Method for measuring glucose using flavin-binding glucose dehydrogenase

Abstract

To accurately measure the amount of 10 mM or less of D-glucose.SOLUTION: A D-glucose is measured by contacting a flavin-binding type GDH with the D-glucose, at 20-40°C, wherein the flavin-binding type GDH has (i) an action: exhibiting GDH activity under the presence of an electron acceptor, (ii) a substrate specificity: having a less reactivity to maltose, D-galactose, and D-xylose as compared with that to D-glucose, (iii) a thermal stability: having 80% or more of a residual activity after a heat treatment at 40°C for 15 minutes, (iv) having a flavin compound as a coenzyme, and (v) a temperature characteristic: having an active value of 74-100% at 20-40°C when the reactivity to D-glucose at a measurement temperature with the highest activity in the range of 20-40°C is 100%.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a simple and accurate method for measuring glucose without temperature correction, using a flavin-binding glucose dehydrogenase derived from the subphylum Mucor, which uses a flavin compound as a coenzyme. [Background technology]

[0002] Blood glucose concentration (blood glucose level) is an important marker for diabetes. Self-monitoring of blood glucose (SMBG) devices using electrochemical biosensors are widely used by diabetic patients to manage their own blood glucose levels. The biosensors used in SMBG devices have traditionally used enzymes that use glucose as a substrate, such as glucose oxidase (GOD). However, because GOD has the property of accepting oxygen as an electron, dissolved oxygen in the measurement sample can affect the measurement value, potentially resulting in inaccurate measurements.

[0003] On the other hand, various glucose dehydrogenases (GDHs) are known as enzymes that use glucose as a substrate but do not use oxygen as an electron acceptor. Specifically, GDHs that use nicotinamide dinucleotide (NAD) or nicotinamide dinucleotide phosphate (NADP) as coenzymes (NAD(P)-GDH) and GDHs that use pyrroloquinoline quinone (PQQ) as coenzymes (PQQ-GDH) have been discovered and are used in biosensors for SMBG devices. However, NAD(P)-GDH has problems such as poor enzyme stability and the need for added coenzymes. Furthermore, PQQ-GDH has low substrate specificity and acts on sugar compounds other than glucose, such as maltose, D-galactose, and D-xylose, resulting in inaccurate measurements.

[0004] Approximately 10 years ago, when an SMBG device using PQQ-GDH as a biosensor was used to measure the blood glucose levels of diabetic patients receiving infusions, PQQ-GDH also reacted with maltose contained in the infusion, resulting in a higher-than-actual blood glucose reading. Treatment based on this reading resulted in the patient developing hypoglycemia and other conditions. 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 Ministry of Health, Labor, and Welfare's Pharmaceuticals and Food Safety Bureau conducted a cross-reactivity test to investigate the effect on blood glucose readings of adding various sugars to glucose solutions. When 600 mg / dL of maltose, 300 mg / dL of D-galactose, or 200 mg / dL of D-xylose were added, the readings of a blood glucose measurement kit using the PQQ-GDH method were 2.5 to 3 times higher than the actual glucose concentration. In other words, it has been found that the presence of maltose, D-galactose, and D-xylose in the measurement sample can cause inaccurate measurements, and there is a strong desire to develop a GDH with high substrate specificity that can specifically measure glucose without being affected by such sugar compounds that cause measurement errors.

[0005] Under the above circumstances, attention has been drawn to GDHs that utilize coenzymes other than those mentioned above, and for example, Patent Documents 1 to 3 disclose a glucose dehydrogenase (FAD-GDH) derived from the genus Aspergillus that uses flavin adenine dinucleotide (FAD) as a coenzyme, and Patent Document 4 discloses an FAD-GDH derived from the genus Aspergillus that has reduced activity toward D-xylose. Patent Documents 1 to 4 disclose FAD-GDHs that have low reactivity toward one or several sugar compounds other than D-glucose, although they cannot be said to have the property of being sufficiently low in reactivity toward maltose, D-galactose, or D-xylose.

[0006] In recent years, FAD-GDH derived from a microorganism belonging to the Mucorales subphylum has been discovered as a flavin-binding GDH that can accurately measure glucose concentrations in the presence of D-glucose, maltose, D-galactose, and D-xylose without being affected by these sugar compounds (see, for example, Patent Document 5). Furthermore, FAD-GDHs derived from Burkholderia cepacia and Circinella, as well as mutant enzymes with improved substrate specificity or improved heat resistance by introducing mutations into the various FAD-GDHs disclosed above, have been actively explored and developed (see, for example, Patent Documents 6 to 10).

[0007] Against this background, the various FAD-GDHs discovered to date are expected to be used as components of liquid reagents or as components immobilized on sensors or strips for the purpose of measuring blood glucose levels. However, in designing such measurement reagents and sensors, in addition to the aforementioned points of "avoiding the measurement of dissolved oxygen in the measurement sample as noise," "avoiding the addition of coenzymes," and "avoiding the measurement of sugars other than D-glucose that are expected to be mixed in the measurement sample as noise," there is another important need: "to minimize fluctuations in measurement values ​​due to temperature during measurement."

[0008] Generally, when measurements are performed using the principles of enzymatic methods, the phenomenon of measured values ​​varying depending on the temperature during measurement is recognized by those skilled in the art as a universal problem unavoidable when enzymes are used. Enzymes have an optimal reaction temperature, and when the reaction is performed at a temperature far from the optimal temperature condition for the reaction, the enzymatic reaction cannot be fully performed. Therefore, it is common technical knowledge among those skilled in the art that, if one wishes to obtain the same measured values ​​with the same performance, the temperature during measurement should be kept constant, and preferably set near the optimal reaction temperature. If the measurement is performed at a temperature far from that temperature, the measured values ​​will vary significantly, which may lead to measurement errors or incorrect diagnosis.

[0009] Temperature settings during measurement can be easily set and managed in laboratory-level measurements where a constant temperature can be easily maintained by pre-adjusting the temperature of the sample in a device such as a thermostatic bath, or when measurements are performed using equipment equipped with a temperature control function such as a heat retention function, so in reality there is often little risk of error occurring. On the other hand, in measurements where it is expected that the temperature control described above cannot be performed sufficiently due to individual circumstances, this problem poses a risk of serious errors occurring.

[0010] Diabetic patients self-monitor their blood glucose levels, known as SMBG, in their daily lives. In such situations, patients have a small, easy-to-use blood glucose monitor at home and collect a small amount of blood several times a day. The patient then soaks the blood onto a test strip and immediately inserts it into the monitor. Because the amount of blood collected is small, it is not possible to adjust the temperature beforehand. Many monitors do not have a temperature adjustment function. Therefore, the measured temperature can fluctuate by several tens of degrees between midsummer and midwinter in cold climates. Furthermore, even in cases where a temperature adjustment function is provided, it has been reported that temperature correction may not be performed properly if the temperatures of the SMBG device and the sensor differ (see, for example, Non-Patent Document 2).

[0011] In fact, it has been confirmed that the effect of temperature on self-monitoring of blood glucose using simple blood glucose measuring devices can result in measurement differences (see, for example, Non-Patent Document 3), and medical professionals, concerned that inaccurate values ​​could lead to incorrect judgments by patients, have suggested that it might be better to store both the SMBG device and test strips at room temperature, or to return them to room temperature before use when measuring, or to use a portable insulated case, etc. These measures are considered effective as on-site countermeasures, but on the other hand, they indicate that the issue of such errors is serious when it comes to self-monitoring of blood glucose using simple blood glucose measuring devices.

[0012] In addition to the guidance to maintain a constant temperature during use, such as performing measurements at room temperature or using an insulated case, a conventional solution that can be implemented on the measurement device itself is temperature compensation. This involves obtaining measurements in advance as the temperature changes, creating a relationship between the two. Then, the ambient temperature during measurement is measured, and the relationship is used to calculate the value that would be obtained if the measurement were performed at (for example) the optimal temperature. This method theoretically makes it possible to approach accurate values ​​through compensation, even if the measurement temperature fluctuates slightly. However, there are limitations to compensation, and it is not possible to fully compensate for enzymes whose activity decreases significantly with temperature fluctuations. Furthermore, incorporating a temperature compensation function would increase the size and cost of the measurement device, making it difficult to implement this method on all measurement devices.

[0013] For the reasons described above, there is a great need for a blood glucose measurement method that can perform measurements in a manner that is as little affected as possible by temperature fluctuations during measurement, or a measurement method that can reduce the need for time-consuming warming procedures or the introduction of a temperature correction mechanism. However, in reality, no measurement reagents or measurement devices capable of achieving such measurements have been known to date, and the only way to deal with this issue has been to devise a method of use based on the currently confirmed errors and to some extent to make temperature corrections. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-289148 [Patent Document 2] International Publication No. 04 / 058958 Brochure [Patent Document 3] International Publication No. 07 / 139013 Brochure [Patent Document 4] Japanese Patent Application Laid-Open No. 2008-237210 [Patent Document 5] Patent No. 4648993 [Patent Document 6] Japanese Patent Application Laid-Open No. 2012-90563 [Patent Document 7] International Publication No. 12 / 169512 Brochure [Patent Document 8] International Publication No. 09 / 084616 Brochure [Patent Document 9] International Publication No. 13 / 051682 Brochure [Patent Document 10] International Publication No. 13 / 065623 Brochure [Non-patent literature]

[0015] [Non-Patent Document 1] Pharmaceuticals and Medical Devices Safety Information No. 206, October 2004, Ministry of Health, Labour and Welfare, Pharmaceuticals and Food Safety Bureau [Non-patent document 2] Sase, M., and Takagi, M., "Effects of Blood Glucose Self-Monitoring Devices on Blood Glucose Levels in Low-Temperature Environments," Journal of the Japanese Society for Clinical Laboratory Automation, 34(4), 770 (2009). [Non-patent document 3] Junko Matsuzaki, Toshiya Okamoto, Yuri Ono, "Effect of temperature on self-monitoring of blood glucose using a simple blood glucose meter," Diabetes, 45(11), 821-824 (2002). Summary of the Invention [Problem to be solved by the invention]

[0016] An object of the present invention is to provide a method for measuring the amount of D-glucose using GDH, which is highly specific to D-glucose over a wide temperature range when measuring D-glucose at 10 mM or less, and can accurately measure the amount of D-glucose even in the coexistence of sugar compounds other than D-glucose. [Means for solving the problem]

[0017] In order to solve the above problems, the inventors conducted extensive research and screened various GDHs. As a result, they discovered that GDH derived from a strain belonging to the Mucorales subphylum can accurately measure glucose even when the substrate concentration is 10 mM or less, even when measurements are performed under a wide range of temperature conditions. They then constructed a new method for measuring D-glucose levels at blood glucose levels of 10 mM or less 2 hours after a meal, which is the blood glucose control index of the Japan Diabetes Society, and completed the present invention.

[0018] That is, the present invention is as follows. (1) The following characteristics (i) to (v): (i) Action: exhibits GDH activity in the presence of an electron acceptor; (ii) Substrate specificity: lower reactivity to maltose, D-galactose, and D-xylose compared to reactivity to D-glucose; (iii) Thermostability: 80% or more of the activity remains after heat treatment at 40°C for 15 minutes. (iv) A flavin compound is used as a coenzyme. (v) temperature characteristics: when the reactivity to D-glucose at the measurement temperature with the highest activity in the range of 20 to 40°C is taken as 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 a flavin-binding GDH comprising the above formula with D-glucose at 20 to 40°C. (2) The method of (1) above, wherein the concentration of D-glucose is 10 mM or less. (3) The method according to (1) above, wherein the flavin-binding GDH is derived from a microorganism classified into the Mucoral subphylum. (4) The method according to (1) above, wherein the flavin-binding GDH is derived from a microorganism classified into the genus Mucor or Circinella. (5) The following characteristics (i) to (v): (i) Action: exhibits GDH activity in the presence of an electron acceptor; (ii) Substrate specificity: lower reactivity to maltose, D-galactose, and D-xylose compared to reactivity to D-glucose; (iii) Thermostability: 80% or more of the activity remains after heat treatment at 40°C for 15 minutes. (iv) A flavin compound is used as a coenzyme. (v) temperature characteristics: when the reactivity to D-glucose at the measurement temperature with the highest activity in the range of 20 to 40°C is taken as 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 a glucose measuring method that does not involve temperature compensation at 20 to 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 Mucor. (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. [Effects of the Invention]

[0019] The measurement method using flavin-binding GDH of the present invention enables accurate blood glucose measurement at 20 to 40°C without a temperature correction function. DETAILED DESCRIPTION OF THE INVENTION

[0020] (Temperature characteristics of flavin-bound GDH) The flavin-bound GDH derived from the genus Mucor is characterized by a decrease in the substrate concentration, which leads to a decrease in fluctuation in activity over a wide measurement temperature range. Specifically, the flavin-bound GDH used in the measurement method of the present invention has an activity value of 74% to 100% at 20 to 40°C, assuming that the reactivity to 10 mM or less D-glucose at the measurement temperature with the highest activity in the range of 20 to 40°C is 100%. Because the measurement method using flavin-bound GDH of the present invention has such excellent temperature characteristics, it is possible to accurately measure the amount of D-glucose without being affected by the temperature environment during measurement.

[0021] (Enzymatic and chemical characteristics of the flavin-binding GDH of the present invention) Examples of enzymes preferred as the flavin-binding GDH of the present invention include those having the following enzymatic characteristics: (1) Action: Exhibits GDH activity in the presence of an electron acceptor (2) Substrate specificity: Compared to reactivity with D-glucose, reactivity with maltose, D-galactose, and D-xylose is low (3) Heat stability: 80% or more of the activity remains after heat treatment at 40°C for 15 minutes. (4) Uses flavin compounds as coenzymes (5) Temperature characteristics: When the reactivity to 10 mM or less of D-glucose at the measurement temperature with the highest activity in the range of 20 to 40°C is taken as 100%, the activity value at 20 to 40°C is 74% to 100%. GDH with the above-described enzymatic characteristics enables accurate measurement of D-glucose levels without being affected by sugar compounds such as maltose, D-galactose, and D-xylose contained in the measurement sample, and without a temperature correction function at measurement temperatures of 20 to 40° C. Furthermore, since it functions well within pH and temperature ranges suitable for application in clinical diagnosis such as blood glucose level measurement, it can be suitably used as a diagnostic measurement reagent (measurement agent), etc. Generally, the smaller the Km value, the better the substrate specificity. However, for the enzyme of the present invention, it is sufficient that the Km value be within a range that allows substantially sufficient substrate selection under specified measurement conditions. The molecular weight can be calculated from primary sequence information using a program such as GENETYX Ver. 11 (Genetyx) or ExPASy (http: / / web.expasy.org / compute_pi / ), or can be measured by SDS-polyacrylamide electrophoresis. For example, when calculated using GENETYX Ver. 11, the molecular weight of flavin-bound GDH having the amino acid sequence shown in SEQ ID NO: 1 is 70 kDa, and the molecular weight of flavin-bound GDH having the amino acid sequence shown in SEQ ID NO: 5 is 69 kDa. Furthermore, when measured by SDS-polyacrylamide electrophoresis, the molecular weight of flavin-bound GDH having the amino acid sequence shown in SEQ ID NO: 1 is approximately 80 kDa, and the molecular weight of flavin-bound GDH having the amino acid sequence shown in SEQ ID NO: 5 is approximately 88 kDa.

[0022] (Substrate specificity of flavin-bound GDH) The flavin-bound GDH that can be used in the present invention is characterized by excellent substrate specificity and extremely high selectivity for D-glucose. Specifically, the flavin-bound GDH that can be used in the present invention has extremely low reactivity with maltose, D-galactose, and D-xylose. Specifically, when the reactivity with D-glucose is taken as 100%, the reactivity with maltose, D-galactose, and D-xylose is each 5% or less, preferably 4% or less, more preferably 3% or less, and even more preferably 2% or less. Because the flavin-bound GDH that can be used in the present invention has such high substrate specificity, it is possible to accurately measure the amount of D-glucose in samples from patients receiving infusions containing maltose or patients undergoing galactose tolerance tests and xylose absorption tests without being affected by sugar compounds such as maltose, D-galactose, and D-xylose contained in the measurement sample.

[0023] The various enzymatic properties described above can be investigated using known techniques for identifying enzyme properties, for example, the methods described in the following Examples. The enzyme properties can be investigated to some extent in the culture medium of a microorganism that produces the flavin-binding GDH of the present invention or during the purification process, and more specifically, can be investigated using the purified enzyme. A purified enzyme refers to an enzyme that has been isolated to a state that is substantially free of components other than the enzyme, particularly proteins other than the enzyme (contaminating proteins). Specifically, for example, the content of contaminating proteins is less than about 20% by weight of the total, preferably less than about 10%, more preferably less than about 5%, and even more preferably less than about 1%. Unless otherwise specified, "MpGDH," as described later in this specification, refers to a purified enzyme.

[0024] In the present invention, the D-glucose concentration during D-glucose measurement is 10 mM or less. This concentration corresponds to the temperature range applicable when measuring D-glucose blood glucose levels 2 hours after a meal. In addition, during such measurements, it may be necessary to measure lower concentrations of D-glucose, such as 6 mM or less, 4 mM or less, or 3 mM or less.

[0025] The electron acceptor used by the flavin-binding GDH of the present invention is not particularly limited, and any electron acceptor known as a suitable reagent component for use in measuring blood glucose levels can be used, for example.

[0026] The coenzyme utilized by the flavin-binding GDH of the present invention is characterized by being a flavin compound, such as flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN).

[0027] A preferred example of the flavin-binding GDH enzyme that can be used in the measurement method of the present invention is a flavin-binding GDH whose activity at 20 to 40° C. is 74% to 100% when the reactivity to 10 mM D-glucose at the measurement temperature with the highest activity in the range of 20 to 40° C. is taken as 100%. More preferred is a flavin-binding GDH whose activity at 20 to 40° C. is 74% to 100% when the reactivity to 10 mM D-glucose at the measurement temperature with the highest activity in the range of 20 to 40° C. is taken as 100% and whose activity at 20° C. is 70% or more.

[0028] (Flavin-binding GDH action principle and activity measurement method) The 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, by utilizing this principle, the activity of the flavin-bound GDH of the present invention can be measured, for example, by the following measurement system using phenazine methosulfate (PMS) and 2,6-dichloroindophenol (DCIP) as electron acceptors. (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 type)

[0029] First, in reaction 1, glucose is oxidized to produce PMS (reduced form). In reaction 2, DCIP is reduced along with the oxidation of PMS, and the disappearance of oxidized DCIP can be measured from the change in absorbance at 600 nm. Specifically, in the present invention, the activity of flavin-bound GDH is measured according to the following procedure. 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. Next, 0.02 mL of 20 mM PMS solution and 0.1 mL of enzyme sample solution are added to initiate the reaction. The absorbance is measured at the start of the reaction and over time, and the decrease in absorbance at 600 nm per minute (ΔA600) associated with the progress of the enzyme reaction is determined. The flavin-bound GDH activity is calculated according to the following formula: 1 U of flavin-bound GDH activity is defined as the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of 50 mM D-glucose at 37°C.

[0030]

number

[0031] In the formula, 2.0 is the volume (mL) of the reaction reagent + enzyme reagent, and 16.3 is the millimolar extinction coefficient (cm) under these activity measurement conditions. 2 / μmol), 0.1 is the volume of the enzyme solution (mL), 1.0 is the optical path length of the cell (cm), ΔA600 blank represents the decrease in absorbance at 600 nm per minute when 10 mM acetate buffer was added instead of the enzyme sample solution to start the reaction, and df represents the dilution factor.

[0032] (Origin of flavin-binding GDH) The flavin-binding GDH of the present invention having the above-mentioned characteristics can be obtained from microorganisms classified into the subphylum Mucor. Examples of microorganisms classified into the subphylum Mucor include the genera Mucor, Absidia, and Actinomucor (Circinella). Specific preferred examples of microorganisms classified into the genus Mucor that produce the flavin-binding GDH of the present invention include Mucor prainii, Mucor javanicus, Mucor circinelloides f. circinelloides, Mucor subtilissimus, Mucor guilliermondii, and Mucor hiemalis. More specific examples include Mucor prainii NISL0103, Mucor javanicus NISL0111, and 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. Specific preferred examples of microorganisms classified into the genus Actinomucor (Circinella) that produce the flavin-binding GDH of the present invention include Actinomucor elegans or Circinella simplex. More specifically, Actinomucor elegans NISL9082 can be mentioned.

[0033] As described above, the flavin-binding GDH that can be used in the measurement method of the present invention is "a flavin-binding GDH derived from a microorganism classified in the subphylum Mucor and having the various properties described above." Furthermore, recombinant flavin-binding GDH produced by using a gene encoding a flavin-binding GDH obtained from such a flavin-binding GDH-producing microorganism by known genetic engineering techniques, partially modifying it if necessary, and introducing it into an appropriate host microorganism by various known techniques is also included in the "flavin-binding GDH derived from a microorganism classified in the subphylum Mucor and having the various properties described above" of the present invention. Similarly, the present invention also encompasses flavin-binding GDHs that have the various properties described above and are obtained based on genetic information derived from "microorganisms classified in the genus Mucor" or "microorganisms classified in the genus Actinomucor (Circinella)," or flavin-binding GDHs designated by the names of specific producing microbial strains.

[0034] (Amino acid sequence of flavin-binding GDH) The flavin-binding GDH of the present invention is characterized by having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5, or an amino acid sequence that is 70% or more homologous to said 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. In addition, GDH having an amino acid sequence that is 70% or more identical, preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, and most preferably 95% or more identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and that has properties similar to those of flavin-binding GDH having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5, is also included in the flavin-binding GDH used in the measurement method of the present invention.

[0035] (Gene sequence encoding flavin-binding GDH) The gene encoding flavin-binding GDH of the present invention refers to DNA encoding flavin-binding GDH having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5, or an amino acid sequence that is 70% or more homologous to said amino acid sequence. Alternatively, the gene encoding 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 flavin-binding GDH of the present invention refers to DNA having a nucleotide sequence that is 70% or more identical, preferably 75% or more identical, more preferably 80% or more identical, more preferably 85%, more preferably 90%, and most preferably 95% or more identical to the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6, and that encodes a protein having flavin-binding GDH enzyme activity.

[0036] (Vector and transformant containing a gene sequence encoding flavin-binding GDH) The gene encoding the flavin-binding GDH of the present invention can be inserted into various suitable known vectors. Furthermore, this vector can be introduced into various suitable known hosts to produce transformants into which recombinant DNA containing the flavin-binding GDH gene has been introduced. Methods for obtaining these genes, methods for obtaining gene sequence and amino acid sequence information, methods for producing various vectors, and methods for producing transformants are known to those skilled in the art, and examples will be described below.

[0037] To obtain a flavin-binding GDH gene from a microorganism that produces flavin-binding GDH, a commonly used gene cloning method is used. For example, chromosomal DNA or mRNA can be extracted from microbial cells or various cells capable of producing flavin-binding GDH by a conventional method, such as the method described in *Current Protocols in Molecular Biology* (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. A chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA obtained in this manner.

[0038] Next, a suitable probe DNA is synthesized based on the amino acid sequence of flavin-binding GDH and used to screen a chromosomal DNA or cDNA library, or a suitable primer DNA is prepared based on the amino acid sequence and a suitable polymerase chain reaction (PCR) such as 5' RACE or 3' RACE is used to amplify DNA containing the target gene fragment, which can then be ligated to obtain DNA containing the full-length target gene.

[0039] A preferred example of the gene encoding the flavin-binding GDH obtained in this manner is a flavin-binding GDH gene derived from the genus Mucor. For ease of handling, these genes are preferably linked to various vectors in the usual manner. For example, a recombinant plasmid containing the isolated gene encoding the flavin-binding GDH derived from the genus Mucor is prepared, and the gene can be extracted and purified from the plasmid using, for example, QIAGEN (Qiagen). Examples of vector DNA that can be used in the present invention include plasmid vector DNA and bacteriophage vector DNA. Specifically, for example, pBluescriptII SK+ (STRATAGENE) is preferred.

[0040] The nucleotide sequence of the flavin-binding GDH gene obtained by the above method can be determined and confirmed using, for example, a multi-capillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter). The flavin-binding GDH gene obtained as described above can be incorporated into a vector such as a bacteriophage, cosmid, or a plasmid used for transforming prokaryotic or eukaryotic cells by a conventional method, and a host corresponding to each vector can be transformed or transduced by a conventional method. Examples of hosts include microorganisms belonging to the genus Escherichia, such as E. coli K-12, preferably E. coli JM109 or DH5α (both manufactured by Takara Bio Inc.), and these hosts can be transformed or transduced to obtain the respective strains. Flavin-binding GDH can be mass-produced by culturing the transformant obtained in this manner. [Example]

[0041] (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, and the GDH activity of the crude enzyme solution was confirmed. The resulting crude enzyme was applied to a Butyl Toyopearl 650C (Tosoh Corporation) column (26φ × 28.5 cm) pre-equilibrated with buffer A (10 mM acetate buffer, 2 M ammonium sulfate, pH 5.0), and eluted 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 (Millipore) and then dialyzed against buffer C (10 mM acetate buffer, pH 4.5). The concentrate was applied to an SP Sepharose FastFlow (GE Healthcare) column (26φ × 28.5 cm) pre-equilibrated with buffer C, and eluted with a linear gradient from buffer C to buffer D (10 mM acetate buffer, 200 mM potassium chloride, pH 4.5). The eluted active fraction was concentrated to obtain the purified enzyme. [Example]

[0042] (Evaluation of the temperature characteristics of MpGDH) The temperature characteristics of the purified MpGDH preparation obtained in Example 1 were examined. In the GDH activity measurement method described above, activity was measured at heating and measurement temperatures of 20°C, 25°C, 30°C, 35°C, or 40°C. The substrate added was 5M glucose, 0.25M, 0.1M, or 0.075M.

[0043] [Table 1]

[0044] The percentages shown in parentheses in Table 1 are relative values ​​based on the activity value at the measurement temperature at which the activity was highest. As shown in Table 1, when the final substrate concentration was 200 mM, the relative activity at 20°C was 57%, assuming that the activity value at the measurement temperature at which MpGDH showed maximum activity was 100%. On the other hand, when the final substrate concentration was 10 mM, the relative activity at 20°C was 78%, assuming that the activity value at the measurement temperature at which MpGDH showed maximum activity was 100%, demonstrating very little variation.

[0045] Next, assuming a hypoglycemic patient, the same temperature characteristics were evaluated as above using 3 mM and 4 mM glucose as the substrate, and the relative activity was calculated, assuming the activity value at the measurement temperature showing maximum activity as 100% (Table 2).

[0046] [Table 2]

[0047] As shown in Table 2, when 3 mM and 4 mM glucose were used as the substrate, the activity fluctuation due to the measurement temperature was smaller than when 10 mM was used. In particular, it was found that the activity value was almost constant between 25°C and 35°C. This indicates that if a good activity value is maintained, accurate glucose measurement is possible even when using a measurement device without a temperature compensation function.

[0048] Thus, it is not common knowledge for those skilled in the art that there are enzymes that exhibit a tendency for activity to vary less with measurement temperature as the substrate concentration decreases. Generally, it is difficult to imagine that activity variations with measurement temperature would be significantly affected by slight variations in substrate concentration during measurement, and no such reports are known. Therefore, even when referring to sales catalogs for so-called enzyme products or descriptions in academic literature describing the properties of enzymes, the substrate concentration condition is almost always fixed at a single point in graphs showing the relationship between temperature and activity. Furthermore, in most cases, the substrate concentration in activity assays used to confirm enzyme properties is set to a sufficiently excessive concentration when calculating the initial reaction velocity, in accordance with the principles of enzyme activity assays (see Biochemical Experimental Chemistry Lectures 5, Enzyme Research Methods (Vol. 1), 1975).

[0049] This recognition by those skilled in the art is also shared in known literature on flavin-binding GDH, in which the measurement temperature and the glucose concentration used to confirm activity are fixed at sufficiently high levels, and the concentration range is far removed from the glucose concentration expected for actually measuring blood glucose levels (see, for example, Japanese Patent No. 4494978, JP-A Nos. 2011-152129, 2011-115156, WO 13 / 051682, WO 13 / 065623, and WO 13 / 118798).

[0050] It was confirmed that under conditions of a high glucose concentration of 200 mM, the activity of MpGDH significantly decreased as the temperature decreased from 30°C or less to 25°C and then to 20°C. However, even when measurements were performed under the same temperature conditions, when measurements were performed under conditions of a low glucose concentration of 10 mM, the decrease in activity was significantly improved, with almost 100% activity being maintained at 30°C, over 90% activity being maintained at 25°C, and approximately 80% activity being maintained at 20°C.

[0051] Comparative Example 1 The same temperature characteristic evaluation as in Example 2 was attempted using glucose dehydrogenase derived from Aspergillus oryzae (hereinafter referred to as AoGDH) (see JP 2008-228740 A). The 1782-bp gene (including the stop codon TAA) shown in SEQ ID NO: 4, which encodes the 593 amino acids of AoGDH shown in SEQ ID NO: 3 and is identical to the base sequence shown in JP 2008-228740 A, was obtained by PCR of a gene fragment using a standard method. At this time, an NdeI site and a BamHI site were added to the 5' and 3' ends of SEQ ID NO: 4, respectively.

[0052] Next, the following procedure was carried out to express the obtained gene shown in SEQ ID NO: 4 in E. coli. First, the gene synthesized above was treated with two restriction enzymes, NdeI and BamHI (Takara Bio Inc.), and inserted into the NdeI-BamHI site of pET22b(+)-Vector (Novagen) to obtain the recombinant plasmid pET22b(+)-AoGDH. This was then transformed under the same conditions as in Example 1, except that the E. coli strain used was the BL21(DE3) strain instead of the JM109 strain, to obtain the E. coli BL21(DE3)(pET22b(+)-AoGDH) strain.

[0053] The AoGDH-producing E. coli BL21(DE3) (pET22b(+)-AoGDH) strain obtained as described above was cultured in 100 ml of LB-amp medium supplemented with 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 25 mM (NH4)2SO4, 100 mM KH2PO4, 100 mM NaHPO4, and 1 mM MgSO4 at 30°C for 24 hours (modified from F. William Studier et al., Protein Expression and Purification (2005)). The cells were then washed with 0.02 M potassium phosphate buffer, pH 6.0, disrupted by sonication, and centrifuged at 9,000 rpm for 10 minutes to prepare 20 ml of AoGDH crude enzyme solution. The resulting crude enzyme solution was used as a sample, and the GDH activity was confirmed using the activity measurement method described in Example 1.

[0054] The crude enzyme was applied to a Butyl Toyopearl 650C (Tosoh) column pre-equilibrated with buffer A (10 mM potassium phosphate buffer, 2 M ammonium sulfate, pH 6.5) and eluted with a linear gradient from buffer A to buffer B (10 mM potassium phosphate, pH 6.5). The eluted active fraction was concentrated with a Centricon Plus-70 (Millipore) and dialyzed against buffer C (10 mM potassium phosphate buffer, pH 6.5). The eluted active fraction was applied to a CaptoQ (GE Healthcare) column pre-equilibrated with buffer C and eluted with a linear gradient from 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 the purified enzyme.

[0055] Using the obtained purified AoGDH enzyme, activity was measured at heating and measurement temperatures of 20°C, 25°C, 30°C, 35°C, or 40°C, as in Example 2. The substrate added was 5 M glucose or 0.25 M.

[0056] [Table 3]

[0057] As shown in Table 3, the temperature characteristics of AoGDH, i.e., the relationship between measurement temperature and activity, were not affected at all by the concentration of glucose, the substrate. This tendency is consistent with the general understanding of those skilled in the art that slight variations in the substrate concentration during measurement are unlikely to significantly affect activity due to the measurement temperature.

[0058] Comparative Example 2 Temperature characteristics were evaluated in the same manner as in Example 2 using glucose dehydrogenase derived from Aspergillus terreus (hereinafter referred to as AtGDH) (see Japanese Patent No. 5020070). A 1779 bp gene (including the stop codon TAA) shown in SEQ ID NO: 6, which encodes the 592 amino acids shown in SEQ ID NO: 5, which is the amino acid sequence of AtGDH shown in Japanese Patent No. 5020070, was obtained by PCR of the gene fragment using standard methods.

[0059] Using the same method as in Example 1, an AtGDH-expressing koji mold strain was transformed with a cassette containing the AtGDH gene to obtain a crude enzyme solution. The resulting crude enzyme was applied to a Butyl Toyopearl 650C (Tosoh Corporation) column pre-equilibrated with buffer A (10 mM potassium phosphate buffer, 2 M ammonium sulfate, pH 6.5) and eluted with a linear gradient from buffer A to buffer B (10 mM potassium phosphate, pH 6.5). The eluted active fraction was concentrated using an Amicon Ultra-15, 30K NMWL (Merck) and dialyzed against buffer C (10 mM potassium phosphate buffer, 150 mM NaCl, pH 6.5). The active fraction was then applied to a HiLoad 26 / 60 Superdex 200 pg (GE Healthcare) column pre-equilibrated with buffer C and eluted. The eluted active fraction was concentrated to obtain the purified enzyme. Using the obtained crude enzyme solution, the temperature characteristics were evaluated in the same manner as in Example 2. However, the activity was measured at heating and measurement temperatures of 20°C, 25°C, 30°C, 35°C, or 40°C. The substrate added was 5M glucose or 0.25M.

[0060] [Table 4]

[0061] As shown in Table 4, the temperature characteristics of AtGDH, i.e., the relationship between measurement temperature and activity, were not affected at all by the concentration of glucose, the substrate. This tendency is consistent with the general understanding of those skilled in the art that slight variations in the substrate concentration during measurement are unlikely to significantly affect activity due to the measurement temperature.

[0062] Comparative Example 3 Using glucose oxidase derived from Aspergillus niger (hereinafter referred to as AnGOD) (BBI, GO3B2), temperature characteristics were evaluated in the same manner as in Example 2. However, activity was measured at heating and measurement temperatures of 20°C, 25°C, 30°C, 35°C, or 40°C. The substrate added was 5M glucose or 0.05M.

[0063] [Table 5]

[0064] As shown in Table 5, the temperature characteristics of AnGOD, i.e., the relationship between measurement temperature and activity, were not affected at all by the concentration of glucose, the substrate. This tendency is consistent with the general understanding of those skilled in the art that slight variations in the substrate concentration during measurement are unlikely to significantly affect activity due to temperature-related changes in activity.

[0065] Example 3 A similar temperature characteristic evaluation as in Example 2 was attempted using glucose dehydrogenase derived from Mucor guilliermondii (hereinafter referred to as MgGDH). First, Mucor guilliermondii NBRC9403 strain was cultured in DPY medium (2% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% monopotassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate) at 30°C for 24 hours. After culture, the cells were harvested, dehydrated, and then disrupted in liquid nitrogen. Genomic DNA was isolated using a DNeasy Plant Mini Kit (QIAGEN) according to the attached protocol. Next, PCR was performed using the isolated genomic DNA as a template and degenerate primers of SEQ ID NOs: 7 and 8 with DNA polymerase PrimeSTAR Max (Takara Bio Inc.). Agarose gel electrophoresis confirmed that approximately 1.9 kb of DNA had been amplified. The amplified DNA fragment was purified and its base sequence analyzed using a multi-capillary DNA analysis system CEQ2000 (Beckman Coulter). Based on the sequence information, primers (SEQ ID NOS: 9 and 10) for the inverse PCR described below were designed. Next, the recovered genomic DNA was treated with the restriction enzyme NcoI, followed by phenol-chloroform treatment and ethanol precipitation. Ligation reactions were then carried out at 16°C for 8 hours using Ligation High ver. 2 (Toyobo Co., Ltd.). Using the ethanol-precipitated reaction solution as a template, inverse PCR was carried out using the primers (SEQ ID NOS: 9 and 10) designed above with DNA polymerase PrimeSTAR Max (Takara Bio Inc.). The amplified DNA fragment was sequenced as described above, and the upstream and downstream base sequences were analyzed, revealing the full-length base sequence of MgGDH (SEQ ID NOS: 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%.

[0066] Using the same method as in Example 1, a MgGDH-expressing koji mold strain was obtained by transformation with a cassette containing the MgGDH gene (SEQ ID NO: 12), and a crude enzyme solution was obtained. Using the obtained crude enzyme solution, temperature characteristics were evaluated in the same manner as in Example 2. However, activity measurements were performed at heating and measurement temperatures of 25°C or 37°C. The substrate added was 5M glucose or 0.25M.

[0067] [Table 6]

[0068] As shown in Table 6, it was also revealed that the activity of MgGDH varied less with the measurement temperature as the substrate concentration decreased. This tendency does not coincide with the general understanding of those skilled in the art that the activity variation due to the measurement temperature is unlikely to be significantly affected by slight variations in the substrate concentration during measurement.

[0069] When MgGDH is measured at a high glucose concentration of 200 mM, there is a difference in activity of nearly 40% between measurements at 37°C and 25°C. However, even when measurements are performed at the same temperature, when measurements are performed at a low glucose concentration of 10 mM, the decrease in activity is significantly improved, and the difference is limited to about 20%. In other words, when measurements are performed using MgGDH at glucose concentrations of around 10 mM or lower, accurate glucose measurements can be performed even with a measuring device without temperature compensation.

[0070] Temperature characteristics were evaluated in the same manner as in Example 2 using glucose dehydrogenase derived from Mucor RD056860 (hereinafter referred to as MrdGDH) having the amino acid sequence shown in SEQ ID NO: 14 (see WO 2013 / 118798).

[0071] Using the same method as in Example 1, an MrdGDH-expressing koji mold strain was obtained by transformation with a cassette containing the MrdGDH gene (SEQ ID NO: 15), and a crude enzyme solution was obtained. The obtained crude enzyme solution was purified using the same method as in Example 1, and temperature characteristics were evaluated in the same manner as in Example 2. However, activity measurements were performed at heating and measurement temperatures of 20°C, 25°C, 30°C, 35°C, or 40°C. The substrate added was 5 M glucose or 0.05 M.

[0072] [Table 7]

[0073] As shown in Table 7, it was also revealed that for MrdGDH, the lower the substrate concentration, the smaller the fluctuation in activity due to measurement temperature.

[0074] Temperature characteristics were evaluated in the same manner as in Example 2 using glucose dehydrogenase derived from Mucor hiemalis f. silvaticus NBRC6754 (hereinafter referred to as MhGDH) (see WO 2013 / 065623), which has the amino acid sequence shown in SEQ ID NO: 16.

[0075] Using the same method as in Example 1, an MhGDH-expressing koji mold strain was obtained by transformation with a cassette containing the MhGDH gene (SEQ ID NO: 17), and a crude enzyme solution was obtained. The obtained crude enzyme solution was purified using the same method as in Example 1, and temperature characteristics were evaluated in the same manner as in Example 2. However, activity measurements were performed at heating and measurement temperatures of 20°C, 25°C, 30°C, 35°C, or 40°C. The substrate added was 5 M glucose or 0.05 M.

[0076] [Table 8]

[0077] As shown in Table 8, it was also revealed that for MhGDH, the lower the substrate concentration, the smaller the activity fluctuation due to measurement temperature.

[0078] It was previously unknown that GDHs from Mucorales are enzymes whose activity is less affected by temperature changes as the substrate concentration decreases. The findings obtained this time have led to the new idea that the Mucorales GDHs MpGDH, MgGDH, MdrGDH, and MhGDH have remarkable properties that are extremely advantageous for the specific practical application of routine, simple blood glucose measurement, and that by utilizing these properties, it may be possible to realize a measurement device without temperature compensation.

[0079] MpGDH, MgGDH, MdrGDH and MhGDH belong to the same Mucorales subphylum and share 73-79% amino acid sequence identity. Therefore, various GDHs from similar Mucorales subphylum, particularly GDHs with sequence identity of 73% or more to MpGDH or MgGDH, are likely to have the favorable property that the lower the glucose concentration, the smaller the fluctuation in activity due to measurement temperature, inferred from their close relationship and high enzymatic structural identity. [Industrial Applicability]

[0080] The method for measuring D-glucose using flavin-binding GDH of the present invention can accurately quantify D-glucose concentrations over a wide temperature range, and is therefore useful in fields such as measuring blood glucose levels and quantifying glucose concentrations in foods.

Claims

1. A method for screening flavin-binding GDH for use in a method for measuring D-glucose at 4 mM or less, comprising: preparing flavin-binding GDH; The prepared flavin-binding GDH has the following properties (i) to (v): (i) Action: exhibits GDH activity in the presence of an electron acceptor; (ii) substrate specificity: lower reactivity to maltose, D-galactose, and D-xylose compared to reactivity to D-glucose; (iii) Thermal stability: 80% or more of the residual activity is retained after heat treatment at 40°C for 15 minutes. (iv) A flavin compound is used as a coenzyme. (v) temperature characteristics: when the reactivity to D-glucose at the measurement temperature with the highest activity in the range of 20 to 40°C is taken as 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 checking whether the When the flavin-binding GDH has the properties (i) to (v), the screening method comprises contacting the flavin-binding GDH with D-glucose at 20 to 40°C to obtain a flavin-binding GDH for use in a method for measuring D-glucose.

2. A method for producing a glucose measuring agent or sensor capable of measuring 4 mM or less of D-glucose, comprising using a flavin-binding GDH capable of measuring 4 mM or less of D-glucose, preparing flavin-binding GDH; The prepared flavin-binding GDH has the following properties (i) to (vi): (i) Action: exhibits GDH activity in the presence of an electron acceptor; (ii) substrate specificity: lower reactivity to maltose, D-galactose, and D-xylose compared to reactivity to D-glucose; (iii) Thermal stability: 80% or more of the residual activity is retained after heat treatment at 40°C for 15 minutes. (iv) A flavin compound is used as a coenzyme. (v) temperature characteristics: when the reactivity to D-glucose at the measurement temperature with the highest activity in the range of 20 to 40°C is taken as 100%, the activity value at 20 to 40°C is 74% to 100%, and the activity value at 20°C is 70% or more; (vi) a molecular weight of 70 kDa or about 80 kDa as measured by SDS-polyacrylamide gel electrophoresis; a step of checking whether the When the flavin-binding GDH has the properties (i) to (vi), a step of incorporating the flavin-binding GDH into a glucose measuring agent for contacting with D-glucose at 20 to 40°C or into a sensor; A method for producing a glucose measuring agent or sensor capable of measuring 4 mM or less of D-glucose, comprising:

3. The method according to claim 2, comprising using a flavin-binding GDH capable of measuring D-glucose at 3 mM or less, wherein the glucose measuring agent or sensor is capable of measuring D-glucose at 3 mM or less.

4. The method according to claim 2, comprising using a flavin-binding GDH capable of measuring D-glucose at 2 mM or less, wherein the glucose measuring agent or sensor is capable of measuring D-glucose at 2 mM or less.

5. The method according to any one of claims 2 to 4, wherein the glucose measuring agent or sensor is for contacting flavin-binding GDH with D-glucose at 20 to 35°C.

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