1.1.5.2 (formerly EC 1.1.99) .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 the complexity of requiring the addition of a coenzyme. On the other hand, the latter PQQ-dependent glucose dehydrogenase is poor in substrate specificity and has a drawback that it impairs the accuracy of measured values because it acts on saccharides other than glucose such as maltose and lactose.

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

  An object of the present invention is to provide a 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.

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

  The inventors of the present invention have sought to find a simpler improvement measure for stability from a viewpoint different from the past measures, and as a result of further earnest research, the GDH composition is stabilized by heating the GDH composition. The present invention has finally been completed by clarifying that the characteristics can be increased.

That is, the present invention has the following configuration.
[Claim 1]
A method for improving the stability of the enzyme, comprising a step of heating the composition in a composition containing a soluble coenzyme-linked glucose dehydrogenase.
[Section 2]
Item 2. The method for improving stability according to Item 1, wherein the coenzyme is pyrroloquinone quinoline or a flavin compound.
[Section 3]
Item 3. The method according to Item 1 or 2, wherein the heating treatment temperature is equal to or lower than a temperature at which a large heat inactivation of the enzyme occurs.
[Claim 4]
A composition comprising a soluble coenzyme-linked glucose dehydrogenase, comprising a soluble coenzyme-linked glucose dehydrogenase with improved thermal stability, produced by a method comprising a step of subjecting the composition to a heating treatment Composition [Item 5]
A method for measuring a glucose concentration using the composition according to Item 4.
[Claim 6]
A glucose sensor comprising the composition of Item 5.
[Claim 7]
A method for producing a composition comprising a soluble coenzyme-bound glucose dehydrogenase with improved stability, comprising a step of subjecting the composition to a heating treatment in a composition comprising a soluble coenzyme-bound glucose dehydrogenase .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  Purification of GDH can be performed by appropriately combining various separation techniques that are usually used depending on the fraction in which GDH activity is present. From the GDH extract, for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denaturing PAGE, SDS-PAGE, ion exchange chromatography, hydroxyapatite chromatography, affinity chromatography, reverse phase high performance liquid chromatography A known separation method such as isoelectric focusing can be selected appropriately. Moreover, various stabilizers etc. can also be added in the extracted GDH or the refined GDH solution. Examples of such substances include, for example, mannitol, trehalose, sucrose, sorbitol, erythritol, amino acids typified by sugars, sugar alcohols, glutamic acid, arginine and the like typified by glycerol, bovine serum albumin, ovalbumin, and various chaperones. And proteins and peptides represented by the above.

  The GDH of the present invention can be provided in a liquid state, but can be pulverized by freeze drying, vacuum drying, spray drying or the like. At this time, GDH dissolved in a buffer solution or the like can be used, and it is preferable to add sugar / sugar alcohols, amino acids, proteins, peptides, etc. as excipients or stabilizers. Further, it can be further granulated after pulverization.

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

  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 at the time of measurement.

The improvement in the stability as used in the present invention means that the residual ratio (%) of the maintained GDH enzyme increases after heat-treating the composition containing the GDH enzyme at a certain temperature for a certain time. For example, in the present invention, when the activity is almost completely maintained, the sample stored at 4 ° C. is regarded as 100%, and this is compared with the activity value of the GDH solution after heat treatment at 55 ° C. for 1 hour. Is calculated. When this residual rate was increased as compared with the case where the heating treatment was not performed, it was determined 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 treatment at 4 ° C. for 16 hours and the GDH activity value (b) after heat treatment were measured, and the measurement value (a) The relative value ((b) / (a) × 100) with respect to the value of 100 was determined. 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 implementation.
In addition, “heat treatment” in the above description refers to a treatment for a test for confirming the stability of the enzyme, and it is noted that it is different from the “warming treatment” of the present invention. The same applies to the explanations other than this paragraph.

In the heating treatment, at least 50% or more of the activity needs to be maintained before and after the heating treatment, preferably 80% or more of the activity is maintained, more preferably 90% or more of the activity. It is desirable that it be maintained.
The temperature at which large heat inactivation of the enzyme in this patent 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. It means a point (processing temperature) that changes more than twice. For example, in FIG. 1, the absolute value of the slope of the approximate expression at 0, 30, and 40 ° C. is 0.233, and the absolute value of the slope of the approximate expression at 40, 50, and 60 ° C. is 3.63. is there. Therefore, under this heat treatment condition, 40 ° C. is a temperature at which large thermal deactivation occurs.
Alternatively, the temperature at which a large heat inactivation of the enzyme in this patent occurs is a temperature at which the residual activity of the enzyme becomes 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”. When it can be determined only by one method, the temperature determined by that method is set as “the temperature at which a large heat inactivation of the enzyme occurs”.

 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 modified PQQGDH used in the study of this patent, when examined at an enzyme concentration of 5 U / ml, the temperature at which large heat inactivation occurs was 40 ° C. (FIG. 1), 20-30 U. When examined at / ml, it was 55 ° C. Therefore, in the examination of the enzyme concentration of 20 to 30 U / ml, considering the safety side below the temperature, the heating treatment is examined at 50 ° C. or less. Further, as a result of the examination of the heating 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.

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

In the present invention, various components can coexist if necessary. For example, you may add surfactant, a stabilizer, an excipient | filler, etc.
For example, PQQGDH can be further stabilized by adding calcium ions or salts thereof, amino acids such as glutamic acid, glutamine, and lysine, and serum albumin.
For example, PQQGDH can be stabilized by containing calcium ions or calcium salts. Examples of calcium salts include calcium chloride, calcium salts of inorganic acids such as calcium acetate and calcium citrate, and organic acids. In the aqueous composition, the calcium ion content is preferably 1 × 10 ⁻⁴ to 1 × 10 ⁻² M.
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. One or more amino acids selected from the group consisting of glutamic acid, glutamine and lysine may be used. It may further contain ovalbumin (OVA).
Or (1) one or more compounds selected from the group consisting of aspartic acid, glutamic acid, α-ketoglutaric acid, malic acid, α-ketogluconic acid, α-cyclodextrin, and salts thereof; and (2) GDH can be stabilized by allowing albumin to coexist.

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

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

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

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

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

Hereinafter, the present invention will be described in more detail based on examples.
Example 1 Construction of PQQ-Dependent Glucose Dehydrogenase Gene Expression Plasmid Wild-type PQQ-dependent glucose dehydrogenase expression plasmid pNPG5 was derived from the Acinetobacter baumannii17 strain NCIMB115 at the multiple cloning site of the vector pBluescript SK (-). A structural gene encoding a PQQ-dependent glucose dehydrogenase is inserted. The base sequence is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of PQQ-dependent glucose dehydrogenase deduced from the base sequence is shown in SEQ ID NO: 1 in the sequence listing.

Example 2: Production of mutant PQQ-dependent glucose dehydrogenase Based on a recombinant plasmid pNPG5 containing a wild-type PQQ-dependent glucose dehydrogenase gene and a synthetic oligonucleotide of about 40 mer containing a triplet encoding the amino acid at the site of mutation introduction , QuickChange ^™ Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) was used to sequentially carry out mutation treatment operations according to the protocol to introduce the mutations of Q168A, 169Y, L169P, A170L, E245D, M342I, N429D, and 430P. Recombinant plasmid encoding mutant PQQ-dependent glucose dehydrogenase with improved (pNPG5-Q168A + 169Y + L169P + A1 0L + E245D + M342I + N429D + 430P) was obtained. The nucleotide sequence of the obtained candidate strain was determined, and the 168th glutamine of the amino acid sequence shown in SEQ ID NO: 1 was alanine, the 169th leucine was proline, the 170th alanine was leucine, and the 245th glutamic acid was It encodes a mutant PQQ-dependent glucose dehydrogenase in which aspartic acid is substituted with 342nd methionine for isoleucine and 429th asparagine for aspartic acid, and 168th for tyrosine and 429th for proline. Confirmed that.
Escherichia coli competent cells (Escherichia coli JM109; manufactured by Toyobo Co., Ltd.) were transformed with the recombinant plasmid to obtain transformants.

Example 3: Construction of an expression vector capable of replicating in Pseudomonas bacteria Recombinant plasmid pNPG5-Q168A + 169Y + L169P + A170L + 5 μg of DNA obtained in Example 2 was cleaved with restriction enzymes BamHI and XhoI (manufactured by Toyobo Co., Ltd.), and dependent on mutant Q The structural gene part of dehydrogenase was isolated. The isolated DNA was reacted with pTM33 (1 μg) cleaved with BamHI and XhoI with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA. Ligated DNA was transformed with Escherichia coli DH5α competent cells. The obtained expression plasmid was designated as pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P.

Example 4 Production of Pseudomonas Bacterium Transformant Pseudomonas putida TE3493 (Microtechnical Research No. 12298) was cultured in LBG medium (LB medium + 0.3% glycerol) at 30 ° C. for 16 hours and centrifuged (12 The bacterial cells were collected by 2,000 rpm for 10 minutes, and 8 ml of 5 mM K-phosphate buffer (pH 7.0) containing 300 mM sucrose ice-cooled was added to the bacterial cells to suspend the bacterial cells. The bacterial cells were collected again by centrifugation (12,000 rpm, 10 minutes), and 0.4 ml of 5 mM K-phosphate buffer (pH 7.0) containing 300 mM sucrose ice-cooled was added to the bacterial cells. Suspended.
0.5 μg of the expression plasmid pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P obtained in Example 3 was added to the suspension and transformed by electroporation. A target transformant was obtained from a colony grown on an LB agar medium containing 100 μg / ml streptomycin.

Example 5: Preparation of PQQ-dependent GDH preparation 500 ml of terrific broth was dispensed into a 2 L Sakaguchi flask, autoclaved at 121 ° C. for 20 minutes, allowed to cool, and then separately sterile filtered streptomycin to 100 μg / ml. Added to. This medium was inoculated with 5 ml of Pseudomonas putida TE3493 (pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P) previously cultured in a PY medium containing 100 μg / ml streptomycin at 30 ° C. for 24 hours, and cultured with aeration 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 in the activity measurement.
The cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.0), disrupted by sonication, and further centrifuged to obtain a supernatant as a crude enzyme solution. . The obtained crude enzyme solution was separated and purified by HiTrap-SP (Amersham-Pharmacia) ion exchange column chromatography. Next, after dialyzing with 10 mM PIPES-NaOH buffer (pH 6.5), calcium chloride was added to a final concentration of 1 mM. Finally, it was separated and purified by HiTrap-DEAE (Amersham-Pharmacia) ion exchange column chromatography to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band by SDS-PAGE.
The purified enzyme thus obtained was used as a PQQ-dependent GLD evaluation sample.

Test Example 1: Method for measuring GDH activity
Measurement principle D-glucose + PMS + PQQGDH → D-glucono-1,5-lactone + PMS (red)
PMS (red) + DCPIP → PMS + DCPIP (red)
The presence of DCPIP (red) formed by the reduction of 2,6-dichlorophenol-indophenol (DCPIP) with phenazine methosulfate (PMS) (red) was measured spectrophotometrically at 600 nm. In the examination of substrate specificity, the D-glucose part was changed to another saccharide, and the specificity for each substrate was measured.
Unit Definition One unit refers to the amount of PQQGDH enzyme that forms 1.0 mmol of DCPIP (red) per minute under the conditions described below.
Methods Reagent A. D-glucose solution: 1.0 M (1.8 g D-glucose (molecular weight 180.16) / 10 ml H ₂ O)
B. PIPES-NaOH buffer, pH 6.5: 50 mM (1.51 g of PIPES (molecular weight 302.36) suspended in 60 mL of water was dissolved in 5N NaOH, and 2.2 ml of 10% Triton X-100 was dissolved. The pH was adjusted to 6.5 ± 0.05 with 5N NaOH at 25 ° C., and water was added to make 100 ml.)
C. PMS solution: 24 mM (73.52 mg phenazine methosulfate (molecular weight 81 7.65) / 10 ml H ₂ O)
D. DCPIP solution: 2.0 mM (6.5 mg of nitrotetrazolium blue (molecular weight 8 17.65) / 10 ml H ₂ O)
E. Enzyme _{diluent: 1mM CaCl 2, 0.1% Triton} X-100, 0. 50 mM PIPES-NaOH buffer (pH 6.5) containing 1% BSA

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

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

2. 3.0 ml of the reaction mixture was put in a test tube (made of plastic) and preheated at 37 ° C. for 5 minutes.
3. 0.1 ml enzyme solution was added and mixed by gentle inversion.
4). The decrease in absorbance for water at 600 nm was recorded on a spectrophotometer while maintaining at 37 ° C. for 4-5 minutes and the ΔOD per minute from the initial linear portion of the curve was calculated (OD test).
At the same time, the same method was repeated except that the enzyme diluent (E) was added instead of the enzyme solution, and a blank (ΔOD blank) was measured.
Immediately before the assay, the enzyme powder was dissolved in ice-cooled enzyme diluent (E) and diluted to 0.05-0.10 U / ml with the same buffer (use of a plastic tube for adhesion of the enzyme) Is preferred).
For the purpose of evaluating substrate specificity, the above activity measurement operation was carried out using a maltose solution as a substrate instead of a glucose solution.

Calculation Activity is calculated using the following formula:
U / ml = {ΔOD / min (ΔOD test−ΔOD blank) × Vt × df} / (16.8 × 1.0 × Vs)
U / mg = (U / ml) × 1 / C
Vt: Total volume (3.1 ml)
Vs: Sample volume (0.1 ml)
16.8: Millimolecular extinction coefficient of DCPIP under the above measurement conditions (cm ² / micromol)
1.0: Optical path length (cm)
df: Dilution factor C: Enzyme concentration in solution (c mg / ml)

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

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

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

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

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

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

Example 7: Examination of optimum temperature and thermal stability of PQQ-dependent GDH Using PQQ-dependent GDH obtained in Examples 1 to 5, the optimum temperature and thermal stability were examined.
As the optimum temperature, the relative activity (%) at each temperature was calculated with the temperature (40 ° C.) showing the maximum activity in 50 mM PIPES-NaOH (pH 6.5) buffer as 100% (FIG. 1). The optimum temperature of the PQQ-dependent GDH used for the study was 40 to 45 ° C.
Thermal stability is 50 mM PIPES-NaOH (pH 6.5), 1 mM CaCl ₂ composition and 5.0 U / ml PQQ-dependent GDH remaining after heat treatment at 30, 40, 50, 60, 70 ° C. for 30 minutes The GDH activity to be performed was compared with the activity value of the sample stored at 4 ° C., and the residual activity (%) at each temperature was calculated (FIG. 2). Until the heat treatment temperature was 40 ° C., the activity was not significantly decreased, and a tendency to be largely deactivated by the 50 ° C. treatment was observed. The thermal stability seems to vary depending on the solution composition and enzyme concentration. For example, if the enzyme concentration is increased, the temperature at which a large heat inactivation is observed is likely to increase (become 50 ° C. or higher).

Example 8: Effect on the stability of PQQ-dependent GDH by heating treatment Using the PQQ-dependent GDH obtained in Examples 1 to 5, the stability of the holo-type enzyme was improved before and after the heating treatment. We examined whether it changed. The PQQ-dependent GDH solution is diluted with 20 mM PIPES buffer (pH 6.5) and 1 mM CaCl _{2 so as} to have an activity of 20-30 U / ml, and this diluted solution is heated at 50 ° C. for 1 hour in a heat bath. went. After the heat treatment, the PQQ-dependent GDH activity (Test Example 1) before and after the heat treatment was compared after treatment under conditions (55 ° C., 1 hour) where heat inactivation occurs. The residual activity after the heat treatment before the heat treatment was 31%, but the residual activity after the heat treatment after the heat treatment was 42% after. It was revealed that the stability of PQQ-dependent GDH is increased by the heating treatment (Table 1). By performing the heating treatment, the holoformation rate of the enzyme was increased, which seemed to lead to an increase in stability.
Table 1 shows the results of studying the thermal stability effect exerted by the 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 heating treatment conditions were extensively examined by the same operation method as in Table 1 (Table 2). Even when the treatment was performed at a treatment temperature of 35 ° C. for about 30 minutes, the effect of improving the stability was recognized although it was weak. However, at a treatment temperature of 35 ° C., a sufficient effect was not seen even when the treatment was performed for a long time. In addition, even when the heating treatment temperature is 50 ° C. for 16 hours, the stability is not impaired, and for stabilization of GDH, treatment is performed for a long time at a temperature slightly lower than the enzyme denatures. Seemed to be the most effective. In this experiment, a PQQ-dependent GDH mutant whose stability was impaired by an amino acid mutation was used to make it easy to understand the effect of the heat treatment, but even if wild-type PQQ-dependent GDH is used, It has been confirmed that the stability is improved by heating treatment. In particular, mutants have reduced PQQ retention capacity due to structural distortion, and it is assumed that the structure of holo-type enzyme is calibrated by heating treatment, and the stability of holo-type enzyme itself is improved. To do. It seems that the same effect may be obtained by adding some energy to the enzyme other than the heating treatment.
Table 2 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%.

In addition, after the heating treatment (50 ° C., 16 hours), it was examined how the thermal stability (55 ° C., 1 hour heat treatment) changes by performing the heating treatment (50 ° C., 16 hours) again. (Table 3). When the heating treatment was insufficient, it was confirmed that the thermal stability was increased by performing the heating treatment again. From these examinations, it became clear that the effect of the heating treatment is proportional to the temperature and time, but does not increase beyond a certain level.
Table 3 shows the examination results of the effects in the reheating treatment after the heating treatment. The residual activity was calculated with the activity value of the sample stored at 4 ° C. after the heating treatment as 100%.

The composition of the present invention is a soluble coenzyme having improved thermostability produced by a method comprising a step of subjecting the composition to a heating treatment in a composition containing soluble coenzyme-bound glucose dehydrogenase It is a composition containing bound glucose dehydrogenase, and is an invention characterized by its production process.
In general, it is difficult to determine whether a certain composition has undergone a heating treatment step. However, in the composition of the present invention, from the results of Table 3 above, the second time at 50 ° C. for 16 hours. When the value of (activity remaining after heat treatment for the first time at 55 ° C. for 1 hour) / (activity remaining ratio after heat treatment for the second time at 55 ° C. for 1 hour) exceeds 60%. It can be estimated that it has been heated.

According to the present invention, it is possible to increase the stability of GDH itself by heating the GDH composition, and to provide a reagent having high storage stability by performing the heating process when producing a glucose measuring reagent. Is possible.

Optimal temperature for PQQ-dependent GDH Thermal stability of PQQ-dependent GDH

Claims (7)

A method for improving the stability of the enzyme, comprising a step of subjecting the composition to a heating treatment in a composition containing soluble coenzyme-linked glucose dehydrogenase. The method according to claim 1, wherein the coenzyme is pyrroloquinone quinoline or a flavin compound. The method for improving stability according to claim 1 or 2, wherein the heating treatment temperature is equal to or lower than a temperature at which a large temperature inactivation of the enzyme occurs. A composition comprising a soluble coenzyme-linked glucose dehydrogenase, comprising a soluble coenzyme-linked glucose dehydrogenase with improved thermal stability produced by a method comprising a step of subjecting the composition to a heating treatment Composition A method for measuring a glucose concentration using the composition of claim 4. A glucose sensor comprising the composition of claim 5. A method for producing a composition comprising a soluble coenzyme-bound glucose dehydrogenase having improved stability, comprising a step of subjecting the composition to a heating treatment in a composition comprising a soluble coenzyme-bound glucose dehydrogenase .