Method for modifying substrate specificity of glucose dehydrogenase and agent for modifying substrate specificity of glucose dehydrogenase

Abstract

The present invention addresses the problem of modifying the substrate specificity of a glucose dehydrogenase.　Provided are: a method for modifying the substrate specificity of a glucose dehydrogenase, by using an agent which is for modifying the substrate specificity of the glucose dehydrogenase and is a low molecular weight compound as a glucose analog; and an agent for modifying the substrate specificity of a glucose dehydrogenase.

Description

[Technical field]

[0001] The present disclosure relates to a method for modifying the substrate specificity of glucose dehydrogenase and a glucose dehydrogenase substrate specificity modifying agent. [Background technology]

[0002] Glucose measurement is used for monitoring blood glucose levels in diabetic patients, etc. Glucose oxidase (hereinafter, also referred to as GOD) and glucose dehydrogenase (hereinafter, also referred to as GDH) are usually used to quantify glucose.

[0003] Glucose oxidase is an oxidoreductase that catalyzes the oxidation of β-D-glucose to D-glucono-1,5-lactone (gluconolactone). Glucose oxidase uses oxygen as the electron acceptor and flavin adenine dinucleotide (FAD) as a cofactor.

[0004] Glucose dehydrogenase is classified as an oxidoreductase, and catalyzes the reaction of glucose and an electron acceptor as substrates to produce gluconolactone and a reduced acceptor. Examples of glucose dehydrogenase include nicotinamide dinucleotide-dependent GDH, nicotinamide dinucleotide phosphate-dependent GDH, pyrroloquinoline quinone (PQQ)-dependent GDH, and FAD-dependent GDH (flavin-binding GDH).

[0005] When measuring blood glucose using an enzyme that uses glucose as a substrate, the substrate specificity of the enzyme can be a problem. In particular, reactivity to maltose can be a problem. For example, wild-type PQQ-GDH is not specific to glucose, and also reacts with other sugars such as maltose, galactose, and xylose. Non-Patent Document 1 describes a problem in which, in glucose measurement using PQQ-GDH, a glucose value that is erroneously increased above the actual value due to maltose and the like present in the system is obtained, which causes hypoglycemia. Therefore, in glucose measurement using an enzyme, a measurement reagent that is highly specific to glucose and has low reactivity to other sugars such as maltose is required.

[0006] In order to increase the substrate specificity for glucose or decrease the reactivity for maltose, approaches have been attempted to search for glucose dehydrogenases (GDHs) with excellent substrate specificity, and various FAD-dependent GDHs have been found (see Non-Patent Documents 2 and 3). In addition, an approach has been attempted in which the gene for isolated GDH is obtained and genetically engineered to modify the substrate specificity.

[0007] On the other hand, apart from the specificity for glucose, the stability of the enzyme can be a problem. Patent Document 1 (JP 2015-139376 A) describes a method for increasing the stability of glucose dehydrogenase by adding smectite, which is a mineral.

[0008] Patent Document 2 (International Publication WO 2005 / 054840) aims to provide a method for measuring blood components that can sufficiently and accurately correct the amount of blood components by measuring hematocrit with high accuracy and high reliability, and a sensor and device used therein, and describes a device containing glucose dehydrogenase. Paragraph 0027 lists sugar alcohols as enzyme stabilizers, and maltitol is preferred. Maltitol is also used in the examples.

[0009] Patent Document 3 (JP Patent Publication 2005-114359) aims to provide a component measuring method and sensor that do not require a complicated correction process, and describes a method for measuring glucose in blood and a sensor used therefor. Paragraph 0016 lists sugar alcohols as enzyme stabilizers, with maltitol being preferred. Maltitol is used in the examples.

[0010] Patent Document 4 (International Publication WO 2001 / 025776) describes a glucose sensor using PQQ-GDH. On page 8, it is described that a stabilizer may be added to the reaction layer of the biosensor, and sugar is disclosed as a stabilizer. Sugars are also listed, including glucose and maltose. There is no description of the use of sugar as a stabilizer in the examples.

[0011] Patent Document 5 (JP 2009-195250 A) describes a method for improving the stability of a composition containing soluble glucose dehydrogenase, and describes a composition containing a recombinant FAD-GDH derived from Aspergillus oryzae or Aspergillus terreus and trehalose.

[0012] Patent Document 6 (JP 2014-018096 A) describes a method for improving the stability of a composition containing flavin-binding glucose dehydrogenase. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] JP 2015-139376 (Patent No. 6402887) [Patent Document 2] International Publication No. 2005 / 054840 Brochure [Patent Document 3] Patent Publication No. 2005-114359 [Patent Document 4] International Publication No. 2001 / 025776 Brochure [Patent Document 5] JP 2009-195250 (Patent No. 5176045) [Patent Document 6] JP 2014-018096 (Patent No. 6101011) [Non-patent literature]

[0014] [Non-Patent Document 1] Frias, et. al., Diabetes Care, Vol. 33, No. 4, April 2010, pp. 728-729 [Non-Patent Document 2] Tsujimura S, et. al., Biosci Biotechnol Biochem., Vol. 70, 2006, pp. 654-659 [Non-Patent Document 3] Satake R, et. al., J Biosci Bioeng., Vol. 120, 2015, pp. 498-503 Summary of the Invention [Problem to be solved by the invention]

[0015] An object of the present disclosure is to provide a method for modifying the substrate specificity of FAD-dependent glucose dehydrogenase by using a small molecular weight compound that is a glucose analog, and a glucose dehydrogenase substrate specificity modifier that is a small molecular weight compound that is a glucose analog. [Means for solving the problem]

[0016] In view of the above problems, the present inventors have intensively investigated a method for modifying the reaction of non-glucose saccharides such as maltose by a method other than the approach of searching for a new GDH enzyme or the approach of modifying the substrate specificity by introducing a mutation into an existing GDH. As a result, it has been surprisingly found that a low-molecular-weight compound that is a specific glucose analogue modifies the reactivity of FAD-dependent glucose dehydrogenase to non-glucose saccharides (e.g., maltose), and the present invention has been completed.

[0017] That is, the present disclosure includes the following embodiments. [1] A method for modifying the substrate specificity of glucose dehydrogenase by using a glucose dehydrogenase substrate specificity modifier, which is a glucose analog and a low molecular weight compound, comprising allowing the glucose dehydrogenase substrate specificity modifier to coexist with glucose dehydrogenase when measuring glucose using FAD-dependent glucose dehydrogenase (FAD-GDH), and wherein the ratio (Mal / Glu) of the reactivity of glucose dehydrogenase to maltose (Mal) and the reactivity (Glu) to glucose of the glucose dehydrogenase in the presence of the modifier is modified compared to the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity (Glu) to glucose of the glucose dehydrogenase in the absence of the modifier. [2] The method of embodiment 1, wherein the glucose dehydrogenase substrate specificity modifier comprises one or more compounds selected from the group consisting of sorbitol, D-iditol, L-iditol, D-glucal, ribitol, L-gulose, trehalose, D-mannitol, xylitol, and glycerol. [3] The method of embodiment 1 or 2, wherein the glucose dehydrogenase is derived from the genus Mucor or Aspergillus. [4] The method according to any one of embodiments 1 to 3, wherein the glucose dehydrogenase is immobilized on a solid surface. [5] The method according to any one of embodiments 1 to 3, wherein the glucose dehydrogenase is not immobilized on a solid surface. [6] A glucose dehydrogenase substrate specificity modifier, which is a low molecular weight compound that is an analog of glucose, and which modifies the ratio (Mal / Glu) of the reactivity of glucose dehydrogenase to maltose (Mal) and the reactivity (Glu) to glucose of FAD-dependent glucose dehydrogenase (FAD-GDH) in the presence of the modifier compared to the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity (Glu) to glucose of FAD-GDH in the absence of the modifier. [7] The glucose dehydrogenase substrate specificity modifying agent of embodiment 6, comprising one or more compounds selected from the group consisting of sorbitol, D-iditol, L-iditol, D-glucal, ribitol, L-gulose, trehalose, D-mannitol, xylitol, and glycerol. [8] The glucose dehydrogenase substrate specificity modifying agent described in embodiment 6, comprising one or more compounds selected from the group consisting of sorbitol, D-iditol, L-iditol, D-glucal, ribitol, L-gulose, D-mannitol, and glycerol. [9] The modifying agent described in any one of embodiments 6 to 8, wherein the glucose dehydrogenase is derived from the genus Mucor or Aspergillus.

[10] A glucose measurement system comprising the glucose dehydrogenase substrate specificity modifier according to any one of embodiments 6 to 9 and glucose dehydrogenase immobilized on a solid surface.

[11] A glucose measurement system comprising the glucose dehydrogenase substrate specificity modifier according to any one of embodiments 6 to 9 and glucose dehydrogenase that is not immobilized on a solid surface.

[12] A composition or reagent for measuring glucose, comprising the glucose dehydrogenase substrate specificity modifier according to any one of embodiments 6 to 9 and glucose dehydrogenase.

[13] A composition or reagent for measuring glucose described in embodiment 12, comprising a glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase as separate reagents.

[14] A composition for measuring glucose or a reagent for measuring glucose as described in embodiment 12, comprising a glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase in the same reagent.

[15] A glucose measurement method using the modifying agent according to any one of embodiments 6 to 9, the system according to embodiment 10 or 11, or the composition or reagent according to any one of embodiments 12 to 14.

[16] A method for screening a glucose dehydrogenase substrate specificity modifying agent, comprising the steps of: i) providing glucose dehydrogenase; ii) determining the ratio (Mal / Glu) of the reactivity of said glucose dehydrogenase towards maltose (Mal) and towards glucose (Glu); iii) contacting the glucose dehydrogenase of i) with a candidate substance which is a low molecular weight compound that is an analog of glucose, and then determining the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity to glucose (Glu) in the presence of the candidate substance; iv) comparing the ratio (Mal / Glu) in ii) with the ratio (Mal / Glu) in the presence of the candidate substance in iii); and v) when the ratio (Mal / Glu) in the presence of the candidate substance in iii) is altered more than the ratio (Mal / Glu) in ii), the candidate substance is selected as a glucose dehydrogenase substrate specificity altering agent.

[17] A method for producing a glucose measuring reagent or a glucose measuring composition, comprising incorporating a glucose dehydrogenase substrate specificity modifier identified by the method described in embodiment 16 into a glucose measuring reagent or a glucose measuring composition.

[18] A glucose measurement reagent comprising a glucose dehydrogenase substrate specificity modifier identified by the method according to embodiment 16 and glucose dehydrogenase.

[19] An electrode comprising a glucose dehydrogenase substrate specificity modifier and FAD-dependent glucose dehydrogenase (FAD-GDH), wherein the glucose dehydrogenase substrate specificity modifier comprises one or more compounds selected from the group consisting of sorbitol, D-iditol, L-iditol, D-glucal, ribitol, L-gulose, trehalose, D-mannitol, xylitol, and glycerol.

[20] A battery comprising a glucose dehydrogenase substrate specificity modifier and FAD-dependent glucose dehydrogenase (FAD-GDH), wherein the glucose dehydrogenase substrate specificity modifier comprises one or more compounds selected from the group consisting of sorbitol, D-iditol, L-iditol, D-glucal, ribitol, L-gulose, trehalose, D-mannitol, xylitol, and glycerol. This specification includes the disclosures of Japanese Patent Application No. 2019-058223 and Japanese Patent Application No. 2019-182592, which are priority documents of this application. Effect of the Invention

[0018] According to the present disclosure, the substrate specificity of glucose dehydrogenase can be modified. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In an embodiment, the present disclosure provides a glucose dehydrogenase substrate specificity modifier. The substrate specificity modifier of the present disclosure may be a glucose analog. In this specification, a glucose analog refers to a sugar compound or a sugar alcohol compound that has a similar chemical structure to glucose but is not recognized as a substrate by glucose dehydrogenase. The glucose analog may be a low molecular weight compound. The low molecular weight compound referred to here refers to a compound having a molecular weight (g / mol) of about 90 to about 380, for example, about 150 to about 380. In addition, "not recognized as a substrate by glucose dehydrogenase" refers to a compound that is not recognized at all or substantially not recognized as a substrate by glucose dehydrogenase. Specifically, "substantially not recognized as a substrate" means that, assuming activity (reactivity) when glucose is used as a substrate is 100%, activity toward a glucose analogue is 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, 0.005% or less, 0.001% or less, for example, 0.0005% or less.

[0020] In one embodiment, the glucose dehydrogenase substrate specificity modifier of the present disclosure comprises one or more compounds selected from the group consisting of L-gulose, D-iditol, L-iditol, sorbitol, ribitol (CAS no. 488-81-3, also known as adonitol), trehalose, and D-glucal, and D-mannitol, xylitol, and glycerol. In another embodiment, the substrate specificity modifier of the present disclosure is L-gulose, D-iditol, L-iditol, sorbitol, ribitol, trehalose, D-glucal, D-mannitol, xylitol, glycerol, or one or more combinations thereof.

[0021] When the substrate specificity modifier of the present disclosure contains L-gulose (CAS No. 6027-89-0), it may be provided as a mixture of L- and D-isomers, for example, a racemate, or it may be provided as the L-isomer. In this specification, the L-isomer of a certain optically active compound means that the L-isomer is present with an optical purity of, for example, 90 to 100% ee, for example, 95 to 100% ee, for example, 97 to 100% ee. For example, L-gulose may have an optical purity of 90 to 100% ee, for example, 95 to 100% ee, for example, 97 to 100% ee.

[0022] When the substrate specificity modifier of the present disclosure contains D-iditol (CAS no. 25878-23-3), it is preferably provided as a D-form. In the present specification, the D-form of a certain optically active compound means that the D-form exists with an optical purity of, for example, 90 to 100% ee, for example, 95 to 100% ee, for example, 97 to 100% ee. For example, D-iditol may have an optical purity of 90 to 100% ee, for example, 95 to 100% ee, for example, 97 to 100% ee.

[0023] When the substrate specificity modifier of the present disclosure contains L-iditol (CAS no. 488-45-9), it is preferably provided as the L-form. For example, the L-iditol may have an optical purity of 90 to 100% ee, for example 95 to 100% ee, for example 97 to 100% ee.

[0024] When the substrate specificity modifier of the present disclosure contains D-glucal (CAS no. 13265-84-4), it is preferably provided as a D-form. For example, the D-glucal may have an optical purity of 90 to 100% ee, for example 95 to 100% ee, for example 97 to 100% ee.

[0025] When the substrate specificity modifier of the present disclosure contains D-mannitol (CAS no. 69-65-8), it is preferably provided as D-mannitol. For example, the D-mannitol may have an optical purity of 90 to 100% ee, for example 95 to 100% ee, for example 97 to 100% ee.

[0026] In certain embodiments, the glucose dehydrogenase substrate specificity modifier of the present disclosure does not comprise a smectite.

[0027] The glucose dehydrogenase substrate specificity modifier of the present disclosure may be blended in a glucose measurement reagent or glucose measurement composition so that the final concentration in the measurement solution during glucose measurement is 0.1 mM to 1000 mM, for example, 0.1 mM to 500 mM, 0.1 mM to 300 mM, 0.1 mM to 100 mM, 0.1 mM to 80 mM, 0.1 mM to 70 mM, 0.1 mM to 60 mM, 0.1 mM to 50 mM, 0.2 mM to 20 mM, 0.25 mM to 10 mM, for example, 5 mM. In an embodiment, an aqueous solution containing the glucose dehydrogenase substrate specificity modifier of the present disclosure and glucose dehydrogenase can be applied to an electrode and dried. In this case, it is assumed that the glucose dehydrogenase substrate specificity modifier is concentrated during drying and becomes a higher concentration than the final concentration before drying. The glucose dehydrogenase substrate specificity modifier at such a high concentration after drying and an electrode to which the modifier is applied are also encompassed by the present disclosure.

[0028] In an embodiment, the present disclosure provides a composition for measuring glucose or a glucose measurement reagent comprising a glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase. In an embodiment, the glucose measurement reagent may be in a solution form or a dry form. In an embodiment, the glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase in the glucose measurement reagent may be included as separate reagents. In another embodiment, the glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase in the glucose measurement reagent may be included in the same reagent. In a further embodiment, the glucose measurement reagent does not include glucose dehydrogenase, in which case glucose dehydrogenase may be added to the measurement solution at the time of glucose measurement. In another embodiment, the glucose measurement reagent does not include glucose dehydrogenase substrate specificity modifier, in which case glucose dehydrogenase substrate specificity modifier may be added to the measurement solution at the time of glucose measurement.

[0029] In an embodiment, the present disclosure provides a glucose measurement method using the substrate specificity modifier and glucose dehydrogenase. The glucose measurement method may be a method for measuring a glucose concentration. The glucose measurement method may be a method for quantifying a glucose concentration. The sample to be measured may contain glucose. The sample to be measured may contain maltose. The glucose dehydrogenase substrate specificity modifier may be contained in advance in the glucose measurement reagent, or may be added to a measurement solution during glucose measurement.

[0030] In the glucose measurement method of the present disclosure, the glucose dehydrogenase substrate specificity modifier may be contained in the measurement solution at a final concentration of 0.1 mM to 1000 mM, for example, 0.1 mM to 500 mM, 0.1 mM to 300 mM, 0.1 mM to 100 mM, 0.1 mM to 80 mM, 0.1 mM to 70 mM, 0.1 mM to 60 mM, 0.1 mM to 50 mM, 0.2 mM to 20 mM, or 0.25 mM to 10 mM, for example, 5 mM.

[0031] In an embodiment, in the glucose measurement method of the present disclosure, the glucose dehydrogenase substrate specificity modifier may be contained in a glucose measurement solution. In this case, glucose dehydrogenase and a sample may be added to the glucose measurement solution to start the measurement. Alternatively, glucose dehydrogenase may be immobilized on an electrode, and the glucose measurement solution and a sample may be brought into contact with the electrode to perform glucose measurement.

[0032] In another embodiment, the glucose dehydrogenase substrate specificity modifier of the present disclosure may be included in advance in a glucose measurement reagent. This measurement reagent may or may not contain glucose dehydrogenase. When the measurement reagent contains glucose dehydrogenase, a sample may be added thereto to start glucose measurement. When the measurement reagent does not contain glucose dehydrogenase, a sample and glucose dehydrogenase may be added thereto (in any order) to start glucose measurement. Alternatively, glucose dehydrogenase may be immobilized on an electrode, and the measurement reagent and a sample may be brought into contact with the electrode to perform glucose measurement.

[0033] In this specification, unless otherwise specified, glucose dehydrogenase refers to FAD-dependent glucose dehydrogenase (FAD-GDH). As glucose dehydrogenase, known FAD-GDH can be used. Suitable examples of microorganisms from which known FAD-GDH is derived include microorganisms classified into the subphylum Mucor, preferably the class Mucorales, more preferably the order Mucorales, and even more preferably the family Mucoraceae. Specific examples include FAD-GDHs derived from the genera Mucor, Absidia, Actinomucor, and Circinella.

[0034] Specific preferred examples of microorganisms classified into the genus Mucor include Mucor prainii, Mucor javanicus, Mucor circinelloides f. circinelloides, Mucor guilliermondii, Mucor hiemalis f. silvaticus, Mucor subtilissimus, Mucor dimorphosporus, etc. More specific examples include Mucor prainii, Mucor javanicus, Mucor circinelloides f. circinelloides, Mucor guilliermondii NBRC9403, Mucor hiemalis, Mucor hiemalis f. silvaticus NBRC6754, Mucor subtilissimus NBRC6338, Mucor RD056860, Mucor dimorphosporus NBRC5395, etc. Specific preferred examples of microorganisms classified into the genus Absidia include Absidia cylindrospora and Absidia hyalospora.Specific preferred examples of microorganisms classified into the genus Actinomucor include Actinomucor elegans.Specific preferred examples of microorganisms classified into the genus Circinella include Circinella minor, Circinella mucoroides, Circinella muscae, Circinella rigida, Circinella simplex, and Circinella umbellata.More specifically, Circinella minor NBRC6448, Circinella mucoroides NBRC4453, Circinella muscae NBRC6410, Circinella rigida NBRC6411, Circinella simplex NBRC6412, Circinella umbellata NBRC4452, Circinella umbellata NBRC5842, Circinella RD055423 and Circinella RD055422 can be mentioned. The NBRC strains and RD strains are stored strains at NBRC (National Institute of Technology and Evaluation, Biotechnology Center). These FAD-GDHs also include their mutants.

[0035] Other known FAD-GDHs include FAD-GDHs derived from the genus Aspergillus, such as GDH derived from Aspergillus oryzae, GDH derived from Aspergillus awamori, GDH derived from Aspergillus aureus, GDH derived from Aspergillus niger, GDH derived from Aspergillus foetidus, GDH derived from Aspergillus isukae, GDH derived from Aspergillus vergicolor, GDH derived from Aspergillus hoenix, GDH derived from Aspergillus bisporus, GDH derived from Aspergillus brunneo-uniseriatus, GDH derived from Aspergillus carneus, GDH derived from Aspergillus malignus, and GDH derived from Aspergillus terreus. These FAD-GDHs also include their mutants.

[0036] Other known FAD-GDHs include FAD-GDHs from the genus Glomerella, such as Glomerella fructigena GDH, for example, Glomerella fructigena NBRC5951 strain GDH, Glomerella cingulata GDH, for example, Glomerella cingulata NBRC107000 strain GDH, Colletotrichum genus FAD-GDH, for example, Colletotrichum gloeosporioides GDH, Colletotrichum chlorophyti GDH, Colletotrichum orbiculare GDH, Botryosphaeria genus FAD-GDH, for example, Botryosphaeria parva GDH. In addition, Burkholderia genus FAD-GDHs include Burkhorderia cepacia GDH, which is a membrane protein, and Burkhorderia cepacia KS1 strain GDH. For example, see JP 2019-000020 (Patent No. 6453385), WO 2017 / 002896, WO 2002 / 036779 (Patent No. 4107386), etc. These FAD-GDHs also include mutants thereof.

[0037] (Obtaining the gene encoding FAD-GDH) The gene encoding FAD-GDH can be obtained by genetic engineering techniques. To obtain the FAD-GDH gene, a commonly used gene cloning method may be used. For example, chromosomal DNA or mRNA can be extracted from known microbial cells or various cells capable of producing FAD-GDH by a conventional method, for example, the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using the 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 publicly known amino acid sequence information of FAD-GDH, and this is used to select an FAD-GDH gene with high substrate specificity from a chromosomal DNA or cDNA library. Alternatively, a suitable primer DNA is prepared based on the above amino acid sequence, and a DNA containing a target gene fragment encoding an FAD-GDH with high substrate specificity is amplified by a suitable polymerase chain reaction (PCR) such as the 5'RACE method or the 3'RACE method, and these DNA fragments are linked to obtain DNA containing the full-length FAD-GDH gene of interest.

[0039] A method can be employed in which a mutation is introduced into the obtained FAD-GDH gene, and selection is performed using the enzymatic properties of FAD-GDH expressed from various mutant genes as an indicator.

[0040] The mutation treatment of the starting material FAD-GDH gene can be carried out by any known method according to the intended mutation form, namely, a method of contacting and reacting the FAD-GDH gene or a recombinant DNA incorporating the gene with a mutagenic agent, ultraviolet irradiation, genetic engineering techniques, or protein engineering techniques can be widely used.

[0041] Examples of mutagenic agents used in the above mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil.

[0042] The conditions for this contact and action can be selected according to the type of drug used, and are not particularly limited as long as the desired mutation can be actually induced in the FAD-GDH gene. In general, the desired mutation can be induced by contact and action at a drug concentration of preferably 0.5 to 12 M at a reaction temperature of 20 to 80° C. for 10 minutes or more, preferably 10 to 180 minutes. When ultraviolet irradiation is performed, it can be performed according to the usual method as described above (Modern Chemistry, pp. 24 to 30, June 1989 issue).

[0043] As a method utilizing protein engineering techniques, a method generally known as Site-Specific Mutagenesis can be used, for example, the Kramer method (Nucleic Acids Res., 12, 9441 (1984): Methods Enzymol., 154, 350 (1987): Gene, 37, 73 (1985)), the Eckstein method (Nucleic Acids Res., 13, 8749 (1985): Nucleic Acids Res., 13, 8765 (1985): Nucleic Acids Res., 14, 9679 (1986)), the Kunkel method (Proc. Natl. Acid. Sci. USA, 82, 488 (1985): Methods Enzymol., 154, 367 (1987)), etc. Specific methods for converting the base sequence in DNA include, for example, using commercially available kits (Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Deletion Kit; Stratagene, Quick Change Site Directed Mutagenesis Kit; Stratagene, etc.).

[0044] Alternatively, a technique known as the general polymerase chain reaction can be used (Technique, 1, 11 (1989)). In addition to the above gene modification methods, the desired FAD-GDH gene can also be directly synthesized by organic synthesis or enzymatic synthesis.

[0045] When determining or confirming the DNA base sequence of the FAD-GDH gene selected by any of the above methods, for example, a multi-capillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter) may be used.

[0046] (Vector and host cell into which FAD-GDH gene has been inserted) The FAD-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 standard methods, and a host cell corresponding to each vector can be transformed or transduced by standard methods.

[0047] Examples of prokaryotic host cells include microorganisms belonging to the genus Escherichia, such as E. coli K-12 strain, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, E. coli W3110, and E. coli C600 (all manufactured by Takara Bio Inc.). These are transformed or transduced to obtain host cells (transformants) into which DNA has been introduced. As a method for transferring a recombinant vector into such host cells, for example, when the host cell is a microorganism belonging to E. coli, a method of transferring recombinant DNA in the presence of calcium ions can be adopted, and electroporation may also be used. Furthermore, commercially available competent cells (for example, ECOS Competent E. coli BL21 (DE3); manufactured by Nippon Gene Co., Ltd.) may also be used.

[0048] An example of a eukaryotic host cell is yeast. Examples of microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Saccharomyces, Pichia, and Candida. The inserted gene may contain a marker gene for enabling the selection of transformed cells. Examples of the marker gene include genes that complement the auxotrophy of the host, such as URA3 and TRP1. In addition, it is desirable for the inserted gene to contain a promoter or other control sequence (e.g., an enhancer sequence, a terminator sequence, a polyadenylation sequence, etc.) that can express the target gene in the host cell. Specific examples of the promoter include the GAL1 promoter and the ADH1 promoter. Methods for transforming yeast include well-known methods, such as a method using lithium acetate (Methods Mol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003) 481-484), but are not limited thereto. Transformation can be performed using any method including the spheroplast method and the glass bead method.

[0049] Other examples of eukaryotic host cells include fungal cells such as those of the genus Aspergillus and Trichoderma. The method for producing a fungal cell transformant is not particularly limited, and for example, a method of inserting a gene encoding FAD-GDH into a host filamentous fungus in a manner that allows the gene to be expressed according to a conventional method can be mentioned. Specifically, a DNA construct is prepared in which a gene encoding FAD-GDH is inserted between an expression-inducing promoter and a terminator, and then a host filamentous fungus is transformed with the DNA construct containing the gene encoding FAD-GDH to obtain a transformant that overexpresses the gene encoding FAD-GDH. In the present specification, a DNA fragment consisting of an expression-inducing promoter-a gene encoding FAD-GDH-terminator and a recombinant vector containing the DNA fragment, which are prepared for transforming a host filamentous fungus, are collectively referred to as a DNA construct.

[0050] The method for inserting the gene encoding FAD-GDH into the host filamentous fungus in such a manner that the gene is expressed is not particularly limited, but examples include a method of directly inserting the gene into the chromosome of the host organism by using homologous recombination; and a method of introducing the gene into the host filamentous fungus by linking the gene onto a plasmid vector.

[0051] In the method using homologous recombination, a DNA construct can be ligated between sequences homologous to the upstream and downstream regions of the recombination site on the chromosome and inserted into the genome of the host filamentous fungus. A transformant can be obtained by self-cloning by overexpressing the gene in the host filamentous fungus under the control of its own high-expression promoter. The high-expression promoter is not particularly limited, and examples thereof include the promoter region of the TEF1 gene (tef1), which is a translation elongation factor, the promoter region of the α-amylase gene (amy), and the promoter region of the alkaline protease gene (alp).

[0052] In the method using a vector, the DNA construct can be inserted into a plasmid vector used for transformation of filamentous fungi by standard methods, and the corresponding host filamentous fungus can be transformed by standard methods.

[0053] Such a suitable vector-host system is not particularly limited as long as it is a system that can produce FAD-GDH in a host filamentous fungus, and examples thereof include a system of pUC19 and a filamentous fungus, and a system of pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and a filamentous fungus.

[0054] The DNA construct is preferably introduced into the chromosome of the host filamentous fungus for use, but as an alternative method, the DNA construct can be incorporated into an autonomously replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)) for use without being introduced into the chromosome.

[0055] The DNA construct may contain a marker gene that allows the selection of transformed cells. The marker gene is not particularly limited, and examples thereof include genes that complement the auxotrophy of the host, such as pyrG, niaD, and adeA; and drug resistance genes against drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably contains a promoter, terminator, and other control sequences (e.g., enhancer, polyadenylation sequence, etc.) that allow the gene encoding FAD-GDH to be overexpressed in the host cell. The promoter is not particularly limited, and examples thereof include appropriate expression-inducing promoters and constitutive promoters, such as tef1 promoter, alp promoter, and amy promoter. The terminator is also not particularly limited, and examples thereof include alp terminator, amy terminator, and tef1 terminator.

[0056] In the DNA construct, the expression control sequence of the gene encoding FAD-GDH is not necessarily required when the DNA fragment containing the gene encoding FAD-GDH to be inserted contains a sequence having an expression control function. In addition, when transformation is performed by co-transformation method, the DNA construct may not need to have a marker gene.

[0057] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, a gene encoding FAD-GDH, an alp gene terminator, and a pyrG marker gene are linked to an In-Fusion Cloning Site in the multiple cloning site of pUC19.

[0058] As a method for transforming a filamentous fungus, a method known to those skilled in the art can be appropriately selected. For example, a protoplast PEG method using polyethylene glycol and calcium chloride after preparing a protoplast of a host filamentous fungus (see, for example, Mol. Gen. Genet. 218, 99-104, 1989, JP 2007-222055 A) can be used. A medium for regenerating a transformed filamentous fungus is used that is appropriate depending on the host filamentous fungus and the transformation marker gene used. For example, when Aspergillus sojae is used as the host filamentous fungus and the pyrG gene is used as the transformation marker gene, the regeneration of the transformed filamentous fungus can be performed, for example, in Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2 M sorbitol.

[0059] Also, for example, in order to obtain a transformed filamentous fungus, the promoter of the gene encoding FAD-GDH originally present on the chromosome of the host filamentous fungus may be replaced with a high expression promoter such as tef1 using homologous recombination. In this case, it is preferable to insert a transformation marker gene such as pyrG in addition to the high expression promoter. For example, for this purpose, referring to Example 1 and FIG. 1 described in JP-A-2011-239681, a transformation cassette consisting of the upstream region of the gene encoding FAD-GDH-transformation marker gene-high expression promoter-all or part of the gene encoding FAD-GDH can be used. In this case, the upstream region of the gene encoding FAD-GDH and all or part of the gene encoding FAD-GDH are used for homologous recombination. All or part of the gene encoding FAD-GDH can be used that includes a region from the start codon to the middle. The length of the region suitable for homologous recombination is preferably 0.5 kb or more.

[0060] The production of a transformed filamentous fungus can be confirmed by culturing the transformed filamentous fungus under conditions in which FAD-GDH enzyme activity is observed, and then confirming the FAD-GDH activity in the culture obtained after cultivation.

[0061] In addition, the production of a transformed filamentous fungus may be confirmed by extracting chromosomal DNA from the transformed filamentous fungus, performing PCR using this as a template, and confirming that an amplifiable PCR product is produced if transformation has occurred.

[0062] For example, PCR is carried out using a combination of a forward primer for the nucleotide sequence of the promoter used and a reverse primer for the nucleotide sequence of the transformation marker gene, and it is confirmed that a product of the expected length is generated.

[0063] (Method of producing FAD-GDH enzyme) The FAD-GDH enzyme may be produced by culturing the FAD-GDH-producing host cells obtained as described above, expressing the FAD-GDH gene contained in the host cells, and then isolating FAD-GDH from the culture.

[0064] The medium for culturing the above-mentioned host cells may contain, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran leachate, to which one or more inorganic salts such as sodium chloride, potassium monophosphate, potassium diphosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and further contains, as necessary, carbohydrate raw materials, vitamins, etc.

[0065] The initial pH of the medium is not limited, but can be adjusted to, for example, pH 6 to 9. Cultivation may be performed at a culture temperature of 10 to 42° C., preferably at about 25° C. for 4 hours to 1 week, and more preferably at about 25° C. for 4 hours to 5 days, by aeration and stirring submerged culture, shaking culture, stationary culture, or the like.

[0066] After the culture is completed, the FAD-GDH enzyme is collected from the culture. For this purpose, a conventional enzyme collection method may be used. For example, the cells may be subjected to ultrasonic destruction, grinding, or the like, or the enzyme may be extracted using a lytic enzyme such as lysozyme or yatalase, or the cells may be shaken or left to stand in the presence of toluene or the like to cause lysis, and the enzyme may be discharged from the cells. The solution may then be filtered, centrifuged, or the like to remove solids, and nucleic acids may be removed as necessary using streptomycin sulfate, protamine sulfate, manganese sulfate, or the like. Ammonium sulfate, alcohol, acetone, or the like may then be added to the solution for fractionation, and the precipitate may be collected to obtain a crude FAD-GDH enzyme.

[0067] The crude enzyme of FAD-GDH can be further purified by any known means. To obtain a purified enzyme preparation, for example, gel filtration using Sephadex, Ultrogel, Biogel, etc.; adsorption elution using an ion exchanger; electrophoresis using polyacrylamide gel, etc.; adsorption elution using hydroxyapatite; sedimentation such as sucrose density gradient centrifugation; affinity chromatography; fractionation using a molecular sieve membrane or hollow fiber membrane, etc., can be appropriately selected or combined to obtain a purified FAD-GDH enzyme preparation.

[0068] In some embodiments, the glucose dehydrogenase may be immobilized on a solid phase. In some embodiments, the present disclosure provides a glucose measurement kit or a glucose measurement system (apparatus) including the substrate specificity modifier and a solid phase on which glucose dehydrogenase is immobilized. Examples of the solid phase include, but are not limited to, beads, particles, polymers, and electrode surfaces. For example, in some embodiments, the glucose dehydrogenase may be immobilized on an electrode. In some embodiments, the present disclosure provides a glucose measurement system (apparatus) including the substrate specificity modifier and an electrode on which glucose dehydrogenase is immobilized.

[0069] (Method for immobilizing glucose dehydrogenase) Glucose dehydrogenase may be immobilized on a solid phase by any known method. Glucose dehydrogenase may be immobilized on beads, membranes, carbon particles, gold particles, platinum particles, polymers, or electrode surfaces. Immobilization methods include a method using a crosslinking reagent, a method of encapsulation in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, and the like, and glucose dehydrogenase may be immobilized in a polymer or adsorbed onto an electrode, or these may be used in combination. Typically, glucose dehydrogenase is immobilized on a carbon electrode using glutaraldehyde, and then the glutaraldehyde is blocked by treating with a reagent having an amine group. In some embodiments, the glucose dehydrogenase substrate specificity modifier of the present disclosure may be present when glucose dehydrogenase is immobilized on a solid phase surface.

[0070] In an embodiment, when glucose dehydrogenase is used by immobilizing it on an electrode or when glucose dehydrogenase is used without immobilizing it, the mediator may be a known mediator, for example, a phenazine compound such as 1-methoxy-5-methylphenazinium methyl sulfate (mPMS), 5-methylphenazinium methyl sulfate (PMS), or 5-ethylphenazinium methyl sulfate (PES), a phenothiazine compound, a ferricyanide such as potassium ferricyanide, a ferrocene compound such as ferrocene, dimethylferrocene, or ferrocene carboxylic acid, or a naphthoquinone, anthraquinone, hydroquinone, or pyrroloquinoline quinone. Quinone compounds, cytochrome compounds, viologen compounds such as benzyl viologen and methyl viologen, indophenol compounds such as dichlorophenol indophenol, ruthenium complexes such as hexaammine ruthenium chloride, osmium complexes such as osmium-2,2'-bipyridine, and phenylenediamine compounds such as p-phenylenediamine, N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), N',N-diphenyl-p-phenylenediamine (DPPD), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, and N,N,N',N'-tetramethyl-1,4-phenylenediamine can be used. These mediators can also be modified with polymers such as polyvinylimidazole, polyethyleneimine, and polyacrylic acid. These mediators can also be immobilized on GDH using a crosslinking agent or the like. These mediators can also be immobilized on the electrode by hydrophobic interactions, with reference to Japanese Patent Nos. 6484741 and 6484742, the entire contents of which are incorporated herein by reference. These mediators can also be immobilized on the electrode after the electrode surface is activated by acid treatment or the like. The electrode can be made of materials such as carbon, gold, platinum, etc.

[0071] The final concentration of the mediator added to the sample solution is not particularly limited, and may be, for example, in the range of 1 pM to 1 M, 1 pM to 100 mM, 1 pM to 20 mM, 1 pM to 10 mM, 1 pM to 5 mM, 2 pM to 1 mM, 3 pM to 800 μM, 4 pM to 600 μM, 5 pM to 500 μM, 6 pM to 400 μM, 7 pM to 300 μM, 8 pM to 200 μM, 9 pM to 100 μM, or 10 pM to 50 μM. The order of addition of the mediator and other reagents is not limited, and they may be added simultaneously or sequentially.

[0072] In some embodiments, the time for performing the redox reaction or the time for performing the electrochemical measurement can be 60 minutes or less, 30 minutes or less, 10 minutes or less, 5 minutes or less, or 1 minute or less. Alternatively, in an enzyme sensor, battery, or the like for long-term measurement, the time for performing the redox reaction can be 60 minutes or more, 120 minutes or more, 1 day or more, 2 days or more, 3 days or more, 1 week or more, 2 weeks or more, or 3 weeks or more.

[0073] Unless otherwise specified, the glucose dehydrogenase contained in the composition or immobilized on the electrode is a purified glucose dehydrogenase enzyme.

[0074] (Measurement of glucose dehydrogenase activity) This section explains how to measure the activity of glucose dehydrogenase (GDH). GDH (EC 1.1.99.10) catalyzes the reaction of oxidizing the hydroxyl group of glucose to produce glucono-δ-lactone. At this time, the electron acceptor receives electrons and becomes a reduced electron acceptor. The activity of GDH can be measured using this principle of action, for example, using the following measurement system that uses 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 (oxidized) + DCIP (reduced)

[0075] Specifically, first, in (Reaction 1), PMS (reduced form) is produced as D-glucose is oxidized. In the subsequent (Reaction 2), DCIP is reduced as PMS (reduced form) is oxidized. The degree of disappearance of this "DCIP (oxidized form)" is detected as the change in absorbance at a wavelength of 600 nm, and the enzyme activity can be calculated based on this change.

[0076] The activity of GDH can be measured according to the following procedure. 2.05 mL of 100 mM phosphate buffer (pH 7.0), 0.6 mL of 1 M D-glucose solution, and 0.15 mL of 2 mM DCIP solution are mixed and incubated at 37°C for 5 minutes. Then, 0.1 mL of 15 mM PMS solution and 0.1 mL of enzyme sample solution are added to start 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) accompanying the progress of the enzyme reaction is determined, and the GDH activity is calculated according to the following formula. In this case, the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of 200 mM D-glucose at 37°C is defined as 1 U of GDH activity.

[0077]

number

[0078] In the formula, 3.0 is the volume of reaction reagent + enzyme reagent (mL), and 16.3 is the millimolar extinction coefficient (cm 2 / μmol), 0.1 is the volume of the enzyme solution (mL), 1.0 is the optical path length of the cell (cm), and ΔA600 blank represents the decrease in absorbance at 600 nm per minute when 100 mM phosphate buffer (pH 7.0) was added instead of the enzyme sample solution to start the reaction, and df represents the dilution factor.

[0079] The glucose measurement kit of the present disclosure includes a substrate specificity modifier in an amount sufficient for at least one assay. Typically, the glucose measurement kit of the present disclosure includes, in addition to the substrate specificity modifier, glucose dehydrogenase, a buffer solution required for the assay, a glucose substrate standard solution for preparing a calibration curve, and a guide. The substrate standard solution may be a glucose standard solution. In addition, when evaluating the reactivity of glucose dehydrogenase to maltose, a maltose substrate standard solution with a known concentration may also be used.

[0080] In one embodiment, the glucose measurement kit of the present disclosure contains a substrate specificity modifier and glucose dehydrogenase as the same reagent. In another embodiment, the glucose measurement kit of the present disclosure contains a substrate specificity modifier and glucose dehydrogenase as separate reagents. In another embodiment, glucose dehydrogenase may be immobilized on an electrode, and the glucose measurement kit of the present disclosure used for such an electrode contains a substrate specificity modifier as a single reagent. However, the term "single reagent" does not mean that the reagent does not contain any substance other than the substrate specificity modifier. The substrate specificity modifier may be in a solution state or in a dry state, for example, in a powder state.

[0081] One example of the colorimetric method is the measurement of glucose concentration. In the case of colorimetric measurement, the measurement of glucose concentration can be performed, for example, as follows. The reaction layer holds a liquid or solid composition containing glucose dehydrogenase (GDH) and one or more substances selected from the group consisting of N-(2-acetamido)imidodiacetic acid (ADA), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), sodium carbonate, and imidazole as a reaction promoter. Here, a pH buffer and a coloring reagent (color changing reagent) are added as necessary. A sample containing glucose is added to this and reacted for a certain period of time. During this period, the absorbance corresponding to the maximum absorption wavelength of the dye polymerized by directly receiving electrons from GDH or the reduced dye is monitored. The glucose concentration in the sample can be calculated based on the rate of change in absorbance per time in the case of the rate method, or based on the change in absorbance until the glucose in the sample is all oxidized in the case of the endpoint method, based on a calibration curve prepared in advance using a glucose solution of a standard concentration.

[0082] As a color-developing reagent (color-changing reagent) that can be used in this method, for example, 2,6-dichloroindophenol (DCIP) can be added as an electron acceptor, and glucose can be quantified by monitoring the decrease in absorbance at 600 nm. Alternatively, nitrotetrazolium blue (NTB) can be added as a color-developing reagent, and the amount of diformazan produced can be determined by measuring the absorbance at 570 nm, and the glucose concentration can be calculated. Needless to say, the color-developing reagent (color-changing reagent) used is not limited to these.

[0083] (Enzyme sensor) In some embodiments, glucose can be detected or measured by an enzyme sensor. The enzyme sensor may include an electrode on which glucose dehydrogenase is immobilized. The above-mentioned mediator may be adsorbed on the electrode. Examples of the electrode of the enzyme sensor include a carbon electrode, a gold electrode, and a platinum electrode, and glucose dehydrogenase can be applied or immobilized on the electrode. Furthermore, the conductive material may include metal fine particles containing at least one element selected from Co, Pd, Rh, Ir, Ru, Os, Re, Ni, Cr, Fe, Mo, Ti, Al, Cu, V, Nb, Zr, Sn, In, Ga, Mg, Pb, Au, Pt, and Ag, and these may be alloys or plated. Carbon also includes carbon nanotubes, carbon black, graphite, fullerene, and derivatives thereof. In some embodiments, the enzyme sensor may be a glucose sensor. That is, in some embodiments, the present disclosure provides a method for detecting or measuring glucose using the substrate specificity modifier and a glucose sensor. The glucose sensor can be used for continuous blood glucose measurement or continuous glucose monitoring.

[0084] The composition or reagent containing the substrate specificity modifier of the present disclosure can be used in various electrochemical measurements using a potentiostat or galvanostat together with an electrode on which glucose dehydrogenase is immobilized, or an enzyme sensor, etc. Electrochemical measurement methods include various techniques such as amperometry, for example, chronoamperometry, potential step chronoamperometry, voltammetry, for example, cyclic voltammetry, differential pulse voltammetry, potentiometry, and coulometry. For example, in glucose measurement, the glucose concentration in a sample can be calculated by measuring the current when glucose is reduced by the amperometry method. The applied voltage depends on the conditions and the settings of the device, but can be, for example, -1000mV to +1000mV (vs. Ag / AgCl).

[0085] Printed electrodes can also be used as the electrodes. This can reduce the amount of solution required for measurement. The electrodes can be formed on an insulating substrate. Specifically, the electrodes can be formed on the substrate by photolithography or printing techniques such as screen printing, gravure printing, and flexography. Materials for the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, and those that are highly resistant to various solvents and chemicals can be used. Carbon cloth, carbon paper, and bucky paper can also be used as the base for the electrodes. The area of ​​the working electrode can be set according to the desired response current. For example, in one embodiment, the area of ​​the working electrode is set to 1 mm 2 Above 1.5mm 2 More than 2mm 2 More than 2.5mm 2 or more, 3mm 2 or more, 4mm 2 More than 5mm 2 Above, 6mm 2 That's it, 7mm 2 That's it, 8mm 2 That's it, 9mm 2 Above 10mm 2 Above, 12mm 2 Above, 15mm 2 Above, 20mm 2 Above, 30mm 2 Above, 40mm 2 Above 50mm 2 More than 1cm 2 More than 2cm 2 More than 3cm 2 Over 4cm 2 More than 5cm 2 More than, for example, 10 cm 2 In one embodiment, the area of ​​the working electrode can be 10 cm 2 Below, 5cm 2 For example, 1 cm 2 The same can be said for the counter electrode. In addition, carbon nanotubes, graphene, Ketjen black, etc. can be immobilized on the working electrode to increase the apparent surface area. In this case, the apparent area can increase by 10 times or more, 50 times or more, 100 times or more, or 1000 times or more.

[0086] In one embodiment, the present disclosure provides a method for screening for glucose dehydrogenase substrate specificity modifiers, the method comprising: i) providing glucose dehydrogenase; ii) determining the ratio (Mal / Glu) of the reactivity of said glucose dehydrogenase towards maltose (Mal) and towards glucose (Glu); iii) contacting the glucose dehydrogenase of i) with a candidate substance, and then determining the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity to glucose (Glu) in the presence of the candidate substance; iv) comparing the ratio (Mal / Glu) in ii) with the ratio (Mal / Glu) in the presence of the candidate substance in iii); and v) when the ratio (Mal / Glu) in the presence of the candidate substance in iii) is modified to be higher than the ratio (Mal / Glu) in ii), the candidate substance is determined to be a glucose dehydrogenase substrate specificity modifier. The candidate substance may be a low molecular weight compound that is an analog of glucose.

[0087] In certain embodiments, the present disclosure provides a method for identifying an effective concentration of a glucose dehydrogenase substrate specificity modifier, the method comprising: i) preparing glucose dehydrogenase and a first concentration, a second concentration, . . . nth concentration of a glucose dehydrogenase substrate specificity modifier (n is a natural number); ii) determining the ratio (Mal / Glu) of the reactivity of glucose dehydrogenase to maltose (Mal) and the reactivity to glucose (Glu) in the presence of the first concentration, the second concentration, ..., and n concentrations of the glucose dehydrogenase substrate specificity modifier; iii) comparing the ratio (Mal / Glu) at each concentration to confirm the effective concentration of the glucose dehydrogenase substrate specificity modifier; For example, in one embodiment, if the ratio Mal / Glu at a specific concentration n is substantially the same as the ratio Mal / Glu when no glucose dehydrogenase substrate specificity modifier is added, the lower limit of the effective concentration of the glucose dehydrogenase substrate specificity modifier may be set to the specific concentration n or higher. In one embodiment, if the ratio Mal / Glu at a specific concentration n is substantially the same as the ratio Mal / Glu when the glucose dehydrogenase substrate specificity modifier is added at a concentration n-1, the upper limit of the effective concentration of the glucose dehydrogenase substrate specificity modifier may be set to the specific concentration n or lower, with the proviso that the concentration n is higher than the concentration n-1.

[0088] In one embodiment, the present disclosure provides a method for producing a glucose measurement reagent or a glucose measurement composition, which comprises blending a glucose dehydrogenase substrate specificity modifier identified by the screening method into a glucose measurement reagent or a glucose measurement composition. In another embodiment, the present disclosure provides a glucose measurement reagent comprising a glucose dehydrogenase substrate specificity modifier identified by the screening method and glucose dehydrogenase.

[0089] The ratio (Mal / Glu) of the reactivity of glucose dehydrogenase to maltose (Mal) and the reactivity to glucose (Glu) can be determined by the following procedure. N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) is adsorbed and immobilized on a carbon printed electrode, and glucose dehydrogenase, for example, glucose dehydrogenase derived from the genus Mucor or glucose dehydrogenase derived from the genus Aspergillus, is crosslinked and immobilized with glutaraldehyde. A potential of +250 mV (Ag / AgCl) is applied using a three-electrode electrode consisting of a working electrode, a counter electrode, and a reference electrode, and chronoamperometry is performed. At this time, after the current value has sufficiently stabilized, maltose is added to a final concentration of 1 mM in phosphate buffered saline (PBS) (for example, added 100 seconds after the start of measurement). Next, after the addition of maltose, and after the current value has sufficiently stabilized again, glucose is added to a final concentration of 1 mM in phosphate buffered saline (PBS) (for example, added 100 seconds after the addition of maltose). The substrate specificity of glucose dehydrogenase can be evaluated by measuring the reactivity to maltose when the reactivity to glucose is taken as 100%. In this specification, the ratio of the reactivity to maltose (Mal) to the reactivity to glucose (Glu) is sometimes expressed as Mal / Glu. When the reactivity is evaluated by the response current, the reactivity ratio can be referred to as the response current ratio.

[0090] For example, if the Mal / Glu ratio of a glucose dehydrogenase is 0.50% in the absence of a glucose dehydrogenase substrate specificity modifier, and if the Mal / Glu ratio of the glucose dehydrogenase is less than 0.50% under the same measurement conditions but in the presence of a glucose dehydrogenase substrate specificity modifier, the reactivity to maltose is reduced, and the substrate specificity of the glucose dehydrogenase is modified. Such a glucose dehydrogenase substrate specificity modifier can reduce the influence of maltose in glucose measurement. The relative value of Mal / Glu of a glucose dehydrogenase in the absence of a glucose dehydrogenase substrate specificity modifier may be defined as 100%. In this case, if the Mal / Glu ratio of the glucose dehydrogenase is less than 100% under the same measurement conditions but in the presence of a glucose dehydrogenase substrate specificity modifier, the reactivity to maltose is reduced, and the substrate specificity of the glucose dehydrogenase is modified. Furthermore, if the Mal / Glu ratio of the glucose dehydrogenase in the presence of a glucose dehydrogenase substrate specificity modifier is 100% or more, it can be said that the reactivity to maltose is increased and the substrate specificity of the glucose dehydrogenase is modified.

[0091] In certain embodiments, when the Mal / Glu ratio of a glucose dehydrogenase in the absence of a glucose dehydrogenase substrate specificity modifier is taken as 100%, the Mal / Glu ratio of the glucose dehydrogenase in the presence of a glucose dehydrogenase substrate specificity modifier of the present disclosure is 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 89% or less, 88% or less, 87% or less, 86% or less, 85% or less, 84% or less, 83% or less, 82% or less, 81% or less, or 80% or less. , 79% or less, 78% or less, 77% or less, 76% or less, 75% or less, 74% or less, 73% or less, 72% or less, 71% or less, 70% or less, 69% or less, 68% or less, 67% or less, 66% or less, 65% or less, 64% or less, 63% or less, 62% or less, 61% or less, 60% or less, 59% or less Lower, 58% or less, 57% or less, 56% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51% or less, 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% Below, 37% or less, 36% or less, 35% or less, 34% or less, 33% or less, 32% or less, 31% or less, 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17 % or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, e.g. 1% or less, e.g. substantially 0%, e.g. 0%, e.g. 99%~0%, 99%~1 %, 98%~2%, 97%~3%, 96%~4%, 95%~5%, 94%~6%, 93%~7%, 92%~8%, 91%~9%, 90%~10%, 89%~11%, 88%~12%, 87%~13%, 86%~14%, 85%~15%, 84%~16%, 83%~17%, 82% ~18%, 81%~19%, 80%~20%, 79%~21%, 78%~22%, 77%~23%, 76%~24%, 75%~25%, 74%~26%, 73%~27%, 72%~28%, 71%~29%, 70%~30%, 69%~31%, 68%~32%, 67%~33%,It may be 66% to 34%, 65% to 35%, 64% to 36%, 63% to 37%, 62% to 38%, 61% to 39%, 60% to 40%, 59% to 41%, 58% to 42%, 57% to 43%, 56% to 44%, 55% to 45%, 54% to 46%, 53% to 47%, 52% to 48%, 51% to 49%, for example, 50%.

[0092] In an embodiment, when the Mal / Glu ratio of a glucose dehydrogenase in the absence of a glucose dehydrogenase substrate specificity modifier is taken as 100%, the Mal / Glu ratio of the glucose dehydrogenase in the presence of a glucose dehydrogenase substrate specificity modifier of the present disclosure can be 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 220% or more, 240% or more, 260% or more, 280% or more, or 300% or more, for example, 110% to 300%, 120% to 280%, 130% to 260%, 140% to 240%, 150% to 220%, 160% to 200%, for example, 170% to 190%. Unless otherwise specified, the numerical ranges in the present disclosure include the upper and lower limit values ​​(for example, the numerical range a to b means a or more and b or less). In addition, the present disclosure includes all combinations of the upper and lower limit values ​​exemplified for the numerical ranges.

[0093] Although not wishing to be bound by a particular theory, it is believed that the glucose dehydrogenase substrate specificity modifier of the present disclosure can modify the substrate specificity of GDH through the following mechanism of action. That is, it is known that glucose dehydrogenase recognizes and acts on glucose through its substrate pocket, and it is believed that a compound having a chemical structure similar to that of glucose (glucose analog) enters the substrate pocket first, preventing contaminating sugars other than glucose, such as maltose, from entering the substrate pocket. Note that glucose is a C6 compound and glycerol is a C3 compound, but it can also be seen that glycerol has a structure identical to that of a portion of glucose, and since the effect of modifying the substrate specificity of glucose dehydrogenase was also observed for glycerol, it is believed that glycerol enters the substrate pocket or interacts with the substrate pocket.

[0094] The present disclosure allows modification of the substrate specificity of glucose dehydrogenase. The present disclosure can also be combined with other methods for modifying the substrate specificity of glucose dehydrogenase. For example, a new glucose dehydrogenase having high substrate specificity can be searched for and found, and used in combination with the GDH substrate specificity modifier of the present disclosure. For example, the substrate specificity of GDH can be modified, and the modified GDH mutant can be used in combination with the GDH substrate specificity modifier of the present disclosure.

[0095] In one embodiment, the glucose dehydrogenase substrate specificity modifier used in the glucose measurement method of the present disclosure may include one or more compounds selected from the group consisting of L-gulose, D-iditol, L-iditol, D-glucal, sorbitol, ribitol, and trehalose, as well as D-mannitol, xylitol, and glycerol, and the glucose dehydrogenase may be an FAD-GDH derived from the genus Mucor, for example, Mucor prainii-derived GDH (MpGDH), and the FAD-GDH may or may not be immobilized on an electrode.

[0096] In one embodiment, the glucose dehydrogenase substrate specificity modifier used in the glucose measurement method of the present disclosure may include one or more compounds selected from the group consisting of L-gulose, D-iditol, D-glucal, sorbitol, ribitol, and trehalose, as well as D-mannitol, xylitol, and glycerol, and the glucose dehydrogenase may be an FAD-GDH derived from the genus Mucor, such as Mucor RD056860 GDH (MrdGDH), and the FAD-GDH may or may not be immobilized on an electrode.

[0097] In one embodiment, the glucose dehydrogenase substrate specificity modifier used in the glucose measurement method of the present disclosure may include one or more compounds selected from the group consisting of L-gulose, D-iditol, D-glucal, ribitol, and trehalose, as well as D-mannitol, xylitol, and glycerol, and the glucose dehydrogenase may be an FAD-GDH derived from the genus Aspergillus, for example, an FAD-GDH derived from Aspergillus oryzae or Aspergillus terreus, or GLD1, and the FAD-GDH may or may not be immobilized on an electrode.

[0098] By using the substrate specificity modifier of the present disclosure, the substrate specificity of glucose dehydrogenase can be modified. This can reduce the influence of maltose when measuring the glucose concentration of a sample using glucose dehydrogenase, enabling more accurate measurement of the glucose concentration. This can be useful for blood glucose level management for diabetes patients.

[0099] (Electrodes of the present disclosure) In some embodiments, the present disclosure provides an electrode comprising a glucose dehydrogenase substrate specificity modifier. In some embodiments, the electrode can be an anode electrode. In some embodiments, the anode electrode of the present disclosure comprises glucose dehydrogenase. In some embodiments, the glucose dehydrogenase can be immobilized on an electrode of a battery. In some embodiments, the anode electrode of the present disclosure can be combined with a cathode electrode and a resistor to provide a battery. In some embodiments, the present disclosure provides a method of generating electricity using the battery. In another embodiment, a battery is provided comprising the glucose dehydrogenase substrate specificity modifier of the present disclosure.

[0100] (Fuel Cell of the Present Disclosure) In some embodiments, the present disclosure provides a fuel cell anode or cathode and a fuel cell including the anode or cathode. The fuel cell includes the glucose dehydrogenase substrate specificity modifier of the present disclosure. In some embodiments, the present disclosure provides a method for generating electricity using a cell including the glucose dehydrogenase substrate specificity modifier, and a method for generating electricity in the presence of the glucose dehydrogenase substrate specificity modifier by immobilizing glucose dehydrogenase on an anode electrode and using a substrate compatible with glucose dehydrogenase, such as glucose, as fuel.

[0101] In an embodiment, the fuel cell of the present disclosure includes the glucose dehydrogenase substrate specificity modifier, an anode or cathode, a fuel tank, a cathode, an anode having glucose dehydrogenase, and an electrolyte. An artificial electron mediator may be adsorbed on the anode or cathode. Examples of the artificial electron mediator include, but are not limited to, the known mediators described above and the phenylenediamine-based compounds described in Japanese Patent Nos. 6484741 and 6484742. In addition, the fuel cell of the present disclosure may be provided with a load resistor between the anode and the cathode as necessary, and may include wiring for this purpose. In an embodiment, the load resistor is a part of the fuel cell of the present disclosure. In an embodiment, the load resistor is not a part of the fuel cell of the present disclosure, and the fuel cell of the present disclosure is configured to be connected to an appropriate load resistor. In the fuel cell of the present disclosure, the oxidoreductase (GDH) constitutes a part of the anode. For example, the oxidoreductase may be in close proximity to or in contact with the anode, may be immobilized, or may be adsorbed. The fuel tank includes a compound that serves as a substrate for the oxidoreductase immobilized on the electrode. For example, when glucose dehydrogenase is immobilized on the electrode, the fuel may be glucose. In an embodiment, the fuel cell of the present disclosure may have an ion exchange membrane separating the anode and the cathode. The ion exchange membrane may have pores of 1 nm to 20 nm. The anode may be a general electrode such as a carbon electrode. For example, an electrode made of conductive carbonaceous material such as carbon black, graphite, activated carbon, or an electrode made of metal such as gold or platinum may be used. Specific examples include carbon paper, carbon cloth, glassy carbon, carbon nanotubes, and HOPG (highly oriented pyrolytic graphite). As the counter cathode, for example, an electrode in which an electrode catalyst generally used in fuel cells, such as platinum or a platinum alloy, is supported on a carbonaceous material such as carbon black, graphite, activated carbon, or a conductor made of gold or platinum, or a conductor made of an electrode catalyst itself, such as platinum or a platinum alloy, may be used as the cathode electrode, and an oxidant (cathode side substrate, oxygen, etc.) may be supplied to the electrode catalyst.

[0102] In another embodiment, a substrate reduction enzyme electrode may be used as a cathode paired with the anode consisting of the substrate oxidation enzyme electrode as described above. Examples of the oxidoreductase that reduces the oxidizing agent include known enzymes such as laccase and bilirubin oxidase. When an oxidoreductase is used as a catalyst for reducing the oxidizing agent, a known electron transfer mediator may be used as necessary. Examples of the oxidizing agent include oxygen and the like.

[0103] In one embodiment, to avoid the influence of impurities (such as ascorbic acid and uric acid) that interfere with the electrode reaction at the cathode, an oxygen-selective membrane (such as a dimethylpolysiloxane membrane) can be placed around the cathode electrode.

[0104] The power generation method of the present disclosure includes a step of supplying a compound that serves as a substrate for the oxidoreductase, which serves as fuel, to an anode having the oxidoreductase. When fuel is supplied to the anode having the oxidoreductase, the substrate is oxidized, and the oxidoreductase transfers the generated electrons to an electron transfer mediator, such as a phenylenediamine compound, that mediates electron transfer between the oxidoreductase and an electrode, and the electrons are transferred to a conductive substrate (anode electrode) by the electron transfer mediator. An electric current is generated when the electrons reach the cathode electrode from the anode electrode through a wiring (external circuit).

[0105] The protons (H + ) travels through the electrolyte solution to the cathode electrode. At the cathode electrode, the protons that have traveled through the electrolyte solution from the anode, the electrons that have traveled from the anode side via an external circuit, and an oxidant (cathode side substrate) such as oxygen or hydrogen peroxide react to produce water. This can be used to generate electricity.

[0106] In some embodiments, glucose in a living body can be used as the fuel for the anode. In this case, if it reacts with maltose in a living body, a current higher than expected may flow, and it is considered that glucose dehydrogenase is required to have substrate specificity that acts only on glucose in order to control the current. The substrate specificity modifier of the present disclosure can also be used for such purposes.

[0107] In some descriptions in the present disclosure, it is expressed that the ratio of reactivity to maltose and reactivity to glucose (Mal / Glu) of glucose dehydrogenase can be "modified." The "modification" here means 「 For example, when it is desired to reduce the influence of maltose in measuring the glucose concentration, it is preferable to reduce the ratio (Mal / Glu). .child In these embodiments, the "modification" in each description is changed to "reduced "and According to the present disclosure, the substrate specificity of GDH can be changed depending on the application by using the above-mentioned substrate specificity modifier, without modifying glucose dehydrogenase by genetic engineering.

[0108] In an embodiment, the present disclosure provides a method for modifying the substrate specificity of glucose dehydrogenase by using a glucose dehydrogenase substrate specificity modifier that is a glucose analog and a small molecule compound. In another embodiment, the present disclosure provides the use of a glucose dehydrogenase substrate specificity modifier that is a glucose analog and a small molecule compound for modifying the substrate specificity of glucose dehydrogenase. In another embodiment, the present disclosure provides a glucose dehydrogenase substrate specificity modifier that is a glucose analog and a small molecule compound for use in modifying the substrate specificity of glucose dehydrogenase. In an embodiment, the method of the present disclosure does not include a medical procedure. EXAMPLES

[0109] The present invention will be further illustrated by the following examples, but the technical scope of the present invention is not limited to these examples in any way.

[0110] [Example 1] Introduction of a Mucor-derived GDH gene into a host and confirmation of GDH activity The amino acid sequence of Mucor genus-derived GDH (MpGDH) described in Japanese Patent No. 4648993 is shown in SEQ ID NO: 1, and the base sequence is shown in SEQ ID NO: 2. With reference to the disclosure of JP Patent Publication No. 2013-176363, a base sequence encoding MrdGDH having the amino acid sequence of SEQ ID NO: 3 was totally synthesized (SEQ ID NO: 4). A DNA construct was prepared by inserting the target gene, MpGDH gene or MrdGDH gene, into the multicloning site of the plasmid pUC19 by a conventional method. Specifically, the pUC19 linearized vector included in the In-Fusion HD Cloning Kit (Clontech) was used as pUC19. The MpGDH gene or MrdGDH gene was ligated to the In-Fusion Cloning Site in the multicloning site of pUC19 using the above-mentioned In-Fusion HD Cloning Kit according to the protocol attached to the kit to obtain plasmids for constructs (pUC19-MpGDH, pUC19-MrdGDH).

[0111] These genes were expressed in Aspergillus sojae and their GDH activities were evaluated.

[0112] Specifically, to obtain MpGDH or MrdGDH, double-joint PCR (Fungal Genetics and Biology, 2004, vol. 41, pp. 973-981) was performed using the GDH gene to construct a cassette consisting of the 5' arm region-pyrG gene-TEF1 promoter gene-flavin-binding GDH gene-3' arm region, and the cassette was used to transform a pyrG-disrupted strain derived from Aspergillus sojae NBRC4239 (a strain lacking 48 bp upstream, 896 bp coding region, and 240 bp downstream of the pyrG gene) as follows. The pyrG gene is a uracil auxotrophy marker. Conidia of the pyrG disruptant derived from Aspergillus sojae NBRC4239 were inoculated into 100 ml of polypeptone dextrin liquid medium containing 20 mM uridine in a 500 ml Erlenmeyer flask, and the mixture was shake-cultured at 30°C for about 20 hours, after which the cells were harvested. Protoplasts were prepared from the harvested cells. Using the obtained protoplasts and 20 μg of the target gene-inserted DNA construct, transformation was performed by the protoplast PEG method, and the resulting cells were incubated at 30°C for more than 5 days in Czapek-Dox minimal medium (Difco; pH 6) containing 0.5% (w / v) agar and 1.2 M sorbitol to obtain transformed Aspergillus sojae with colony-forming ability.

[0113] The resulting transformed Aspergillus sojae was able to grow in uridine-free medium due to the introduction of pyrG, a gene that complements the uridine requirement, and was therefore selected as a strain into which the target gene had been introduced. The desired transformants were selected from the resulting strains by PCR.

[0114] Aspergillus sojae transformed with the MpGDH or MrdGDH gene was used to produce the respective GDHs.

[0115] Conidia of each strain were inoculated into 40 ml of DPY liquid medium (1% (w / v) polypeptone, 2% (w / v) dextrin, 0.5% (w / v) yeast extract, 0.5% (w / v) KH2PO4, 0.05% (w / v) MgSO4·7H20; pH unadjusted) in a 200 ml Erlenmeyer flask, and cultured at 30°C for 4 days with shaking at 160 rpm. The culture was then filtered, and the resulting medium supernatant fraction was concentrated and desalted to 10 ml using an Amicon Ultra-15, 30K NMWL (Millipore), and replaced with 20 mM potassium phosphate buffer (pH 6.5) containing 150 mM NaCl. Next, the mixture was applied to HiLoad 26 / 60 Superdex 200pg (GE Healthcare) equilibrated with 20mM potassium phosphate buffer (pH 6.5) containing 150mM NaCl, and eluted with the same buffer to collect fractions showing GDH activity, thereby obtaining a purified preparation of MpGDH. In the same manner, a purified preparation of MrdGDH was obtained. Note that these enzymes are in a state where they are bound to FAD via their FAD binding sites (holoenzymes).

[0116] [Example 2] Effect of substrate specificity modifier when immobilized enzyme is used 10μL of 12mg / ml MpGDH purified preparation, 10μL of 15mM glucose analogues, and 10μL of 20mM K-phosphate buffer were mixed and left to stand for more than 2 hours. N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) was adsorbed and immobilized on SCREEN-PRINTED ELECTRODES (DropSens, product number DRP-C110), 12μg of each MpGDH was applied, and dried at room temperature. Finally, the electrode was placed in 25% glutaraldehyde vapor, and the resulting electrode was washed with pure water to obtain an IPPD·MpGDH-immobilized electrode. Next, the electrode was connected to an ALS electrochemical analyzer 814D using a dedicated connector (DRP-CAC), and the IPPD·GDH-immobilized electrode, Ag / AgCl reference electrode, and platinum counter electrode were immersed in PBS, and chronoamperometric measurements were performed. The applied voltage was +250 mV (Ag / AgCl). At this time, after the current value was sufficiently stabilized (about 100 seconds after the start of measurement), maltose was added to a final concentration of 1 mM in PBS. Next, after the addition of the maltose solution, after the current value was sufficiently stabilized again (about 100 seconds after the addition of maltose), a glucose solution was added to a final concentration of 1 mM. The difference in the response current before and after the addition of maltose was recorded as the response current value of maltose, and the difference in the response current before and after the addition of glucose was recorded as the response current value of glucose. The value obtained by dividing the response current value of maltose by the response current value of glucose was calculated as maltose / glucose (%). The results are shown in Table 1. Sorbitol, ribitol, L-gulose, and D-iditol modified the substrate specificity. Furthermore, when the maltose / glucose of MpGDH without the addition of a substrate specificity modifier was taken as 100%, the addition of D-glucal resulted in a modification of the substrate specificity to 69%.

[0117] [Table 1]

[0118] Similarly, the effect of a substrate specificity modifier was examined using Glucose Dehydrogenase (FAD-dependent) (manufactured by BBI, Product Code: GLD1, hereafter referred to as GLD1). The results are shown in Table 2. L-gulose, D-iditol, L-iditol, trehalose, and D-glucal modified the substrate specificity.

[0119] [Table 2]

[0120] Similarly, the effects of substrate specificity modifiers were examined using MrdGDH. The results are shown in Table 3. Sorbitol, D-iditol, trehalose, and D-glucal modified the substrate specificity.

[0121] [Table 3]

[0122] [Example 3] Effect of substrate specificity modifiers when non-immobilized enzymes are used 10μL of 12mg / ml MpGDH purified preparation, 10μL of each 15mM glucose analogue, and 10μL of 20mM K-phosphate buffer were mixed and left to stand for more than 2 hours. 80μL of PBS, 5μL of each MpGDH solution, and 10μL of 2mg / ml mPMS solution were applied and mixed on SCREEN-PRINTED ELECTRODES (DropSens, product number DRP-C110). The applied voltage was +100mV (Ag / Ag+), and chronoamperometry measurement was performed. After the current value was sufficiently stabilized (about 100 seconds after the start of measurement), maltose solution was added to a final concentration of 5mM and mixed by pipetting. When the current value was sufficiently stabilized, the measurement was stopped, and the difference before and after the addition of maltose was taken as the response current value of maltose. When an equal amount of pure water was added instead of the maltose solution, no increase in the response current was observed. The printed electrode used was thoroughly washed with ultrapure water, dried, and chronoamperometry was performed in the same manner as above. A glucose solution was added instead of the maltose solution, and the response current value of glucose was measured. The response current value of maltose was divided by the response current value of glucose to calculate maltose / glucose (%). When the maltose / glucose ratio of MpGDH was taken as 100%, the ratio was 67% when sorbitol was added, 58% when ribitol was added, 56% when L-gulose was added, 84% when D-iditol was added, 68% when L-iditol was added, and 72% when trehalose was added, modifying the substrate specificity. It was found that L-iditol acts as a substrate specificity modifier when the enzyme is not immobilized.

[0123] Similarly, the effects of substrate specificity modifiers were examined using GLD1. The results are shown in Table 4. Ribitol, L-gulose, L-iditol, trehalose, and D-glucal modified the substrate specificity.

[0124] [Table 4]

[0125] Similarly, the effects of substrate specificity modifiers were examined using MrdGDH. The results are shown in Table 5. Sorbitol, ribitol, L-gulose, D-iditol, and trehalose modified the substrate specificity.

[0126] [Table 5]

[0127] [Example 4] Effect of substrate specificity modifier when using a carbon cloth electrode 80μl of multi-walled carbon nanotube solution (1.4 wt%, MWCNT solution) with N'N-diphenyl-p-phenylenediamine (DPPD) adsorbed and fixed was applied to a 5 mm square carbon cloth (manufactured by Toyo Corporation) in multiple applications. After drying thoroughly, it was washed with ultrapure water. Next, 20μl of a solution in which 40mg / ml wild-type MpGDH and 20μl of 100 mM various glucose analogs dissolved in ultrapure water were mixed in equal amounts was applied to the MWCNT / DPPD immobilized electrode, dried at room temperature, and then placed in 25% glutaraldehyde vapor for 20 minutes to crosslink and fix MpGDH. Then, it was washed with ultrapure water in the same manner as above to obtain a MWCNT / DPPD / MpGDH immobilized electrode. Next, this was connected to an ALS electrochemical analyzer 814D, and the MWCNT / DPPD / MpGDH immobilized electrode, Ag / AgCl reference electrode, and platinum counter electrode were immersed in PBS, and chronoamperometry measurement was performed. The applied voltage was +250 mV (Ag / AgCl). At this time, after the current value was sufficiently stabilized (about 100 seconds after the start of measurement), maltose was added to a final concentration of 1 mM in PBS. Next, after the addition of the maltose solution, the current value was again sufficiently stabilized (about 50 seconds after the addition of maltose), and a glucose solution was added to a final concentration of 1 mM. The difference in the response current before and after the addition of maltose was recorded as the response current value of maltose, and the difference in the response current before and after the addition of glucose was recorded as the response current value of glucose. The value obtained by dividing the response current value of maltose by the response current value of glucose was calculated as maltose / glucose (%). These results are shown in the table below. L-gulose, L-iditol, D-mannitol, glycerol, and xylitol altered the substrate specificity of GDH. In addition, it was shown that various glucose analogs could alter the substrate specificity of GDH regardless of the electrode shape.

[0128] [Table 6]

[0129] [Example 5] Effect of combination of substrate specificity modifiers on carbon cloth electrodes Similarly to Example 4, an MWCNT / DPPD / MpGDH immobilized electrode was prepared. However, the only step changed was to apply 20 μl of a solution prepared by mixing 20 μl of 40 mg / ml wild-type MpGDH with 10 μl each of 100 mM glycerol and 100 mM L-iditol dissolved in ultrapure water to the MWCNT / DPPD immobilized electrode. Thereafter, chronoamperometry was performed by the method described in Example 4, and maltose / glucose (%) was calculated. As a result, when the maltose / glucose response current ratio of MpGDH without the addition of a substrate specificity modifier was taken as 100%, the ratio was 8% when both glycerol and L-iditol were added, and the substrate specificity was modified. Therefore, when 25 mM L-iditol and glycerol were added together, the substrate specificity modification effect was greater than when 50 mM of each substance was used alone, and it was found that the substrate specificity modification effect can be modified synergistically by combining different glucose analogs.

[0130] [Example 6] Construction of a fuel cell The carbon cloth electrode prepared in Example 4 using a solution containing purified GDH and D-mannitol was used as the anode electrode. However, IPPD was used instead of DPPD as the mediator. A cathode electrode was prepared by adsorbing and immobilizing 10 mg / ml of bilirubin oxidase (BOD, manufactured by Sigma) on a carbon cloth adsorbed with a multi-walled carbon nanotube solution (1.4 wt%, MWCNT solution). A variable resistor was connected between the wiring connecting the anode electrode and the cathode electrode, and an ALS electrochemical analyzer 814D was also connected. The fuel tank was an electrolyte solution containing PBS (pH 7.4) and 200 mM D-glucose, and the above anode electrode and cathode electrode were immersed in the electrolyte solution. Measurements were performed at room temperature using the open circuit potential, which is a potentiostat technique. As a result, 0.125 mA / cm was obtained when connected at 10 kΩ. 2 The current value was obtained.

[0131] Although not wishing to be bound by a particular theory, the fuel cell of the present disclosure is considered as follows. That is, in Example 4, by using an electrode on which GDH was immobilized in the presence of various glucose analogs, it was possible to react with glucose with high substrate specificity. Therefore, for example, in a fuel cell using the anode electrode of the present disclosure, when a component in a living body in which maltose and glucose coexist is used as fuel, for example, even when the maltose concentration temporarily increases after ingestion of food or infusion, it is expected to be possible to pass a constant current. In order to modify the substrate specificity, the enzyme may be modified through genetic engineering, but this is time-consuming and cumbersome. According to the present disclosure, the substrate specificity of GDH can be modified according to the application. This is advantageous in various applications. [Industrial Applicability]

[0132] According to the present disclosure, the substrate specificity of glucose dehydrogenase can be modified. This can reduce the influence of maltose when measuring the glucose concentration of a sample using glucose dehydrogenase, enabling more accurate measurement of the glucose concentration. This can be useful for managing the blood glucose level of diabetic patients. Furthermore, if the substrate specificity of glucose dehydrogenase is modified so as to increase the reactivity to maltose, a higher current value can be obtained when using a fuel cell that contains a mixture of glucose and maltose as fuel.

[0133] All documents mentioned herein are hereby incorporated by reference in their entirety.

[0134] A brief description of the sequence SEQ ID NO: 1 Mucor prainii derived GDH (MpGDH) aa SEQ ID NO: 2 MpGDH gene DNA SEQ ID NO: 3 Mucor RD056860 GDH (MrdGDH) aa SEQ ID NO: 4 MrdGDH gene DNA

Claims

1. A method for modifying the substrate specificity of glucose dehydrogenase derived from the genus Mucor or Aspergillus by using a glucose dehydrogenase substrate specificity modifier which is a glucose analog and a low molecular weight compound, comprising one or more compounds selected from the group consisting of sorbitol, D-iditol, L-iditol, D-glucal, ribitol, L-gulose, trehalose, D-mannitol, xylitol, and glycerol, the method comprising allowing the glucose dehydrogenase substrate specificity modifier to coexist with glucose dehydrogenase when glucose is measured using FAD-dependent glucose dehydrogenase (FAD-GDH), and modifying the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity (Glu) to glucose of the glucose dehydrogenase in the presence of the modifier compared to the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity (Glu) to glucose of the glucose dehydrogenase in the absence of the modifier.

2. The method of claim 1, wherein the glucose dehydrogenase substrate specificity modifier comprises one or more compounds selected from the group consisting of D-glucal, L-gulose, and trehalose.

3. The method according to claim 1 or 2, wherein the glucose dehydrogenase is derived from the genus Mucor.

4. The method according to any one of claims 1 to 3, wherein the glucose dehydrogenase is immobilized on a solid surface.

5. The method according to any one of claims 1 to 3, wherein the glucose dehydrogenase is not immobilized on a solid surface.

6. A glucose dehydrogenase substrate specificity modifying agent comprising one or more compounds selected from the group consisting of D-glucal, L-gulose, and trehalose, wherein the modifying agent is a low molecular weight compound which is an analogue of glucose, and the ratio (Mal / Glu) of the reactivity of glucose dehydrogenase to maltose (Mal) and the reactivity (Glu) to glucose of FAD-dependent glucose dehydrogenase (FAD-GDH) in the presence of the modifying agent is modified compared to the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity (Glu) to glucose of FAD-GDH in the absence of the modifying agent, the composition for glucose measurement or the glucose measurement reagent comprising the glucose dehydrogenase substrate specificity modifying agent and a glucose dehydrogenase derived from the genus Mucor or Aspergillus.

7. A method for screening a glucose dehydrogenase substrate specificity modifying agent, comprising the steps of: i) providing a glucose dehydrogenase derived from the genus Mucor or Aspergillus; ii) determining the ratio of reactivity of said glucose dehydrogenase towards maltose (Mal) to reactivity towards glucose (Glu) (Mal / Glu); iii) contacting the glucose dehydrogenase of i) with a candidate substance which is a low molecular weight compound that is an analog of glucose, and then determining the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity to glucose (Glu) in the presence of the candidate substance; iv) comparing the ratio (Mal / Glu) in ii) with the ratio (Mal / Glu) in the presence of the candidate substance in iii); and v) when the ratio (Mal / Glu) in the presence of the candidate substance in iii) is altered more than the ratio (Mal / Glu) in ii), the candidate substance is selected as a glucose dehydrogenase substrate specificity altering agent.

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