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

Abstract

To modify the substrate specificity of glucose dehydrogenase.SOLUTION: Provided are a method of modifying the substrate specificity of glucose dehydrogenase by using a glucose dehydrogenase substrate specificity modifier, the modifier being a glucose analog and a low molecular weight compound, as well as a glucose dehydrogenase substrate specificity modifier.SELECTED DRAWING: None

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 to monitor 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 an electron acceptor and flavin adenine dinucleotide (FAD) as a cofactor.

[0004] Glucose dehydrogenase is a type of oxidoreductase that 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. Reactivity to maltose, in particular, has been a problem. For example, wild-type PQQ-GDH is not glucose-specific and also reacts with other sugars, such as maltose, galactose, and xylose. Non-Patent Document 1 describes the problem that, in glucose measurements using PQQ-GDH, glucose values ​​that are erroneously increased above the actual level due to maltose and other substances present in the system can cause hypoglycemia. Therefore, there has been a demand for measuring glucose using an enzyme that has high specificity for glucose and low reactivity to other sugars, such as maltose.

[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 discovered (see Non-Patent Documents 2 and 3). In addition, approaches have been attempted to alter the substrate specificity of isolated GDHs by obtaining their genes and genetically modifying them.

[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 the mineral smectite.

[0008] Patent Document 2 (International Publication No. 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, as well as a sensor and device used therein, and describes a device containing glucose dehydrogenase. Paragraph 0027 lists sugar alcohols as enzyme stabilizers, with maltitol being preferred. Maltitol is also used in the examples.

[0009] Patent Document 3 (JP 2005-114359 A) aims to provide a component measurement method and sensor that do not require a complicated calibration 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 No. 2001 / 025776) describes a glucose sensor using PQQ-GDH. On page 8, it is stated 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. However, there is no mention 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 Pamphlet [Patent Document 3] Patent Publication No. 2005-114359 [Patent Document 4] International Publication No. 2001 / 025776 Pamphlet [Patent Document 5] JP 2009-195250 (Patent No. 5176045) [Patent Document 6] Patent Publication No. 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] The present disclosure aims to provide a method for modifying the substrate specificity of FAD-dependent glucose dehydrogenase by using a low molecular weight compound that is a glucose analog, and a glucose dehydrogenase substrate specificity modifier that is a low 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 methods for modifying the reactivity of FAD-dependent glucose dehydrogenase toward saccharides other than glucose, such as maltose, by methods other than those for searching for novel GDH enzymes and those for modifying the substrate specificity of existing GDHs by introducing mutations into them. As a result, they have surprisingly found that low-molecular-weight compounds that are specific glucose analogs can modify the reactivity of FAD-dependent glucose dehydrogenase toward saccharides other than glucose (e.g., maltose), thereby completing the present invention.

[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 and glucose dehydrogenase to coexist when glucose is measured 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 a glucose dehydrogenase 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 a glucose analog, and which modifies the ratio (Mal / Glu) of the reactivity of FAD-dependent glucose dehydrogenase (FAD-GDH) to maltose (Mal) and glucose (Glu) in the presence of the modifier, compared to the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and glucose (Glu) in the absence of the modifier. [7] The glucose dehydrogenase substrate specificity modifying agent according to embodiment 6, wherein the modifying agent 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. [8] The glucose dehydrogenase substrate specificity modifying agent of embodiment 6, wherein the modifying agent comprises 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 according to any one of embodiments 6 to 8, wherein the glucose dehydrogenase is derived from the genus Mucor or Aspergillus.

[10] A system for measuring glucose, 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 system for measuring glucose, 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 according to 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 according to embodiment 12, which comprises a glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase in the same reagent.

[15] A method for measuring glucose 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 the glucose dehydrogenase to maltose (Mal) and glucose (Glu); iii) contacting the glucose dehydrogenase of i) with a candidate substance which is a low molecular weight compound that is a glucose analog, and then determining the ratio (Mal / Glu) of the reactivity of the glucose dehydrogenase to maltose (Mal) and the reactivity to glucose (Glu) of the glucose dehydrogenase 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) if 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 designated as a glucose dehydrogenase substrate specificity modifier.

[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 of 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 Nos. 2019-058223 and 2019-182592, from which the present application claims priority. [Effects of the Invention]

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

[0019] In certain embodiments, the present disclosure provides a glucose dehydrogenase substrate specificity modifier. The substrate specificity modifier of the present disclosure may be a glucose analog. As used herein, a glucose analog refers to a sugar compound or 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 with a molecular weight (g / mol) of about 90 to about 380, for example, about 150 to about 380. Furthermore, "not recognized as a substrate by glucose dehydrogenase" means that the compound is not recognized at all or substantially not recognized as a substrate by glucose dehydrogenase. Specifically, "substantially not recognized as a substrate" means that, when the activity (reactivity) when glucose is used as a substrate is taken as 100%, the activity against 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 some embodiments, 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 other embodiments, 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 the L- and D-isomers, for example, a racemate, or it may be provided as the L-isomer. As used herein, the L-isomer of a certain optically active compound refers to the presence of the L-isomer 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 the D-isomer. As used herein, the D-isomer of an optically active compound refers to the presence of the D-isomer with an optical purity of, for example, 90 to 100% ee, such as 95 to 100% ee, or 97 to 100% ee. For example, D-iditol may have an optical purity of 90 to 100% ee, such as 95 to 100% ee, or 97 to 100% ee.

[0023] When the substrate specificity modifying agent of the present disclosure contains L-iditol (CAS no. 488-45-9), it is preferably provided in 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 modifying agent 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 modifying agent of the present disclosure contains D-mannitol (CAS no. 69-65-8), it is preferably provided as the D-isomer. 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 smectite.

[0027] The glucose dehydrogenase substrate specificity modifier of the present disclosure can be incorporated into 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, or 0.25 mM to 10 mM, for example, 5 mM. In certain embodiments, 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, the glucose dehydrogenase substrate specificity modifier is expected to be concentrated upon drying, resulting in a concentration higher than the final concentration before drying. The present disclosure also encompasses such a glucose dehydrogenase substrate specificity modifier at a high concentration after drying and an electrode coated with the modifier.

[0028] In certain embodiments, 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 certain embodiments, the glucose measurement reagent may be in the form of a solution or a dry form. In certain embodiments, the glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase in the glucose measurement reagent may be contained as separate reagents. In another embodiment, the glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase in the glucose measurement reagent may be contained in the same reagent. In a further embodiment, the glucose measurement reagent does not contain glucose dehydrogenase, in which case glucose dehydrogenase may be added to the measurement solution during glucose measurement. In another embodiment, the glucose measurement reagent does not contain a glucose dehydrogenase substrate specificity modifier, in which case glucose dehydrogenase substrate specificity modifier may be added to the measurement solution during glucose measurement.

[0029] In certain embodiments, 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 a 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, during measurement.

[0031] In some embodiments, 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, the measurement may be initiated by adding glucose dehydrogenase and a sample to the glucose measurement solution. 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 contained 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 sample may be brought into contact with the electrode to perform glucose measurement.

[0033] Unless otherwise specified, glucose dehydrogenase herein refers to FAD-dependent glucose dehydrogenase (FAD-GDH). Known FAD-GDHs can be used as glucose dehydrogenases. Suitable examples of microorganisms from which known FAD-GDHs are derived include those classified in 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, examples of FAD-GDHs include 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. The NBRC strains and RD strains are stored at the NBRC (National Institute of Technology and Evaluation, National Biotechnology Center). These FAD-GDHs also encompass their mutants.

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

[0036] Other known FAD-GDHs include FAD-GDHs from the genus Glomerella, such as Glomerella fructigena GDH (e.g., Glomerella fructigena NBRC5951 strain GDH), Glomerella cingulata GDH (e.g., Glomerella cingulata NBRC107000 strain GDH), Colletotrichum FAD-GDHs (e.g., Colletotrichum gloeosporioides GDH, Colletotrichum chlorophyti GDH, Colletotrichum orbiculare GDH), Botryosphaeria FAD-GDH (e.g., Botryosphaeria parva GDH), and Burkholderia FAD-GDHs, such as Burkhorderia cepacia GDH and Burkhorderia cepacia KS1 strain GDH, which are membrane proteins. See, for example, JP 2019-000020 A (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 can be used. For example, chromosomal DNA or mRNA can be extracted from known microbial cells or various cells capable of producing FAD-GDH by standard methods, such as the method described in *Current Protocols in Molecular Biology* (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. A chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA obtained in this manner.

[0038] Next, based on the publicly known amino acid sequence information of FAD-GDH, an appropriate probe DNA is synthesized and used to select an FAD-GDH gene with high substrate specificity from a chromosomal DNA or cDNA library. Alternatively, based on the above amino acid sequence, appropriate primer DNA is prepared and an appropriate polymerase chain reaction (PCR) such as the 5'RACE method or the 3'RACE method is used to amplify DNA containing the desired gene fragment encoding an FAD-GDH with high substrate specificity. These DNA fragments are then linked to obtain DNA containing the full-length FAD-GDH gene of interest.

[0039] A method can be employed in which mutations are introduced into the obtained FAD-GDH gene, and selection is carried out using as an index the enzymatic properties of FAD-GDH expressed from various mutant genes.

[0040] The starting material, the FAD-GDH gene, can be mutated by any known method depending on the intended mutation type, including 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.

[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 reaction can be adjusted depending on the type of drug used, and are not particularly limited as long as the desired mutation can actually be induced in the FAD-GDH gene. Generally, the desired mutation can be induced by contact and reaction 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. Ultraviolet irradiation can also be performed according to the standard method described above (Gendai Kagaku, pp. 24-30, June 1989).

[0043] Protein engineering techniques can be used, generally known as site-specific mutagenesis, such as 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)), and the Kunkel method (Proc. Natl. Acid. Sci. USA, 82, 488 (1985); Methods Enzymol., 154, 367 (1987)). 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 general technique known as 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) or the like may be used.

[0046] (Vector and host cells into which the 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 conventional methods, and host cells corresponding to each vector can be transformed or transduced by conventional methods.

[0047] Examples of prokaryotic host cells include microorganisms belonging to the genus Escherichia, such as E. coli K-12, E. coli BL21(DE3), E. coli JM109, E. coli DH5α, E. coli W3110, and E. coli C600 (all manufactured by Takara Bio Inc.). These cells can be transformed or transduced to obtain host cells (transformants) carrying DNA. For example, when the host cell is a microorganism belonging to the Escherichia coli species, methods such as transfection of recombinant DNA in the presence of calcium ions can be used. Electroporation can also be used. Commercially available competent cells (e.g., ECOS Competent E. coli BL21(DE3); manufactured by Nippon Gene) can also be used.

[0048] An example of a eukaryotic host cell is yeast. Microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Saccharomyces, Pichia, and Candida. The inserted gene may contain a marker gene that enables the selection of transformed cells. Examples of marker genes include genes that complement the auxotrophy of the host, such as URA3 and TRP1. It is also desirable that the inserted gene contain a promoter or other regulatory sequences (e.g., enhancer sequence, terminator sequence, polyadenylation sequence, etc.) that can express the target gene in the host cell. Specific examples of promoters 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 of a variety of methods, including the spheroplast method and the glass bead method.

[0049] Other examples of eukaryotic host cells include fungal cells such as those of the genera Aspergillus and Trichoderma. Methods for producing fungal cell transformants are not particularly limited, and include, for example, inserting a gene encoding FAD-GDH into a host filamentous fungus in a manner that allows its expression, according to standard methods. Specifically, a DNA construct is prepared in which a gene encoding FAD-GDH is inserted between an expression-inducible 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. Herein, a DNA fragment consisting of an expression-inducible promoter-the gene encoding FAD-GDH-terminator, and a recombinant vector containing the DNA fragment, 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 it into the chromosome of the host organism by using homologous recombination; or a method of introducing it into the host filamentous fungus by linking it onto a plasmid vector.

[0051] In the homologous recombination method, a DNA construct is ligated between sequences homologous to the upstream and downstream regions of a recombination site on a chromosome and inserted into the genome of a host filamentous fungus. Self-cloning transformants can be obtained by overexpressing the construct in the host filamentous fungus under the control of its own high-expression promoter. Examples of high-expression promoters include, but are not limited to, the promoter region of the translation elongation factor TEF1 gene (tef1), 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 transforming filamentous fungi by a conventional method, and the corresponding host filamentous fungus can be transformed by a conventional method.

[0053] Such a suitable vector-host system is not particularly limited as long as it is a system that allows FAD-GDH to be produced 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; however, as an alternative, 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 to enable the selection of transformed cells. The marker gene is not particularly limited, and examples include genes that complement auxotrophy of the host, such as pyrG, niaD, and adeA; and drug resistance genes for drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably contains a promoter, terminator, or other regulatory sequence (e.g., enhancer, polyadenylation sequence, etc.) that enables overexpression of the gene encoding FAD-GDH in the host cell. The promoter is not particularly limited, and examples include appropriate inducible promoters and constitutive promoters, such as the tef1 promoter, alp promoter, and amy promoter. The terminator is also not particularly limited, and examples include the alp terminator, amy terminator, and tef1 terminator.

[0056] In the DNA construct, an expression control sequence for the gene encoding FAD-GDH is not necessarily required if the DNA fragment containing the gene encoding FAD-GDH to be inserted contains a sequence with an expression control function. Furthermore, when transformation is performed by cotransformation, the DNA construct may not necessarily 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, the alp gene terminator, and the pyrG marker gene are linked to the In-Fusion Cloning Site in the multiple cloning site of pUC19.

[0058] Methods for transforming filamentous fungi can be selected from those known to those skilled in the art. For example, the protoplast PEG method, which involves preparing protoplasts of the host filamentous fungus and then using polyethylene glycol and calcium chloride (see, for example, Mol. Gen. Genet. 218, 99-104, 1989; JP 2007-222055 A), can be used. The medium for regenerating the transformed filamentous fungus is appropriate depending on the host filamentous fungus and 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 transformed filamentous fungus can be regenerated in, for example, Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2 M sorbitol.

[0059] Alternatively, for example, to obtain a transformed filamentous fungus, homologous recombination may be used to replace the promoter of the FAD-GDH-encoding gene originally present on the chromosome of the host filamentous fungus with a high-expression promoter such as tef1. In this case, it is also preferable to insert a transformation marker gene such as pyrG in addition to the high-expression promoter. For example, for this purpose, a transformation cassette consisting of the upstream region of the FAD-GDH-encoding gene, a transformation marker gene, a high-expression promoter, and all or part of the FAD-GDH-encoding gene can be used, as described in Example 1 and Figure 1 of Japanese Patent Application Laid-Open No. 2011-239681. In this case, the upstream region of the FAD-GDH-encoding gene and all or part of the FAD-GDH-encoding gene are used for homologous recombination. All or part of the FAD-GDH-encoding gene can be used, including the region from the initiation codon to the intermediate region. 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 the enzyme activity of FAD-GDH is observed, and then confirming the activity of FAD-GDH in the culture obtained after culturing.

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

[0062] For example, PCR is performed 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 produced.

[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 infusion, to which one or more inorganic salts such as sodium chloride, potassium diphosphate, potassium diphosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and which may further contain carbohydrate raw materials, vitamins, etc. as needed.

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

[0066] After the culture is completed, the FAD-GDH enzyme is collected from the culture. Conventional, well-known enzyme collection methods can be used. For example, the cells can be subjected to ultrasonic disruption, grinding, or the like, or the enzyme can be extracted using a lytic enzyme such as lysozyme or ytalase, or the cells can be lysed by shaking or standing in the presence of toluene, etc., to excrete the enzyme from the cells. The solution can then be filtered, centrifuged, or the like to remove solids. If necessary, nucleic acids can be removed using streptomycin sulfate, protamine sulfate, manganese sulfate, or the like. After this, the solution is fractionated by adding ammonium sulfate, alcohol, acetone, or the like, and the precipitate is collected to obtain crude FAD-GDH enzyme.

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

[0068] In certain embodiments, glucose dehydrogenase may be immobilized on a solid phase. In certain embodiments, the present disclosure provides a glucose measurement kit or a glucose measurement system (device) comprising 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 certain embodiments, glucose dehydrogenase may be immobilized on an electrode. In certain embodiments, the present disclosure provides a glucose measurement system (device) comprising the substrate specificity modifier and an electrode on which glucose dehydrogenase is immobilized.

[0069] (Method for immobilizing glucose dehydrogenase) Glucose dehydrogenase can 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 using a crosslinking reagent, encapsulating in a polymer matrix, coating with a dialysis membrane, using a photocrosslinkable polymer, a conductive polymer, or a redox polymer. Glucose dehydrogenase may be immobilized in a polymer or adsorbed onto an electrode, or a combination of these methods may be used. Typically, glucose dehydrogenase is immobilized on a carbon electrode using glutaraldehyde, and then treated with a reagent containing an amine group to block the glutaraldehyde. In certain 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 immobilization, known mediators may be used, for example, phenazine compounds such as 1-methoxy-5-methylphenazinium methyl sulfate (mPMS), 5-methylphenazinium methyl sulfate (PMS), and 5-ethylphenazinium methyl sulfate (PES), phenothiazine compounds, ferricyanides such as potassium ferricyanide, ferrocene compounds such as ferrocene, dimethylferrocene, and ferrocenecarboxylic acid, naphthoquinone, anthraquinone, hydroquinone, and 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 hexaammineruthenium 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 crosslinking agents. These mediators can also be immobilized on electrodes through hydrophobic interactions, as described in Japanese Patent Nos. 6,484,741 and 6,484,742, the entire contents of which are incorporated herein by reference. These mediators can also be immobilized on electrodes after activating the electrode surface by acid treatment or the like. Materials such as carbon, gold, and platinum can be used for the electrodes.

[0071] The final concentration of the mediator added to the sample solution is not particularly limited and may be, for example, in the ranges 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 glucose dehydrogenase (GDH) activity. GDH (EC 1.1.99.10) catalyzes the oxidation of the hydroxyl group of glucose to produce glucono-δ-lactone. During this reaction, the electron acceptor accepts electrons and becomes a reduced electron acceptor. GDH activity can be measured using this principle of action, for example, with the following assay system using phenazine methosulfate (PMS) and 2,6-dichloroindophenol (DCIP) as electron acceptors. (Reaction 1) D-glucose + PMS (oxidized form) → D-glucono-δ-lactone + PMS (reduced form) (Reaction 2) PMS (reduced form) + DCIP (oxidized form) → PMS (oxidized) + DCIP (reduced)

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

[0076] GDH activity 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. Next, 0.1 mL of 15 mM PMS solution and 0.1 mL of enzyme sample solution are added to initiate the reaction. Absorbance is measured at the start of the reaction and over time. The decrease in absorbance at 600 nm per minute (ΔA600) associated with the progress of the enzyme reaction is calculated, and GDH activity is calculated according to the following formula: 1 U of GDH activity is defined as the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of 200 mM D-glucose at 37°C.

[0077]

number

[0078] In the formula, 3.0 is the volume (mL) of the reaction reagent + enzyme reagent, and 16.3 is the millimolar extinction coefficient (cm) under these activity measurement conditions. 2 / μmol), 0.1 is the volume of the enzyme solution (mL), 1.0 is the optical path length of the cell (cm), ΔA600 blank represents the decrease in absorbance at 600 nm per minute when 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 contains a substrate specificity modifier in an amount sufficient for at least one assay. Typically, the glucose measurement kit of the present disclosure contains, in addition to the substrate specificity modifier, glucose dehydrogenase, a buffer solution necessary 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. When evaluating the reactivity of glucose dehydrogenase to maltose, a maltose substrate standard solution of known concentration may also be used.

[0080] In certain embodiments, the glucose measurement kit of the present disclosure comprises a substrate specificity modifier and glucose dehydrogenase as the same reagent. In another embodiment, the glucose measurement kit of the present disclosure comprises 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 for use with such an electrode comprises a substrate specificity modifier as a single reagent. However, the term "single reagent" does not mean that the reagent does not contain any substances other than the substrate specificity modifier. The substrate specificity modifier may be in a solution state or in a dry state, for example, a powder.

[0081] An example of a colorimetric method is the measurement of glucose concentration. For colorimetric measurements, glucose concentration can be measured, for example, as follows. A liquid or solid composition containing glucose dehydrogenase (GDH) and one or more reaction promoters selected from the group consisting of N-(2-acetamido)imidodiacetic acid (ADA), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), sodium carbonate, and imidazole is retained in the reaction chamber. A pH buffer and a color-changing reagent are added as needed. A glucose-containing sample is added and allowed to react for a certain period of time. During this time, the absorbance corresponding to the maximum absorption wavelength of the dye polymerized by directly accepting electrons from GDH or the reduced dye is monitored. The glucose concentration in the sample can be calculated from the rate of change in absorbance over time (rate method), or from the change in absorbance up to the point at which all the glucose in the sample is oxidized (endpoint method) based on a calibration curve previously prepared using glucose solutions of standard concentrations.

[0082] As a color-developing reagent (color-changing reagent), 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 using an enzyme sensor. The enzyme sensor may include an electrode on which glucose dehydrogenase is immobilized. Furthermore, the mediator described above may be adsorbed onto the electrode. Examples of electrodes for enzyme sensors include carbon electrodes, gold electrodes, and platinum electrodes, on which glucose dehydrogenase can be coated or immobilized. Furthermore, the conductive material may include metal particles containing at least one element selected from the group consisting of 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. These may be alloys or plated. Examples of carbon include carbon nanotubes, carbon black, graphite, fullerenes, 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] Compositions or reagents containing the substrate specificity modifiers of the present disclosure can be used in various electrochemical measurements using a potentiostat, galvanostat, or the like, together with an electrode on which glucose dehydrogenase is immobilized or an enzyme sensor. Electrochemical measurement methods include various techniques such as amperometry (e.g., chronoamperometry, potential step chronoamperometry), voltammetry (e.g., 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 generated when glucose is reduced using amperometry. The applied voltage varies depending on the conditions and device settings, but can be, for example, −1000 mV to +1000 mV (vs. Ag / AgCl).

[0085] Printed electrodes can also be used as electrodes. This reduces the amount of solution required for measurement. Electrodes can be formed on an insulating substrate. Specifically, electrodes can be formed on a substrate using photolithography or printing techniques such as screen printing, gravure printing, and flexographic printing. 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 buckypaper can also be used as the electrode substrate. 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 can be set to 1 mm 2 Over 1.5mm 2 More than 2mm 2 Over 2.5mm 2 or more, 3mm 2 or more, 4mm 2 More than 5mm 2 More than 6mm 2 More than 7mm 2 Above, 8mm 2 That's it, 9mm 2 More than 10mm 2 Above, 12mm 2 Above, 15mm 2 Over 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 or more, for example, 10 cm 2 In one embodiment, the area of ​​the working electrode can be 10 cm or more. 2 Below, 5cm 2 For example, 1cm 2 The same can be said for the counter electrode. Furthermore, 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 certain embodiments, 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 the glucose dehydrogenase to maltose (Mal) and 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) of the glucose dehydrogenase 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) above, the candidate substance is used as a glucose dehydrogenase substrate specificity modifier. The candidate substance may be a low molecular weight compound that is a glucose analog.

[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 glucose dehydrogenase substrate specificity modifier at a first concentration, a second concentration, ..., an nth concentration (n is a natural number); ii) determining the ratio (Mal / Glu) of the reactivity of glucose dehydrogenase to maltose (Mal) to the reactivity to glucose (Glu) in the presence of the glucose dehydrogenase substrate specificity modifier at each of the first, second, ..., and n concentrations; 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 can 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 can be set to the specific concentration n or lower, provided that concentration n is higher than concentration n-1.

[0088] In certain embodiments, the present disclosure provides a method for producing a glucose measurement reagent or a glucose measurement composition, which comprises incorporating a glucose dehydrogenase substrate specificity modifier identified by the screening method into a glucose measurement reagent or a glucose measurement composition.In addition, in certain embodiments, 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 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, such as glucose dehydrogenase derived from the genus Mucor or glucose dehydrogenase derived from the genus Aspergillus, is crosslinked and immobilized using glutaraldehyde. A potential of +250 mV (Ag / AgCl) is applied to a three-electrode electrode consisting of a working electrode, a counter electrode, and a reference electrode, and chronoamperometry is performed. After the current value has sufficiently stabilized, maltose is added to a final concentration of 1 mM in phosphate-buffered saline (PBS) (e.g., 100 seconds after the start of measurement). Next, after the current value has sufficiently stabilized again after the addition of maltose, glucose is added to a final concentration of 1 mM in phosphate-buffered saline (PBS) (e.g., 100 seconds after the addition of maltose). The substrate specificity of glucose dehydrogenase can be evaluated by measuring its reactivity to maltose when its reactivity to glucose is taken as 100%. Herein, the ratio of the reactivity to maltose (Mal) to the reactivity to glucose (Glu) is sometimes referred to as Mal / Glu. When reactivity is evaluated based on a 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 under the same measurement conditions but in the presence of a glucose dehydrogenase substrate specificity modifier is less than 0.50%, then the reactivity to maltose is reduced, and the substrate specificity of the glucose dehydrogenase can be said to be modified. Such a glucose dehydrogenase substrate specificity modifier can reduce the influence of maltose on glucose measurement. Note that the relative value of Mal / Glu of a glucose dehydrogenase under 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 under the same measurement conditions but in the presence of a glucose dehydrogenase substrate specificity modifier is less than 100%, then the reactivity to maltose is reduced, and the substrate specificity of the glucose dehydrogenase can be said to be 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, the reactivity to maltose is increased, and it can be said that the substrate specificity of the glucose dehydrogenase has been 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 can 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 certain embodiments, when the Mal / Glu ratio of a certain 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 this disclosure include the upper and lower limit values ​​(for example, the numerical range a to b means not less than a and not more than b). In addition, the present disclosure encompasses all combinations of the upper and lower limit values ​​exemplified for the numerical ranges.

[0093] Without wishing to be bound by any 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, glucose dehydrogenase is known to recognize and act 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 glycerol can also be considered to have a structure identical to a portion of glucose. Furthermore, since glycerol was also observed to have the effect of modifying the substrate specificity of glucose dehydrogenase, it is believed that glycerol enters or interacts with the substrate pocket.

[0094] The substrate specificity of glucose dehydrogenase can be modified according to the present disclosure. The present disclosure can also be combined with other methods for modifying the substrate specificity of glucose dehydrogenase. For example, a new glucose dehydrogenase discovered by searching for a glucose dehydrogenase with high substrate specificity can be used in combination with the GDH substrate specificity modifier of the present disclosure. Furthermore, 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 certain embodiments, the glucose dehydrogenase substrate specificity modifier used in the glucose measurement method of the present disclosure may comprise 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 certain embodiments, the glucose dehydrogenase substrate specificity modifier used in the glucose measurement method of the present disclosure may comprise 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), which may or may not be immobilized on an electrode.

[0097] In certain embodiments, the glucose dehydrogenase substrate specificity modifier used in the glucose measurement method of the present disclosure may comprise one or more compounds selected from the group consisting of L-gulose, D-iditol, D-glucal, ribitol, and trehalose, and D-mannitol, xylitol, and glycerol, and the glucose dehydrogenase may be an FAD-GDH derived from the genus Aspergillus, such as 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 glucose concentration measurement. This can be useful for managing blood glucose levels in diabetic 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 may 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 for 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 comprises 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 comprising 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 one 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 containing glucose dehydrogenase, and an electrolyte. An artificial electron mediator may be adsorbed on the anode or cathode. Examples of artificial electron mediators include, but are not limited to, the known mediators described above and the phenylenediamine-based compounds described in Japanese Patent Nos. 6,484,741 and 6,484,742. Furthermore, the fuel cell of the present disclosure may optionally include a load resistor between the anode and the cathode, and may include wiring for this purpose. In one embodiment, the load resistor is part of the fuel cell of the present disclosure. In one embodiment, the load resistor is not 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) forms part of the anode. For example, the oxidoreductase may be close to or in contact with the anode, may be immobilized, or may be adsorbed. The fuel tank contains a compound that serves as a substrate for the oxidoreductase immobilized on the electrode. For example, when glucose dehydrogenase is immobilized on an electrode, the fuel can be glucose. In some embodiments, the fuel cell of the present disclosure can have an ion exchange membrane separating the anode and the cathode. The ion exchange membrane can have pores of 1 nm to 20 nm. The anode can be a common electrode such as a carbon electrode. For example, an electrode made of a conductive carbonaceous material such as carbon black, graphite, or activated carbon, or an electrode made of a metal such as gold or platinum can be used. Specific examples include carbon paper, carbon cloth, glassy carbon, carbon nanotubes, and HOPG (highly oriented pyrolytic graphite). The opposing cathode can be, for example, an electrode in which an electrocatalyst commonly used in fuel cells, such as platinum or a platinum alloy, is supported on a carbonaceous material such as carbon black, graphite, or activated carbon, or on a conductor made of gold or platinum, or a conductor made of the electrocatalyst itself, such as platinum or a platinum alloy, and a configuration in which an oxidant (cathode-side substrate, oxygen, etc.) is supplied to the electrocatalyst can be used.

[0102] In another embodiment, a substrate-reducing enzyme electrode may be used as a cathode paired with the anode formed of the substrate-oxidizing enzyme electrode described above. Examples of oxidizing-reductase enzymes that reduce oxidants include known enzymes such as laccase and bilirubin oxidase. When an oxidizing-reductase enzyme is used as a catalyst for reducing oxidants, a known electron transfer mediator may be used as needed. Examples of oxidizing agents include oxygen.

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

[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 the 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 oxidizer and the electrode. The electrons are then transferred to a conductive substrate (anode electrode) by the electron transfer mediator. Current is generated when the electrons travel from the anode electrode through wiring (external circuit) to the cathode electrode.

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

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

[0107] In some explanations of the present disclosure, it has been stated that the ratio of reactivity to maltose to reactivity to glucose (Mal / Glu) of glucose dehydrogenase can be "modified." Here, "modification" can also encompass "reduction." For example, if it is desired to reduce the effect of maltose on measuring glucose concentration, it is preferable to reduce the ratio (Mal / Glu). In these embodiments, "modification" in each explanation should be read as "reduction." According to the present disclosure, the substrate specificity of GDH can be changed depending on the application using the substrate specificity modifier described above, without modifying glucose dehydrogenase by genetic engineering.

[0108] In certain embodiments, 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 molecular weight compound. In another embodiment, the present disclosure provides use of a glucose dehydrogenase substrate specificity modifier that is a glucose analog and a small molecular weight 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 molecular weight compound for use in modifying the substrate specificity of glucose dehydrogenase. In certain embodiments, the methods of the present disclosure do not involve medical intervention. [Example]

[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 GDH (MpGDH) described in Japanese Patent No. 4648993 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. A nucleotide sequence encoding MrdGDH having the amino acid sequence of SEQ ID NO: 3 was totally synthesized (SEQ ID NO: 4) with reference to the disclosure of JP 2013-176363 A. A DNA construct was prepared by inserting the target gene, MpGDH gene or MrdGDH gene, into the multicloning site of the plasmid pUC19 using standard methods. Specifically, the pUC19 linearized vector included in the In-Fusion HD Cloning Kit (Clontech) was used for pUC19. The MpGDH gene or MrdGDH gene was ligated into the In-Fusion Cloning Site in the multicloning site of pUC19 using the In-Fusion HD Cloning Kit described above according to the protocol provided with the kit to obtain construct plasmids (pUC19-MpGDH, pUC19-MrdGDH).

[0111] These genes were expressed in Aspergillus sojae and their GDH activity was 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, and 3' arm region. This cassette was then 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 auxotrophic marker. Conidia of the pyrG-disrupted strain of Aspergillus sojae NBRC4239 were inoculated into 100 ml of polypeptone dextrin liquid medium containing 20 mM uridine in a 500 ml Erlenmeyer flask and cultured at 30°C for approximately 20 hours with shaking. After incubation, the cells were harvested. Protoplasts were prepared from the harvested cells. The resulting protoplasts and 20 μg of the target gene-inserted DNA construct were transformed by the protoplast PEG method. The resulting transformants were then incubated at 30°C for at least 5 days in Czapek-Dox minimal medium (Difco; pH 6) containing 0.5% (w / v) agar and 1.2 M sorbitol. Transformed Aspergillus sojae strains were identified as those capable of forming colonies.

[0113] The resulting transformed Aspergillus sojae strains were selected as strains carrying the target gene because they were able to grow on uridine-free medium due to the introduction of pyrG, a gene that complements the uridine requirement. Transformants were then selected from the resulting strains by PCR.

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

[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) MgSO4·7H2O; 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 supernatant was concentrated and desalted to 10 ml using an Amicon Ultra-15, 30K NMWL (Millipore). The supernatant was then eluted with 20 mM potassium phosphate buffer (pH 6.5) containing 150 mM NaCl. The resulting mixture was then applied to a HiLoad 26 / 60 Superdex 200 pg column (GE Healthcare) equilibrated with 20 mM potassium phosphate buffer (pH 6.5) containing 150 mM NaCl, and eluted with the same buffer. Fractions exhibiting GDH activity were collected to obtain purified MpGDH. Purified MrdGDH samples were obtained in the same manner. 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 modifiers when immobilized enzymes are used A mixture of 10 μL of 12 mg / mL purified MpGDH, 10 μL of 15 mM glucose analogs, and 10 μL of 20 mM potassium phosphate buffer was incubated for at least 2 hours. N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) was immobilized on screen-printed electrodes (DropSens, product number DRP-C110), and 12 μg of each MpGDH was applied and dried at room temperature. Finally, the electrode was immersed in 25% glutaraldehyde vapor and washed with pure water to obtain the IPPD-MpGDH-immobilized electrode. The electrode was then connected to an ALS Electrochemical Analyzer 814D using a dedicated connector (DRP-CAC). 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). After the current had stabilized sufficiently (approximately 100 seconds after the start of measurement), maltose was added to a final concentration of 1 mM in PBS. After the maltose solution was added and the current had stabilized sufficiently again (approximately 100 seconds after maltose addition), glucose 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 maltose response current, and the difference in the response current before and after the addition of glucose was recorded as the glucose response current. The maltose response current was divided by the glucose response current to calculate the maltose / glucose ratio (%). The results are shown in Table 1. Sorbitol, ribitol, L-gulose, and D-iditol altered substrate specificity. Furthermore, the maltose / glucose ratio of MpGDH without any substrate specificity modifier was 100%, and the addition of D-glucal resulted in a 69% change in substrate specificity.

[0117] [Table 1]

[0118] Similarly, the effects of substrate specificity modifiers were 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 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 A mixture of 10 μL of 12 mg / mL purified MpGDH preparation, 10 μL of 15 mM glucose analogs, and 10 μL of 20 mM potassium phosphate buffer was incubated for at least 2 hours. 80 μL of PBS, 5 μL of each MpGDH solution, and 10 μL of 2 mg / mL mPMS solution were applied to screen-printed electrodes (DropSens, product number DRP-C110) and mixed. Chronoamperometry measurements were performed at an applied voltage of +100 mV (Ag / Ag+). After the current value had stabilized sufficiently (approximately 100 s after the start of measurement), maltose solution was added to a final concentration of 5 mM and mixed by pipetting. The measurement was stopped once the current value had stabilized sufficiently, and the difference between before and after the addition of maltose was taken as the response current to maltose. Note that adding an equal volume of pure water instead of the maltose solution did not result in an increase in the response current. The printed electrode used was thoroughly washed with ultrapure water, dried, and subjected to chronoamperometry as described above. A glucose solution was added instead of the maltose solution, and the glucose response current was measured. The maltose / glucose ratio (%) was calculated by dividing the maltose response current by the glucose response current. When the maltose / glucose ratio of MpGDH was taken as 100%, the ratio was 67% with the addition of sorbitol, 58% with the addition of ribitol, 56% with the addition of L-gulose, 84% with the addition of D-iditol, 68% with the addition of L-iditol, and 72% with the addition of trehalose, thereby modifying 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 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 modifiers when using a carbon cloth electrode Eighty microliters of a 1.4 wt% N'N-diphenyl-p-phenylenediamine (DPPD)-immobilized multiwalled carbon nanotube solution (MWCNT solution) was applied to a 5 mm square carbon cloth (Toyo Corporation) in multiple applications. After thorough drying, the cloth was washed with ultrapure water. Next, 20 μl of a solution consisting of equal volumes of 40 mg / ml wild-type MpGDH and 20 μl of 100 mM glucose analogs dissolved in ultrapure water was applied to the MWCNT / DPPD-immobilized electrode. After drying at room temperature, the electrode was immersed in 25% glutaraldehyde vapor for 20 minutes to crosslink and immobilize MpGDH. The electrode was then washed with ultrapure water as described above to obtain the 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 measurements were performed. The applied voltage was +250 mV (Ag / AgCl). After the current value had sufficiently stabilized (approximately 100 seconds after the start of measurement), maltose was added to a final concentration of 1 mM in PBS. Next, after the maltose solution was added, and after the current value had sufficiently stabilized again (approximately 50 seconds after maltose addition), 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 maltose response current, and the difference in the response current before and after the addition of glucose was recorded as the glucose response current. The maltose response current value was divided by the glucose response current value to calculate 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. Furthermore, various glucose analogs were shown to be able to alter the substrate specificity of GDH regardless of the electrode configuration.

[0128] [Table 6]

[0129] [Example 5] Effect of combination of substrate specificity modifiers on carbon cloth electrodes An MWCNT / DPPD / MpGDH-immobilized electrode was prepared in the same manner as in Example 4. The only difference was that 20 μl of a solution containing 20 μl of 40 mg / ml wild-type MpGDH mixed with 10 μl each of 100 mM glycerol and 100 mM L-iditol dissolved in ultrapure water was applied to the MWCNT / DPPD-immobilized electrode. Chronoamperometry was then performed as described in Example 4, and the maltose / glucose ratio (%) 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, indicating that substrate specificity was modified. Therefore, the addition of 25 mM L-iditol and glycerol together produced a greater substrate specificity modification effect than when 50 mM of each substance was used alone, demonstrating that combining different glucose analogs can synergistically modify substrate specificity.

[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. The cathode electrode was prepared by adsorbing 10 mg / ml bilirubin oxidase (BOD, Sigma) onto 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 and cathode electrodes, and an ALS 814D electrochemical analyzer was also connected. The fuel tank was an electrolyte solution containing PBS (pH 7.4) and 200 mM D-glucose, and the anode and cathode electrodes were immersed in the fuel. Measurements were performed at room temperature using the open-circuit potential technique of a potentiostat. The result was 0.125 mA / cm when connected to 10 kΩ. 2 The current value was obtained.

[0131] Without wishing to be bound by any particular theory, the fuel cell of the present disclosure is considered as follows. Specifically, 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, a fuel cell using the anode electrode of the present disclosure is expected to be able to flow a constant current even when using a biological component in which maltose and glucose coexist as fuel, such as when maltose concentration temporarily increases after ingestion of food or intravenous fluids. To alter substrate specificity, enzymes can be modified through genetic engineering, but this is time-consuming and complicated. According to the present disclosure, the substrate specificity of GDH can be modified to suit the application, which is advantageous in various applications. [Industrial Applicability]

[0132] The present disclosure makes it possible to modify the substrate specificity of glucose dehydrogenase. This reduces the influence of maltose when measuring the glucose concentration of a sample using glucose dehydrogenase, enabling more accurate glucose concentration measurements. This may be useful for managing blood glucose levels in diabetic patients. Furthermore, modifying the substrate specificity of glucose dehydrogenase to increase its reactivity to maltose makes it possible to obtain a higher current value when using a fuel cell that contains a mixture of glucose and maltose as fuel.

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

[0134] A brief description of arrays 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

[0135] The present disclosure includes the following sequence information: SEQUENCE LISTING <110> Kikkoman Corporation <120> A Method for modifying the substrate specificity of glucose dehydrogenase and glucose dehydrogenase substrate specificity modifying agent <130> P25-0061 <150> JP 2019-182592 <151> 2019-10-03 <150> JP 2019-058223 <151> 2019-03-26 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 641 <212> PRT <213> Mucor prainii <400> 1 Met Lys Ile Thr Ala Ala Ile Ile Thr Val Ala Thr Ala Phe Ala Ser 1 5 10 15 Phe Ala Ser Ala Gln Gln Asp Thr Asn Ser Ser Ser Thr Asp Thr Tyr 20 25 30 Asp Tyr Val Ile Val Gly Gly Gly Val Ala Gly Leu Ala Leu Ala Ser 35 40 45 Arg Ile Ser Glu Asn Lys Asp Val Thr Val Ala Val Leu Glu Ser Gly 50 55 60 Pro Asn Ala Asn Asp Arg Phe Val Val Tyr Ala Pro Gly Met Tyr Gly 65 70 75 80 Gln Ala Val Gly Thr Asp Leu Cys Pro Leu Ile Pro Thr Thr Pro Gln 85 90 95 Glu Asn Met Gly Asn Arg Ser Leu Thr Ile Ala Thr Gly Arg Leu Leu 100 105 110 Gly Gly Gly Ser Ala Ile Asn Gly Leu Val Trp Thr Arg Gly Gly Leu 115 120 125 Lys Asp Tyr Asp Ala Trp Glu Glu Leu Gly Asn Pro Gly Trp Asn Gly 130 135 140 Ala Asn Leu Phe Lys Tyr Phe Lys Lys Val Glu Asn Phe Thr Pro Pro 145 150 155 160 Thr Pro Ala Gln Ile Glu Tyr Gly Ala Thr Tyr Gln Lys Ser Ala His 165 170 175 Gly Lys Lys Gly Pro Ile Asp Val Ser Phe Thr Asn Tyr Glu Phe Ser 180 185 190 Gln Ser Ala Ser Trp Asn Ala Ser Leu Glu Thr Leu Asp Phe Thr Ala 195 200 205 Leu Pro Asp Ile Leu Asn Gly Thr Leu Ala Gly Tyr Ser Thr Thr Pro 210 215 220 Asn Ile Leu Asp Pro Glu Thr Val Gln Arg Val Asp Ser Tyr Thr Gly 225 230 235 240 Tyr Ile Ala Pro Tyr Thr Ser Arg Asn Asn Leu Asn Val Leu Ala Asn 245 250 255 His Thr Val Ser Arg Ile Gln Phe Ala Pro Lys Asn Gly Ser Glu Pro 260 265 270 Leu Lys Ala Thr Gly Val Glu Trp Tyr Pro Thr Gly Asn Lys Asn Gln 275 280 285 Lys Gln Ile Ile Lys Ala Arg Tyr Glu Val Ile Ile Ser Ser Gly Ala 290 295 300 Ile Gly Ser Pro Lys Leu Leu Glu Ile Ser Gly Ile Gly Asn Lys Asp 305 310 315 320 Ile Val Ser Ala Ala Gly Val Glu Ser Leu Ile Asp Leu Pro Gly Val 325 330 335 Gly Ser Asn Met Gln Asp His Val His Ala Ile Thr Val Ser Thr Thr 340 345 350 Asn Ile Thr Gly Tyr Thr Thr Asn Ser Val Phe Val Asn Glu Thr Leu 355 360 365 Ala Gln Glu Gln Arg Glu Glu Tyr Glu Ala Asn Lys Thr Gly Ile Trp 370 375 380 Ala Thr Thr Pro Asn Asn Leu Gly Tyr Pro Thr Pro Glu Gln Leu Phe 385 390 395 400 Asn Gly Thr Glu Phe Val Ser Gly Lys Glu Phe Ala Asp Lys Ile Arg 405 410 415 Asn Ser Thr Asp Glu Trp Ala Asn Tyr Tyr Ala Ser Thr Asn Ala Ser 420 425 430 Asn Val Glu Leu Leu Lys Lys Gln Tyr Ala Ile Val Ala Ser Arg Tyr 435 440 445 Glu Glu Asn Tyr Leu Ser Pro Ile Glu Ile Asn Phe Thr Pro Gly Tyr 450 455 460 Glu Gly Ser Gly Asn Val Asp Leu Gln Asn Asn Lys Tyr Gln Thr Val 465 470 475 480 Asn His Val Leu Ile Ala Pro Leu Ser Arg Gly Tyr Thr His Ile Asn 485 490 495 Ser Ser Asp Val Glu Asp His Ser Val Ile Asn Pro Gln Tyr Tyr Ser 500 505 510 His Pro Met Asp Ile Asp Val His Ile Ala Ser Thr Lys Leu Ala Arg 515 520 525 Glu Ile Ile Thr Ala Ser Pro Gly Leu Gly Asp Ile Asn Ser Gly Glu 530 535 540 Ile Glu Pro Gly Met Asn Ile Thr Ser Glu Asp Asp Leu Arg Ser Trp 545 550 555 560 Leu Ser Asn Asn Val Arg Ser Asp Trp His Pro Val Gly Thr Cys Ala 565 570 575 Met Leu Pro Lys Glu Leu Gly Gly Val Val Ser Pro Ala Leu Met Val 580 585 590 Tyr Gly Thr Ser Asn Leu Arg Val Val Asp Ala Ser Ile Met Pro Leu 595 600 605 Glu Val Ser Ser His Leu Met Gln Pro Thr Tyr Gly Ile Ala Glu Lys 610 615 620 Ala Ala Asp Ile Ile Lys Asn Phe Tyr Lys Thr Gln His Lys Asn Gln 625 630 635 640 Asn <210> 2 <211> 1926 <212> DNA <213> Mucor prainii <400> 2 atgaagatca cagctgccat tatcactgtt gccacagcat ttgcttcttt tgcttctgct 60 caacaagaca caaattcttc ctcaactgat acttatgatt atgttatcgt tggcggcggt 120 gtagctggtt tggctttggc tagtcgtatc tctgaaaaca aggatgtcac tgttgctgtt 180 ctcgagtccg gtcctaatgc caatgataga tttgttgttt atgctcctgg catgtatggc 240 caagctgttg gcactgatct ctgtcctctc attcctacta ctcctcaaga aaatatgggc 300 aacagaagtc tcacaatcgc tactggtaga ttgctcggtg gtggcagtgc tattaatggt 360 ctcgtttgga cccgtggtgg cttgaaggat tacgatgctt gggaggagct cggtaaccct 420 ggatggaacg gtgccaactt gttcaagtac tttaagaagg tcgaaaactt cactcctcct 480 actcctgccc aaattgaata cggcgctact tatcagaaaa gtgctcatgg caagaaggga 540 cctattgatg tctctttcac gaactacgag ttctctcaat ctgctagctg gaacgcctca 600 ctcgaaaccc ttgatttcac tgcacttcct gatatcttga acggtacttt ggccggttac 660 tctaccactc ccaacatttt ggaccctgag actgttcaac gtgttgattc ctatactggt 720 tacattgctc cttacactag ccgtaacaac ctcaatgttt tggccaacca taccgtctcc 780 cgcattcaat ttgctcccaa gaatggtagc gaacctctca aggctaccgg tgttgaatgg 840 tatccactg gcaacaagaa tcaaaagcaa attatcaagg cccgttatga agttatcatc 900 tcatctggtg ccattggtag tcctaagctt ttggaaatct ctggtatcgg taataaggat 960 atcgtctctg ctgctggtgt cgagtccttg attgacttgc ctggcgttgg ttccaacatg 1020 caagatcacg ttcatgctat cactgtctct actaccaata ttactggcta tactaccaac 1080 agcgtctttg tcaatgaaac ccttgcccaa gaacaaagag aagaatatga agccaacaag 1140 actggtatct gggctactac tcccaacaac ctcggttatc ctacgcccga acaactcttc 1200 aatggcaccg aattcgtttc tggaaaggag tttgctgaca agattcgtaa ctctactgat 1260 gaatgggcca actattatgc ttccaccaac gcctccaatg tcgagttatt aaagaagcaa 1320 tatgctattg tcgcctctcg ttacgaagag aactacttgt ctcctattga aatcaacttc 1380 actcctggtt atgagggtag cggtaatgtc gatttgcaaa acaacaagta ccaaactgtc 1440 aaccatgtct tgattgctcc tttaagtcgt ggttatactc acattaactc ttctgatgtg 1500 gaggatcatt ctgtcattaa tccccaatac tactctcatc ctatggatat tgatgtccat 1560 atcgcttcca ctaaacttgc tcgcgaaatc atcactgcct ctcccggtct tggtgacatt 1620 aacagtggcg aaatcgaacc cggtatgaat attacttctg aagacgacct tagatcttgg 1680 ttgagtaata atgtccgttc tgactggcat cctgttggta cttgtgctat gcttcccaag 1740 gaattaggtg gtgttgtcag ccccgctctc atggtttacg gcacttccaa cttgcgtgtt 1800 gttgatgctt cgattatgcc cctcgaagtc tctctcatt tgatgcacc cacctacggt 1860 attgctgaga aggctgctga cattattag aatttctaca agactcaca cacaaccaa 1920 Attack 1926 <210> 3 <211> 631 <212> PRT <213> Mucor RD056860 <400> 3 Met Arg Leu Ser Val Ala Ile Leu Thr Leu Thr Ser Ala Leu Ala Ser 1 5 10 15 Val Thr Will Be Only Gln Gln Asn Asn Thr Asp Thr Tyr Asp Tyr Val Ile 20 25 30 Val Gly Gly Gly Val Gly Gly Leu Ala Leu Ala Ser Arg Leu Ser Glu 35 40 45 Asp Lys Asn Val Thr Val Ala Val Leu Glu Ser Gly Pro Tyr Ala Asp 50 55 60 Asp Lys Phe Val Val Tyr Ala Pro Gly Met Tyr Gly Gln Ala Val Gly 65 70 75 80 Thr Asp Leu Cys Pro Leu Leu Pro Thr Val Pro Gln Pro Ser Met Asn 85 90 95 Asn Arg Thr Ile Thr Ile Ala Thr Gly Arg Leu Leu Gly Gly Gly Ser 100 105 110 Ala Val Asn Gly Leu Val Trp Thr Arg Gly Ala Met Lys Asp Phe Asp 115 120 125 Ala Trp Gln Glu Leu Gly Asn Pro Gly Trp Asn Gly Thr Thr Met Phe 130 135 140 Lys Tyr Phe Lys Lys Ile Glu Asn Phe His Pro Pro Thr Glu Glu Gln 145 150 155 160 Ile Gln Tyr Gly Ala Thr Tyr Asn Lys Ser Val His Gly Phe Asn Gly 165 170 175 Pro Ile Asp Ile Ala Phe Pro Val Phe Glu Phe Pro Gln Ser Ala Asn 180 185 190 Trp Asn Ala Ser Leu Ala His Leu Asn Phe Thr Arg Arg Gln Asp Leu 195 200 205 Leu Asp Gly Ser Leu His Gly Tyr Ser Thr Thr Pro Asn Thr Leu Asn 210 215 220 Pro Gln Thr Ala Arg Arg Ala Asp Ala Tyr Ala Gly Tyr Ile Gln Pro 225 230 235 240 Asn Val Asn Arg Thr Asn Leu Ala Val Leu Ala Asn His Thr Val Ser 245 250 255 Arg Ile Gln Phe Glu Ala Arg Asn Gly Ser Gln Pro Leu Lys Ala Ile 260 265 270 Gly Val Glu Trp Tyr Thr Thr Gly Gly Asp Lys Thr Ser Lys Gln Thr 275 280 285 Ile Lys Ala Arg Arg Glu Ile Ile Leu Ser Ser Gly Ala Ile Gly Ser 290 295 300 Pro Lys Leu Leu Glu Val Ser Gly Ile Gly Asn Lys Ala Ile Val Thr 305 310 315 320 Ala Ala Gly Val Gln Ser Leu Ile Asp Leu Pro Gly Val Gly Ser Asn 325 330 335 Met Gln Asp His Val His Ala Val Thr Val Ser Thr Thr Asn Ile Asp 340 345 350 Gly Tyr Thr Thr Asn Ser Val Phe Thr Asn Glu Thr Leu Ala Gln Glu 355 360 365 Gln Lys Asp Leu Tyr Tyr Asn Asn Lys Thr Gly Ile Trp Thr Thr Thr 370 375 380 Pro Asn Asn Leu Gly Tyr Pro Ser Pro Ser Gln Leu Phe Thr Asn Thr 385 390 395 400 Thr Phe Lys Ser Gly Lys Glu Phe Ala Ala Met Ile Arg Asn Ser Thr 405 410 415 Asp Lys Tyr Ala Gln Tyr Tyr Ala Ala Asn Asn Ala Thr Asn Val Glu 420 425 430 Leu Leu Lys Lys Gln Tyr Ser Ile Val Ala Arg Arg Tyr Glu Glu Asn 435 440 445 Tyr Ile Ser Pro Ile Glu Ile Asn Phe Thr Pro Gly Tyr Gly Gly Thr 450 455 460 Gly Met Ala Asp Leu Gln Asn Lys Lys Tyr Gln Thr Val Asn His Val 465 470 475 480 Leu Val Ala Pro Leu Ser Arg Gly Tyr Thr His Ile Asn Ser Ser Asp 485 490 495 Ile Glu Asp Pro Val Val Ile Asp Pro Gln Tyr Tyr Ser His Pro Leu 500 505 510 Asp Val Asp Val His Val Ala Ser Thr Gln Leu Ala Arg Ser Ile Leu 515 520 525 Asn Ala Pro Gly Leu Ala Ser Ile Asn Ser Gly Glu Val Glu Pro Gly 530 535 540 Glu Lys Val Gln Ser Asp Glu Asp Val Arg Lys Trp Leu Ser Asp Asn 545 550 555 560 Val Arg Ser Asp Trp His Pro Val Gly Thr Cys Ala Met Leu Pro Arg 565 570 575 Lys Leu Gly Gly Val Val Asp Ser Lys Leu Lys Val Tyr Gly Thr Ala 580,585,590 Asn Leu Arg Ile Val Asp Ala Ser Ile Ile Pro Leu Glu Ile Ser Ser 595,600,605 His Leu Met Gln Pro Val Tyr Ala Val Ser Glu Arg Ala Ala Asp Ile 610 615 620 Ile Lys Dear Dear Dear Lys Lys 625 630 <210> 4 <211> 1896 <212> DNA <213> Artificial <220> <223> synthetic <400> 4 atgcgtctct ctgtggcgat cctcactctc acttcggctc tgcttcggt tacctcggcc 60 caacaaaaca atactgatac ttatgactac gtgatcgtcg gaggaggagt gggtggactg 120 gctctcgctt cgcgcctc cgaggataag aacgttaccg tgctgtcct ggaatcgggc 180 ccttatgcgg atgacaaatt cgtggtctac gccccaggga tgtatggtca ggctgtcgga 240 actgacctgt gtcctctgct cccaacggtt cctcaaccat ctatgaacaa tcgaaccatc 300 actattgcta cgggacgtct gctcggagga ggttcagctg tgaacggact ggtctggacc 360 cgtggagcta tgaaggattt cgacgcttgg caggagctgg gaaacccagg atggaatggg 420 accactatgt tcaagtactt caagaaaatc gaaaacttcc atcccccgac cgaggaacag 480 attcaatacg gcgctactta taacaagtct gtccacggtt tcaatggccc gatcgatatt 540 gcctttcccg tgttcgagtt tccgcagtct gctaactgga atgcgtcact ggcccatctc 600 aacttcaccc gccggcaaga tctgctcgac ggtagtctcc acggctacag cacgacccct 660 aacaccctga atccacagac tgcccgacgt gcggatgcct acgctggata tatccaacct 720 aacgtcaatc gaacgaacct ggctgtcctc gcgaatcata ccgttagtcg catccagttt 780 gaggcgcgga acggtagcca accactgaag gccattggcg tggaatggta tactacggggc 840 gagachaga ctagtaaca gacgatcaag gcgcgccggg agatcattct gagtagcgga 900 gccattgggt cgcctaagct gctcgaagtg tccgggatcg gtaacaaagc cattgttacc 960 gccgctggag tgcagtctct gatcgatctc ccaggcgttg gatcaacat gcaagaccat 1020 gtgcacgctg ttaccgtgtc gaccactaat atcgatgggt acacgaccaa ctccgtgttc 1080 acaatgaga ccctcgccca ggcaaaag gacctgtact acaacaa gactggaatc 1140 tggactacga cccctaacaa tctcggtat cccagtccga gccagctgtt caccacact 1200 acgtttaagt ctggcaaaga gtttgcggcc atgatccgca acagtactga taagtacgcc 1260 cagtactatg ctgcgaacaa tgctacgaac gtcgagctgc tcagaaca atatagtatc 1320 gtggcccgac gttacgagga aaactacatc agcctacg aaatcaactt cacgccagga 1380 tacgggggta ccgggatggc tgatctgcag aacaagaaat atcaaaccgt gaatcatgtc 1440 ctggttgccc ccctcagtcg gggctacact cacatcaact cgtccgatat tgaggacccc 1500 gttgtgatcg acccgcagta ctatagccat ccgctggatg tggacgtcca cgttgcgagt 1560 acccaactgg cccgaagcat cctcaacgcc cccggactgg cttctattaa ttcaggcgag 1620 gtggaaccgg gcgagaaggt ccagagcgat gaagacgttc gcaaatggct gtcggataac 1680 gtgcgttccg actggcatcc agtcggaacc tgcgctatgc tgccacgaaa gctcggagga 1740 gtcgttgatt cgaagctcaa agtctacggc accgcgaatc tgcgtatcgt tgacgcctcc 1800 atcattccgc tcgagatttc ttcacacctg atgcaaccag tctatgcggt ctccgaacgg 1860 gctgccgaca tcatcaaatc ctcctctaaa aaataa 1896

Claims

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 glucose is measured using FAD-dependent glucose dehydrogenase (FAD-GDH), and modifying 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, compared with 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 sorbitol, D-iditol, L-iditol, D-glucal, ribitol, L-gulose, trehalose, D-mannitol, xylitol, and glycerol.

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

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 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 with 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.

7. The glucose dehydrogenase substrate specificity modifying agent according to claim 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 according to claim 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 according to any one of claims 6 to 8, wherein the glucose dehydrogenase is derived from the genus Mucor or Aspergillus.

10. 10. A system for measuring glucose, comprising the glucose dehydrogenase substrate specificity modifier according to any one of claims 6 to 9 and glucose dehydrogenase immobilized on a solid surface.

11. 10. A system for measuring glucose, comprising the glucose dehydrogenase substrate specificity modifier according to any one of claims 6 to 9 and glucose dehydrogenase that is not immobilized on a solid surface.

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

13. The composition or reagent for measuring glucose according to claim 12, comprising a glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase as separate reagents.

14. The composition for measuring glucose or the reagent for measuring glucose according to claim 12 , which comprises a glucose dehydrogenase substrate specificity modifier and glucose dehydrogenase in the same reagent.

15. A method for measuring glucose, comprising using the modifying agent according to any one of claims 6 to 9, the system according to claim 10 or 11, or the composition or reagent according to any one of claims 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 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.

17. A method for producing a glucose measuring reagent or a glucose measuring composition, comprising blending a glucose dehydrogenase substrate specificity modifier identified by the method according to claim 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 of claim 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 an 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.

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