JP7109155B2 - Method for measuring glucose using flavin-binding glucose dehydrogenase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a simple and accurate glucose measurement method that does not require temperature correction, using a flavin-binding glucose dehydrogenase derived from the subphylum Mucoralida using a flavin compound as a coenzyme.

Blood glucose concentration (blood sugar level) is an important marker of diabetes. Self-monitoring of blood glucose (SMBG) devices using electrochemical biosensors are widely used as devices for diabetic patients to manage their own blood sugar levels. Biosensors used in SMBG devices conventionally utilize enzymes that use glucose as a substrate, such as glucose oxidase (GOD). However, since GOD has the property of using oxygen as an electron acceptor, in SMBG equipment using GOD, dissolved oxygen in the measurement sample affects the measured value, and accurate measured value may not be obtained. can happen.

On the other hand, various glucose dehydrogenases (hereinafter referred to as GDH) are known as other enzymes that use glucose as a substrate but do not use oxygen as an electron acceptor. Specifically, a type of GDH (NAD (P)-GDH) with nicotinamide dinucleotide (NAD) or nicotinamide dinucleotide phosphate (NADP) as a coenzyme, and pyrroloquinoline quinone (PQQ) as a coenzyme A similar GDH (PQQ-GDH) has been found and used in biosensors in SMBG devices. However, NAD(P)-GDH has the problem of poor enzyme stability and the need to add a coenzyme. also acts on sugar compounds such as maltose, D-galactose, and D-xylose, so sugar compounds other than glucose in the measurement sample affect the measured value, and accurate measured values cannot be obtained. A point exists.

About 10 years ago, PQQ-GDH was used as a biosensor to measure the blood glucose level of diabetic patients who were receiving infusions. As a result, it has been reported that a measured value higher than the actual blood sugar level was obtained, and a patient developed hypoglycemia or the like due to treatment based on this value. It has also been found that similar events can occur in patients undergoing galactose tolerance tests and xylose absorption tests (see, for example, Non-Patent Document 1). In response to this, the Pharmaceutical and Food Safety Bureau of the Ministry of Health, Labor and Welfare conducted a cross-reactivity test for the purpose of investigating the effects on blood glucose measurement values when each sugar was added to a glucose solution. of D-galactose or 200 mg/dL of D-xylose, the measured value of the blood glucose measurement kit using the PQQ-GDH method is 2.5 to 3 times higher than the actual glucose concentration. showed the value. That is, it was found that maltose, D-galactose, and D-xylose that may be present in the measurement sample make the measurement value inaccurate. Development of GDH with high substrate specificity that can be specifically measured is earnestly desired.

Under the above background, GDH of the type that utilizes a coenzyme other than the above has been attracting attention. is disclosed, and Patent Document 4 discloses FAD-GDH derived from the genus Aspergillus with reduced activity on D-xylose. In Patent Documents 1 to 4, although it cannot be said that it has a sufficiently low reactivity to any of maltose, D-galactose, and D-xylose, one or several types that are not D-glucose FAD-GDH is disclosed that is less reactive to sugar compounds of

Subsequently, in recent years, D-glucose, maltose, D-galactose, under conditions where D-xylose is present, flavin-binding GDH capable of accurately measuring glucose concentration without being affected by those sugar compounds As such, FAD-GDH derived from a microorganism belonging to the subphylum Mucoralcoidea has been found (see, for example, Patent Document 5). Furthermore, FAD-GDH derived from Burkholderia cepacia and Circinella, mutant enzymes with improved substrate specificity by introducing mutations into the disclosed FAD-GDH, and mutant enzymes with improved heat resistance. etc. have been vigorously explored and developed (see, for example, Patent Documents 6 to 10).

Against this background, various FAD-GDHs that have been found so far have been used as components of liquid reagents or as components immobilized in sensors or strips for the purpose of measuring blood sugar levels. However, in designing such measurement reagents and measurement sensors, the above-mentioned "I do not want to measure dissolved oxygen in the measurement sample as noise", "I do not want to add coenzyme", and "Measurement I do not want to measure sugars other than D-glucose that are assumed to be mixed in the sample as noise”, there is another important need. That is the need to "avoid as much as possible fluctuations in measured values due to temperature during measurement."

In general, when measuring using the principle of the enzymatic method, the phenomenon that the measured value fluctuates depending on the temperature at the time of measurement is recognized by those skilled in the art as an unavoidable universal problem as long as enzymes are used. . Enzymes have an optimum reaction temperature, and when the reaction is carried out at a temperature far from the optimum temperature condition for the reaction, the enzymatic reaction cannot be sufficiently exhibited. Therefore, if one wants to obtain the same measured value with the same performance, it is common technical knowledge for those skilled in the art that the temperature during measurement should be kept constant and preferably set near the optimum reaction temperature. If the measurement is performed at a temperature that deviates from that temperature, the measured value may fluctuate greatly, leading to measurement errors and erroneous diagnoses.

The temperature setting at the time of measurement should be done at the laboratory level where it is easy to keep the temperature constant during measurement by adjusting the temperature of the sample in advance in a device such as a constant temperature bath. If the measurement is performed using a device equipped with a temperature adjustment function such as a heat retention function, it is easy to set and manage, so in reality, there are many cases where the risk of error occurrence is unlikely to occur. .
On the other hand, this problem poses a serious risk of error occurrence in measurements where it is expected that the above temperature adjustment cannot be performed sufficiently due to individual circumstances.

The so-called SMBG scene in which a diabetic self-monitors blood glucose is a daily life. In such situations, the patient has a small and convenient measuring device at home, and several times a day, he/she collects a small amount of blood by himself and puts it on a piece of paper (test strip) for testing. After soaking it, it is immediately inserted into the measuring device and the measurement is performed. Due to the small amount of blood to be collected, it is not possible to adjust the temperature in advance. Many models of measuring devices do not have a temperature adjustment function, so it is assumed that the measured temperature fluctuates by several tens of degrees between measurements in midsummer and in cold regions in midwinter. Furthermore, even when the temperature adjustment function is provided, it has been reported that temperature correction may not be properly performed when the temperature of the SMBG device differs from that of the sensor (see, for example, Non-Patent Document 2).

In fact, regarding the effect of temperature on self-monitoring of blood glucose by a simple blood glucose measuring device, it has been confirmed that there is a difference in measurement (see, for example, Non-Patent Document 3), and the value is unacceptable by medical personnel. Due to concerns that inaccuracies may lead to patient misjudgment, both the SMBG device and test strips should be stored at room temperature, or allowed to cool to room temperature before use, or carried in a portable thermal case. It has also been suggested that devising such as using These ideas are thought to be effective as countermeasures taken from the field, but on the other hand, it shows that the problem of such errors is serious in self-monitoring of blood glucose using a simple blood glucose measuring device. .

Along with the instruction to keep the temperature constant during use, such as measuring at room temperature and using a heat insulating case, there is temperature correction as a conventional measure that can be taken on the side of the measuring device. That is, a relational expression between the two is created by obtaining measurement values based on temperature changes in advance. Then, the ambient temperature at the time of measurement is measured, and a relational expression is used to calculate from the actual measured value (for example) the value when measured at the optimum temperature. If this method is used, even if the measured temperature fluctuates a little, it is theoretically possible to approximate an accurate value by correction. However, there is a limit to correction, and for enzymes whose activity is significantly reduced by temperature fluctuations, the correction cannot be completed, and by incorporating a temperature correction function, the size and cost of the measurement device are increased. There is a problem that it is difficult to adopt this method for all measuring devices due to the fact that it is difficult to store them.

Due to the circumstances described above, in blood sugar level measurement, it is necessary to introduce a measurement method that is less susceptible to the effects of temperature fluctuations during measurement as much as possible, or introduce a time-consuming task of keeping warm and a temperature correction mechanism. It can be said that there is a great need for a measurement method that can reduce the need to perform
However, in reality, no measurement reagents or measurement devices capable of achieving such measurements have been known to date. The reality was that there was no other way to deal with it than to do

JP 2007-289148 A WO 04/058958 Pamphlet WO 07/139013 pamphlet Japanese Patent Application Laid-Open No. 2008-237210 Patent No. 4648993 JP 2012-90563 A WO 12/169512 pamphlet WO 09/084616 pamphlet WO 13/051682 pamphlet WO 13/065623 pamphlet

Pharmaceuticals and Medical Devices Safety Information No. 206, October 2004, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare Shojiro Sase, Masayoshi Takagi, "Influence of blood glucose self-monitoring device on blood glucose level in low temperature environment", Journal of Japanese Society of Clinical Laboratory Automation, 34(4), 770 (2009). Junko Matsuzaki, Toshiya Okamoto, Yuri Ono, ``Influence of temperature on self-measurement of blood glucose by a simple blood glucose meter'', Diabetes, 45 (11), 821-824 (2002).

In the present invention, when measuring D-glucose of 10 mM or less, in a wide temperature range, it is highly specific to D-glucose, and the amount of D-glucose can be accurately measured even under the coexistence conditions of sugar compounds other than D-glucose. An object of the present invention is to provide a method for measuring the amount of D-glucose using measurable GDH.

In order to solve the above problems, the present inventors have made extensive studies and conducted various GDH screenings. We newly found that glucose can be measured accurately even when measured under conditions, and constructed a new method for measuring the amount of D-glucose at 2-hour postprandial blood sugar levels of 10 mM or less, which is the blood sugar control index of the Japan Diabetes Society. , completed the present invention.

That is, the present invention is as follows.
(1) the following properties (i) to (v):
(i) action: showing GDH activity in the presence of an electron acceptor;
(ii) substrate specificity: low reactivity to maltose, D-galactose, and D-xylose compared to reactivity to D-glucose;
(iii) thermal stability: having a residual activity of 80% or more after heat treatment at 40° C. for 15 minutes;
(iv) with a flavin compound as a coenzyme,
(V) Temperature characteristics: When the reactivity to D-glucose at the measurement temperature where the activity is highest in the range of 20 to 40 ° C. is 100%, the activity value at 20 to 40 ° C. is 74% to 100%. And the activity value at 20 ° C. is 70% or more,
A method for measuring D- glucose, comprising contacting flavin-binding GDH with D- glucose at 20 to 40 ° C.
(2) The measuring method according to (1) above, wherein the concentration of D-glucose is 10 mM or less.
(3) Flavin-binding GDH is derived from microorganisms classified in the subphylum Malocoria, the measurement method according to (1) above.
(4) Flavin-binding GDH is derived from a microorganism classified into the genus Mucor or Circinella, the measurement method according to (1) above.
(5) the following properties (i) to (v):
(i) action: showing GDH activity in the presence of an electron acceptor;
(ii) substrate specificity: low reactivity to maltose, D-galactose, and D-xylose compared to reactivity to D-glucose;
(iii) thermal stability: having a residual activity of 80% or more after heat treatment at 40° C. for 15 minutes;
(iv) with a flavin compound as a coenzyme,
(V) Temperature characteristics: When the reactivity to D-glucose at the measurement temperature where the activity is highest in the range of 20 to 40 ° C. is 100%, the activity value at 20 to 40 ° C. is 74% to 100%. And the activity value at 20 ° C. is 70% or more,
A glucose measuring agent or sensor for glucose measurement methods without temperature correction at 20-40° C. comprising flavin-binding GDH comprising:
(6) The glucose measuring agent or sensor according to (5) above, wherein the concentration of D-glucose in the measurement sample is 10 mM or less.
(7) The glucose-measuring agent or sensor according to (5) above, wherein the flavin-binding GDH is derived from a microorganism classified into the subphylum Mucormophyta.
(8) The glucose measuring agent or sensor according to (5) above, wherein the flavin-binding GDH is derived from a microorganism classified into the genus Mucor or Circinella.

By the measurement method using the flavin-binding GDH of the present invention, accurate blood glucose measurement is possible at 20 to 40 ° C. without a temperature correction function.

(Temperature characteristics of flavin-binding GDH)
Mucor genus-derived flavin-binding GDH is characterized in that the lower the substrate concentration, the smaller the activity fluctuation in a wide measurement temperature range. Specifically, the flavin-binding GDH used in the measurement method of the present invention has the highest activity in the range of 20 to 40 ° C. When the reactivity to 10 mM or less D-glucose at the measurement temperature is 100%, 20 Activity values at ~40°C range from 74% to 100%. Measurement method using flavin-binding GDH of the present invention has such excellent temperature characteristics, without being affected by the temperature environment to be measured, it is possible to accurately measure the amount of D- glucose.

(Enzymechemical characteristics of flavin-binding GDH of the present invention)
Examples of preferred enzymes as flavin-binding GDH of the present invention include those having the following enzymatic chemical characteristics.
(1) Action: GDH activity in the presence of an electron acceptor (2) Substrate specificity: Low reactivity to maltose, D-galactose, and D-xylose compared to reactivity to D-glucose (3) Thermal stability: 80% or more residual activity after heat treatment at 40 ° C. for 15 minutes (4) Uses flavin compound as a coenzyme (5) Temperature characteristics: At the measurement temperature where the activity is highest in the range of 20 to 40 ° C. When the reactivity to D-glucose of 10 mM or less is defined as 100%, the activity value at 20 to 40°C is 74% to 100%.
If GDH has the above enzymatic chemical characteristics, it is not affected by sugar compounds such as maltose, D-galactose, and D-xylose contained in the measurement sample, and the measurement temperature is 20 to 40 ° C. It is possible to accurately measure the amount of D-glucose without having a temperature correction function. In addition, since it works well in a suitable pH range and temperature range for application to clinical diagnosis such as measurement of blood sugar level, it can be suitably used for applications such as a diagnostic measurement reagent (measuring agent).
It is generally said that the smaller the Km value, the better the substrate specificity. However, the enzyme of the present invention has a value within a range in which substantially sufficient substrate selection is realized under predetermined measurement conditions. All you have to do is
For molecular weight, for example, GENETYX Ver. 11 (manufactured by Genetics) or ExPASy (http://web.expasy.org/compute_pi/) or other programs, it can be calculated from primary sequence information or measured by SDS-polyacrylamide electrophoresis. For example, GENETYX Ver. 11, the molecular weight of flavin-binding GDH having the amino acid sequence shown by SEQ ID NO: 1 is 70 kDa, and the molecular weight of flavin-binding GDH having the amino acid sequence shown by SEQ ID NO: 5 is 69 kDa. be. Further, for example, when measured by SDS-polyacrylamide electrophoresis, the molecular weight of the flavin-binding GDH having the amino acid sequence represented by SEQ ID NO: 1 is about 80 kDa, and the flavin binding having the amino acid sequence represented by SEQ ID NO: 5 The molecular weight of type GDH is approximately 88 kDa.

(Substrate specificity of flavin-binding GDH)
Flavin-binding GDH that can be used in the present invention is characterized by excellent substrate specificity and extremely high selectivity for D-glucose. Specifically, flavin-binding GDH that can be used in the present invention, maltose, D- galactose, D- xylose very low reactivity. Specifically, when the reactivity to D-glucose is 100%, the reactivity to maltose, D-galactose and D-xylose is 5% or less, preferably 4% or less, more preferably 3% or less. , and more preferably 2% or less. Flavin-binding GDH that can be used in the present invention, because it has such a high substrate specificity, and patients receiving an infusion containing maltose, galactose load test and xylose absorption test of the patient during the As for the sample, it is possible to accurately measure the amount of D-glucose without being affected by sugar compounds such as maltose, D-galactose, and D-xylose contained in the measurement sample.

The various enzymatic properties described above can be examined using known techniques for specifying various properties of enzymes, such as the methods described in the Examples below. Various properties of the enzyme can be examined to some extent in the culture solution of the microorganism that produces the flavin-binding GDH of the present invention, or in the middle of the purification process, and more specifically, can be examined using a purified enzyme.
A purified enzyme refers to an enzyme separated in a state substantially free of components other than the enzyme, particularly proteins other than the enzyme (contaminant proteins). Specifically, for example, the content of contaminating proteins is less than about 20%, preferably less than about 10%, more preferably less than about 5%, and even more preferably less than about 1% by weight. “MpGDH” described later in this specification refers to a purified enzyme unless otherwise specified.

In the present invention, the D-glucose concentration when measuring D-glucose is 10 mM or less. This concentration corresponds to a temperature range that corresponds to the measurement of D-glucose in the blood sugar level 2 hours after a meal. Moreover, in such measurements, D-glucose at a lower concentration may be measured in some cases. For example, it may be 6 mM or less, 4 mM or less, or 3 mM or less.

The electron acceptor utilized by the flavin-binding GDH of the present invention is not particularly limited, for example, any electron acceptor known as a reagent component suitable for use in blood glucose measurement can be used.

The coenzyme utilized by the flavin-binding GDH of the present invention is characterized by being a flavin compound. Flavin compounds include, for example, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and the like.

Examples of preferred enzymes as flavin-binding GDH that can be used in the measurement method of the present invention, when the reactivity to 10mM D-glucose at the measurement temperature with the highest activity in the range of 20 ~ 40 ° C. is 100% , flavin-binding GDH with an activity value of 74% to 100% at 20 to 40 ° C. More preferably, the activity value at 20 to 40 ° C. is 74% to 100% when the reactivity to 10 mM D-glucose at the measurement temperature where the activity is highest in the range of 20 to 40 ° C. is 100%, and Flavin-binding GDH having an activity value of 70% or more at 20°C.

(Principle of action and activity measurement method of flavin-binding GDH)
Flavin-binding GDH of the present invention catalyzes the reaction of oxidizing the hydroxyl group of glucose in the presence of an electron acceptor to produce glucono-δ-lactone.
Therefore, using this principle, for example, the following measurement system using phenazine methosulfate (PMS) and 2,6-dichloroindophenol (DCIP) as electron acceptors, the activity of flavin-binding GDH of the present invention can be measured.
(Reaction 1) D-glucose + PMS (oxidized form)
→ D-glucono-δ-lactone + PMS (reduced form)
(Reaction 2) PMS (reduced form) + DCIP (oxidized form)
→ PMS + DCIP (reduced form)

First, in (Reaction 1), PMS (reduced form) is produced as glucose is oxidized. Since DCIP is reduced along with the oxidation of PMS in the subsequent reaction (reaction 2), the disappearance of oxidized DCIP can be measured from the amount of change in absorbance at a wavelength of 600 nm.
Specifically, in the present invention, the activity of flavin-binding GDH is measured according to the following procedures. 1.79 mL of 100 mM phosphate buffer (pH 7.0), 0.08 mL of 5.0 M D-glucose solution and 0.01 mL of 20 mM DCIP solution are mixed and incubated at 37° C. for 5 minutes. 0.02 mL of 20 mM PMS solution and 0.1 mL of enzyme sample solution are then added to initiate the reaction. At the start of the reaction, and, to measure the absorbance over time, the amount of decrease in absorbance per minute at 600 nm with the progress of the enzyme reaction (ΔA600) is calculated, and the flavin-binding GDH activity is calculated according to the following formula. At this time, the flavin-binding GDH activity defines 1U as the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of D-glucose at a concentration of 50 mM at 37°C.

In the formula, 2.0 is the liquid volume (mL) of the reaction reagent + enzyme reagent, 16.3 is the millimole molecular extinction coefficient (cm ² /μmol) under the activity measurement conditions, and 0.1 is the liquid volume of the enzyme solution. (mL), 1.0 is the optical path length of the cell (cm), ΔA600 _blank is the amount of decrease per minute in absorbance at 600 nm when 10 mM acetate buffer is added instead of the enzyme sample solution to start the reaction, df represents the dilution factor.

(Origin of flavin-binding GDH)
The flavin-binding GDH of the present invention having the characteristics described above can be obtained from microorganisms classified in the subphylum Mucoralida. Examples of microorganisms classified into the subphylum Mucor include the genus Mucor, the genus Absidia, the genus Actinomucor (Circinella), and the like. Examples of preferred specific microorganisms that are classified into the genus Mucor and produce the flavin-binding GDH of the present invention include Mucor brainii, Mucor javanicus, and Mucor circinelloides f. circinelloides, Mucor subtilissimus, Mucor guilliermondii or Mucor hiemalis. More specifically, Mucor prainii NISL0103, Mucor javanicus NISL0111 or Mucor circinelloides f. circinelloides NISL0117. Specific preferred examples of microorganisms classified into the genus Absidia that produce the flavin-binding GDH of the present invention include Absidia cylindrospora and Absidia hyalospora. More specifically, Absidia cylindrospora NISL0211 and Absidia hyalospora NISL0218 can be mentioned. Actinomucor (Circinella) microorganisms classified into the genus, specific preferred examples of microorganisms that produce the flavin-binding GDH of the present invention, Actinomucor elegans or Circinella simplex can be mentioned. More specifically, Actinomucor elegans NISL9082 can be mentioned.

Flavin-binding GDH that can be used in the measurement method of the present invention is, as described above, "flavin-binding GDH derived from microorganisms classified in the subphylum Mucoralida and having the above-described various properties". Furthermore, using a gene encoding flavin-binding GDH obtained by known genetic engineering techniques from these flavin-binding GDH-producing microorganisms, partially modifying it as necessary, Various known to appropriate host microorganisms Recombinant flavin-binding GDH introduced and produced by the technique of is also included in the "flavin-binding GDH derived from microorganisms classified in the subphylum Mucoralida and having the above-described various properties" of the present invention. Similarly, "microorganisms classified in the genus Mucor" or "microorganisms classified in the genus Actinomucor (Circinella)", or for flavin-binding GDH that describes a specific production microorganism strain name, genetic information derived from each The present invention also includes flavin-binding GDH having the above-described various properties obtained based on.

(Amino acid sequence of flavin-binding GDH)
The flavin-binding GDH of the present invention is characterized by having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 5, or an amino acid sequence that is 70% or more homologous to the amino acid sequence. Flavin-binding GDH having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5 has the various properties described above. Also, 70% or more identity, preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, most preferably 95%, with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 GDH having an amino acid sequence having the above identity and having properties similar to those of the flavin-binding GDH having the amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 5 is also a flavin-binding type used in the measurement method of the present invention. Included in GDH.

(Gene sequence encoding flavin-binding GDH)
The gene encoding the flavin-binding GDH of the present invention, the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5, or DNA encoding flavin-binding GDH having an amino acid sequence that is 70% or more homologous to the amino acid sequence Say. Alternatively, the gene encoding the flavin-binding GDH of the present invention refers to DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6. Alternatively, the gene encoding the flavin-binding GDH of the present invention, 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6, preferably 75% or more identity, more preferably, 80% or more identity, more preferably 85%, more preferably 90%, most preferably having a nucleotide sequence with 95% or more identity, and DNA encoding a protein having flavin-binding GDH enzymatic activity Say.

(Vector containing gene sequence encoding flavin-binding GDH, transformant)
The gene encoding the flavin-binding GDH of the present invention can be inserted into various known appropriate vectors. Furthermore, this vector can be introduced into each known suitable host to prepare a transformant into which a recombinant DNA containing a flavin-binding GDH gene has been introduced. Methods for obtaining these genes, methods for obtaining information on gene sequences and amino acid sequences, methods for producing various vectors, and methods for producing transformants are known to those skilled in the art, and examples thereof will be described later.

To obtain the flavin-binding GDH gene from microorganisms that produce flavin-binding GDH, commonly used gene cloning method is used. For example, chromosomal DNA or mRNA can be extracted from microbial cells and various cells having flavin-binding GDH-producing ability by a conventional method, for example, the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. The chromosomal DNA or cDNA thus obtained can be used to construct a library of chromosomal DNA or cDNA.

Then, based on the amino acid sequence of flavin-binding GDH, a method of synthesizing an appropriate probe DNA and using it to screen from a chromosomal DNA or cDNA library, or based on the amino acid sequence, an appropriate primer DNA. DNA containing the target gene fragment is amplified by an appropriate polymerase chain reaction (PCR method) such as the 5'RACE method or the 3'RACE method, and these are ligated to obtain a DNA containing the full-length target gene. Obtainable.

A preferred example of a gene encoding flavin-binding GDH thus obtained is a flavin-binding GDH gene derived from the genus Mucor. These genes are preferably ligated to various vectors in a conventional manner for handling, for example, to prepare a recombinant plasmid containing a gene encoding an isolated flavin-binding GDH derived from the genus Mucor, where can be obtained by extraction and purification from, for example, QIAGEN (manufactured by Qiagen). Vector DNA that can be used in the present invention includes, for example, plasmid vector DNA, bacteriophage vector DNA, and the like. Specifically, for example, pBluescriptII SK+ (manufactured by STRATAGENE) and the like are preferable.

Determination and confirmation of the base sequence of the flavin-binding GDH gene obtained by the above method can be performed, for example, by using a multi-capillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter, Inc.).
The flavin-binding GDH gene obtained as described above is incorporated into a vector such as a plasmid used for transforming bacteriophage, cosmid, or prokaryotic cells or eukaryotic cells by a conventional method, and the host corresponding to each vector can be transformed or transduced by conventional methods. Examples of the host include microorganisms belonging to the genus Escherichia, such as Escherichia coli K-12, preferably Escherichia coli JM109, DH5α (both manufactured by Takara Bio Inc.), and the like. to obtain the respective strains. By culturing the transformant thus obtained, flavin-binding GDH can be produced in large amounts.

(Production and purification of MpGDH)
An Aspergillus sojae strain (see Japanese Patent No. 4648993) transformed with a cassette containing the MpGDH gene (SEQ ID NO: 2) was cultured to confirm the GDH activity of the crude enzyme solution. The obtained crude enzyme was applied to a Butyl Toyopearl 650C (manufactured by Tosoh Corporation) column (26φ×28.5 cm) pre-equilibrated with buffer A (10 mM acetate buffer, 2 M ammonium sulfate, pH 5.0), Elution was performed with a linear gradient from buffer A to buffer B (10 mM acetate buffer, pH 5.0). The eluted active fraction was concentrated with Centricon Plus-70 (manufactured by Millipore), dialyzed against buffer C (10 mM acetate buffer, pH 4.5), and pre-equilibrated with buffer C with SP Sepharose FastFlow (GE Healthcare) column (26φ×28.5 cm) and eluted with a linear gradient of buffer C to buffer D (10 mM acetate buffer, 200 mM potassium chloride, pH 4.5). The eluted active fraction was concentrated to obtain a purified enzyme.

(Temperature characteristic evaluation of MpGDH)
The temperature characteristics of the purified MpGDH preparation obtained in Example 1 were examined.
In the GDH activity measurement method shown above, the activity was measured at a heating temperature and a measurement temperature of 20°C, 25°C, 30°C, 35°C or 40°C. The substrate to be added was 5M glucose, 0.25M, 0.1M or 0.075M.

The ratio shown in parentheses in Table 1 is a relative value based on the activity value at the measurement temperature at which the activity was highest. As shown in Table 1, when the final concentration of the substrate was 200 mM, the relative activity at 20° C. was 57% when the activity value at the measurement temperature at which MpGDH exhibits maximum activity was taken as 100%. On the other hand, when the final concentration of the substrate is 10 mM, the relative activity at 20 ° C. is 78% when the activity value at the measurement temperature where MpGDH exhibits the maximum activity is 100%, and it is clear that the fluctuation is very small. became.

Subsequently, assuming a hypoglycemic patient, the same temperature characteristic evaluation as above was performed using 3 mM and 4 mM glucose as a substrate, and the relative activity was calculated when the activity value at the measurement temperature showing the maximum activity was taken as 100% ( Table 2).

As shown in Table 2, when 3 mM and 4 mM glucose were used as substrates, the activity fluctuation due to measurement temperature was further reduced compared to when 10 mM was used. In particular, it was found that the activity value was almost constant between 25°C and 35°C. It was shown that accurate glucose levels can be measured even when

Thus, it is not common knowledge for those skilled in the art that there are enzymes that exhibit a tendency that the lower the substrate concentration is, the smaller the activity fluctuations due to the measurement temperature are. In general, it is difficult to think that activity changes due to measurement temperature are greatly affected by slight changes in substrate concentration during measurement, and such reports are not known. Therefore, even with reference to sales catalogs of so-called enzyme products and descriptions in academic literature showing various properties of enzymes, the substrate concentration condition is fixed at one point in the graph showing the relationship between temperature and activity. is mostly In most cases, the substrate concentration in the activity measurement method for confirming various properties of the enzyme is set to a sufficiently excessive concentration when calculating the initial reaction rate, in accordance with the principle of the enzyme activity measurement method. (See Biochemical Experimental Chemistry Course 5, Enzyme Research Method (1), 1975).

Even in the literature known to the public flavin-binding GDH, this recognition of those skilled in the art is the same, the glucose concentration at the time of confirming the measurement temperature and activity is fixed to a sufficiently high, the concentration range is actually blood glucose It was far from the glucose concentration assumed to measure the value (for example, Japanese Patent No. 4494978, Japanese Unexamined Patent Publication No. 2011-152129, Japanese Unexamined Patent Publication No. 2011-115156, International Publication No. 13/ 051682 pamphlet, WO 13/065623 pamphlet, WO 13/118798 pamphlet).

It was confirmed that the activity of MpGDH decreased greatly as the temperature was lowered to 30°C or lower, 25°C, and 20°C under a high glucose concentration condition of 200 mM. However, even when the measurement was performed under the same temperature conditions, the decrease in activity was remarkably improved when the measurement was performed under the low glucose concentration condition of 10 mM, and almost 100% activity was maintained at 30°C and 25°C. It was found that more than 90% of the activity was retained even at 20°C, and about 80% of the activity was retained even at 20°C.

Comparative example 1
The same temperature characteristic evaluation as in Example 2 was attempted using Aspergillus oryzae-derived glucose dehydrogenase (hereinafter referred to as AoGDH) (see JP-A-2008-228740).
The 1782 bp gene shown in SEQ ID NO: 4, which is the amino acid sequence of AoGDH, encodes 593 amino acids shown in SEQ ID NO: 3, and is exactly the same as the nucleotide sequence shown in JP-A-2008-228740 (stop codon TAA ) was obtained by PCR of the gene fragment, which is a standard method. At this time, an NdeI site and a BamHI site were added to the 5' end and 3' end of SEQ ID NO: 4, respectively.

Subsequently, the following procedure was carried out in order to express the obtained gene shown in SEQ ID NO: 4 in E. coli. First, the gene synthesized above was treated with two types of restriction enzymes, NdeI site BamHI (manufactured by Takara Bio Inc.), and inserted into the NdeI-BamHI site of pET22b(+)-Vector (manufactured by Novagen). A recombinant plasmid pET22b(+)-AoGDH was obtained, and under the same conditions as in Example 1, E. coli to be used was transformed as BL21(DE3) strain instead of JM109 strain, and E. coli BL21(DE3) (pET22b(+ )-AoGDH) strain was obtained.

Escherichia coli BL21 (DE3) (pET22b(+)-AoGDH) strain having AoGDH-producing ability obtained as described above was added to LB-amp medium with 0.5% glycerol, 0.05% glucose, and 0.2% α. - cultured for 24 hours at 30°C in 100 ml of medium supplemented with lactose, 25 mM (NH4)2SO4, 100 mM KH2PO4, 100 mM NaHPO4, 1 mM MgSO4 (F. William Studier et al., Protein Expression and Purification (2005) modification). Thereafter, the cells were washed with 0.02 M potassium phosphate buffer of pH 6.0, sonicated, and centrifuged at 9,000 rpm for 10 minutes to prepare 20 ml of AoGDH crude enzyme solution. Using the obtained crude enzyme solution as a sample, the activity of GDH was confirmed according to the activity measurement method according to Example 1.

Using the obtained crude enzyme, it is applied to a Butyl Toyopearl 650C (manufactured by Tosoh Corporation) column pre-equilibrated with buffer A (10 mM potassium phosphate buffer, 2 M ammonium sulfate, pH 6.5), and buffer is removed from buffer A. Elution was performed with a linear gradient of solution B (10 mM potassium phosphate, pH 6.5). The eluted active fraction was concentrated with Centricon Plus-70 (manufactured by Millipore), dialyzed against buffer C (10 mM potassium phosphate buffer, pH 6.5), and CaptoQ (GE Healthcare Co.) column and eluted with a linear gradient of buffer C to buffer D (10 mM potassium phosphate buffer, 1 M potassium chloride, pH 6.5). The eluted active fraction was concentrated to obtain a purified enzyme.

Using the obtained AoGDH-purified enzyme, the activity was measured in the same manner as in Example 2 at a heating temperature of 20°C, 25°C, 30°C, 35°C or 40°C. Also, the substrate to be added was 5M glucose or 0.25M.

As shown in Table 3, the temperature characteristics of AoGDH, that is, the relationship between measured temperature and activity were not affected by the substrate glucose concentration at all. Such a trend is consistent with the general recognition of those skilled in the art that it is difficult to imagine that activity changes due to measurement temperature are greatly affected by slight changes in substrate concentration during measurement.

Comparative example 2
Aspergillus terreus-derived glucose dehydrogenase (hereinafter referred to as AtGDH) (
(See Japanese Patent No. 5020070), and the same temperature characteristic evaluation as in Example 2 was attempted.
The 1779 bp gene (including the stop codon TAA) shown in SEQ ID NO: 6, which encodes 592 amino acids shown in SEQ ID NO: 5, which is the amino acid sequence of AtGDH shown in Patent No. 5020070, was obtained by PCR of the gene fragment, which is a standard method. , cDNA was obtained.

By the same method as in Example 1, an AtGDH-expressing koji strain was obtained by transformation using a cassette containing the AtGDH gene, and a crude enzyme solution was obtained. Using the obtained crude enzyme, it is applied to a Butyl Toyopearl 650C (manufactured by Tosoh Corporation) column pre-equilibrated with buffer A (10 mM potassium phosphate buffer, 2 M ammonium sulfate, pH 6.5), and buffer is removed from buffer A. Elution was performed with a linear gradient of solution B (10 mM potassium phosphate, pH 6.5). The eluted active fraction was concentrated with Amicon Ultra-15, 30K NMWL (manufactured by Merck), dialyzed against buffer C (10 mM potassium phosphate buffer, 150 mM NaCl, pH 6.5), and then dialyzed against buffer C in advance. It was applied to an equilibrated HiLoad 26/60 Superdex 200 pg (manufactured by GE Healthcare) column and eluted. The eluted active fraction was concentrated to obtain a purified enzyme.
Temperature characteristics were evaluated in the same manner as in Example 2 using the obtained crude enzyme solution. However, the activity was measured at a heating temperature and a measuring temperature of 20°C, 25°C, 30°C, 35°C or 40°C. Moreover, the substrate to be added was 5M glucose or 0.25M.

As shown in Table 4, the temperature characteristics of AtGDH, that is, the relationship between measurement temperature and activity, were not affected by the substrate glucose concentration at all. Such a trend is consistent with the general recognition of those skilled in the art that it is difficult to imagine that activity changes due to measurement temperature are greatly affected by slight changes in substrate concentration during measurement.

Comparative example 3
Temperature characteristics were evaluated in the same manner as in Example 2 using Aspergillus niger-derived glucose oxidase (hereinafter referred to as AnGOD) (GO3B2 manufactured by BBI). However, the activity was measured at a heating temperature and a measuring temperature of 20°C, 25°C, 30°C, 35°C or 40°C. Also, the substrate to be added was 5M glucose or 0.05M.

As shown in Table 5, the temperature characteristics of AnGOD, that is, the relationship between measured temperature and activity, were not affected by the substrate glucose concentration at all. Such a trend is consistent with the general recognition of those skilled in the art that it is difficult to imagine that activity changes due to measurement temperature are greatly affected by slight changes in substrate concentration during measurement.

Example 3
The same temperature characteristic evaluation as in Example 2 was attempted using Mucor guilliermondii-derived glucose dehydrogenase (hereinafter referred to as MgGDH). First, Mucor guilliermondii NBRC9403 strain was added to DPY medium (2% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% monopotassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate), 30 C. for 24 hours. After culturing, the cells were collected, water was removed from the cells, the cells were disrupted in liquid nitrogen, and genomic DNA was obtained using DNeasy Plant Mini Kit (QIAGEN) according to the attached protocol. Next, using the obtained genomic DNA as a template, PCR is performed using DNA polymerase PrimeSTAR Max (manufactured by Takara Bio Inc.) using degenerate primers of SEQ ID NOs: 7 and 8, and about 1.9 kb of DNA is subjected to agarose gel electrophoresis. was confirmed to be amplified. This amplified DNA fragment was purified, and the base sequence was analyzed using a multi-capillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter). Based on the sequence information, primers (SEQ ID NOS: 9 and 10) for inverse PCR used below were designed.
Next, the genomic DNA collected above was treated with a restriction enzyme NcoI, followed by phenol/chloroform treatment and ethanol precipitation, followed by Ligation High ver. 2 (manufactured by Toyobo Co., Ltd.), the ligation reaction was carried out at 16° C. for 8 hours. Using the ethanol-precipitated reaction solution as a template, inverse PCR was performed using the primers of SEQ ID NOs: 9 and 10 designed above and DNA polymerase PrimeSTAR Max (manufactured by Takara Bio Inc.). Sequence analysis of the amplified DNA fragment was performed in the same manner as described above, and the upstream and downstream nucleotide sequences were analyzed, revealing the full-length nucleotide sequence of MgGDH (SEQ ID NO: 11). Furthermore, based on the sequence information of MpGDH, the exon sequence (SEQ ID NO: 12) excluding the intron sequence was predicted, and the amino acid sequence of MgGDH (SEQ ID NO: 13) was determined. The amino acid sequence identity between MgGDH and MpGDH was 78%.

By the same method as in Example 1, a MgGDH-expressing koji strain was obtained by transformation using a cassette containing the MgGDH gene (SEQ ID NO: 12) to obtain a crude enzyme solution. Temperature characteristics were evaluated in the same manner as in Example 2 using the obtained crude enzyme solution. However, the activity was measured at a heating temperature and a measuring temperature of 25°C or 37°C. Also, the substrate to be added was 5M glucose or 0.25M.

As shown in Table 6, it was also revealed that the lower the substrate concentration, the smaller the activity fluctuation due to the measurement temperature for MgGDH. Such a trend does not agree with the general recognition of those skilled in the art that it is difficult to imagine that activity changes due to measurement temperature are greatly affected by slight changes in substrate concentration during measurement.

MgGDH produces a nearly 40% difference in activity between measurements at 37°C and 25°C under conditions of a high glucose concentration of 200 mM. However, even when the measurement was performed under the same temperature conditions, the decrease in activity was remarkably improved when the measurement was performed under the low glucose concentration condition of 10 mM, and the difference was only about 20%. That is, it was shown that when measuring glucose concentration of about 10 mM or lower using MgGDH, it is possible to accurately measure the amount of glucose even when using a measuring device that does not have a temperature compensation function. was done.

The same temperature characteristic evaluation as in Example 2 was attempted using Mucor RD056860-derived glucose dehydrogenase (hereinafter referred to as MrdGDH) (see International Publication 2013/118798) having the amino acid sequence shown in SEQ ID NO: 14.

A MrdGDH-expressing koji strain was obtained by transformation using a cassette containing the MrdGDH gene (SEQ ID NO: 15) in the same manner as in Example 1, and a crude enzyme solution was obtained. Using the obtained crude enzyme solution, purification was performed by the same method as in Example 1, and the temperature characteristics were evaluated in the same manner as in Example 2. However, the activity was measured at a heating temperature and a measuring temperature of 20°C, 25°C, 30°C, 35°C or 40°C. Also, the substrate to be added was 5M glucose or 0.05M.

As shown in Table 7, it was also revealed that the lower the substrate concentration, the smaller the variation in the activity of MrdGDH due to the measurement temperature.

Evaluation of temperature characteristics in the same manner as in Example 2 using Mucor hiemalis f. silvaticus NBRC6754-derived glucose dehydrogenase (hereinafter referred to as MhGDH) having the amino acid sequence represented by SEQ ID NO: 16 (see International Publication 2013/065623) tried.

By the same method as in Example 1, an MhGDH-expressing koji strain was obtained by transforming with a cassette containing the MhGDH gene (SEQ ID NO: 17) to obtain a crude enzyme solution. Using the obtained crude enzyme solution, purification was performed by the same method as in Example 1, and the temperature characteristics were evaluated in the same manner as in Example 2. However, the activity was measured at a heating temperature and a measuring temperature of 20°C, 25°C, 30°C, 35°C or 40°C. Also, the substrate to be added was 5M glucose or 0.05M.

As shown in Table 8, it was also revealed that the lower the substrate concentration, the smaller the activity fluctuation due to the measurement temperature for MhGDH.

Until now, it was not known that the lower the substrate concentration, the less susceptible the activity of GDH derived from the subphylum Mucophyta is to the change in activity due to the measurement temperature. Based on the findings obtained this time, MpGDH, MgGDH, MdrGDH and MhGDH derived from Mucophyta have remarkable properties that are very advantageous in specific practical applications such as daily simple measurement of blood sugar levels, and they are utilized. This led to a new idea that it would be possible to implement a measuring device that does not have a temperature compensation function.

MpGDH, MgGDH, MdrGDH and MhGDH belong to the same subphylum Mucoral phylum, and their amino acid sequence identity is 73-79%. is 73% or more, presumed from its closeness and high structural identity as an enzyme, the lower the glucose concentration, the smaller the activity fluctuation due to the measurement temperature. There is a high probability that there will be.

Using the flavin-binding GDH of the present invention, the method for measuring D- glucose can accurately quantify D- glucose concentration in a wide temperature range, so measurement of blood sugar level and quantification of glucose concentration in food It is useful in fields such as

Claims (1)

A screening method for flavin-binding GDH for use in a method for measuring D-glucose of 10 mM or less,
A step of preparing a flavin-binding GDH,
The prepared flavin-binding GDH has the following properties (i) to (v):
(i) action: showing GDH activity in the presence of an electron acceptor;
(ii) substrate specificity: low reactivity to maltose, D-galactose, and D-xylose compared to reactivity to D-glucose;
(iii) thermal stability: having a residual activity of 80% or more after heat treatment at 40° C. for 15 minutes;
(iv) with a flavin compound as a coenzyme,
(V) Temperature characteristics: When the reactivity to D-glucose at the measurement temperature where the activity is highest in the range of 20 to 40 ° C. is 100%, the activity value at 20 to 40 ° C. is 74% to 100%. And the activity value at 20 ° C. is 70% or more,
and when the flavin-binding GDH has the properties of (i) to (v), the flavin-binding GDH is contacted with D- glucose at 20 to 40 ° C. , A screening method as a flavin-binding GDH for use in a method for measuring D-glucose.