Direct electron transfer-type enzyme and use thereof

Abstract

The purpose of the present invention is to provide a technique for monitoring a measurement target substance in real time. This direct electron transfer-type enzyme is modified with a modifying substance. There is provided an electron transfer-type enzyme which includes a direct electron transfer site and a measurement target substance detection site, wherein: the direct electron transfer site has both a catalytic reaction activity for achieving conversion from a substrate to a product and a direct electron transfer activity associated with the catalytic reaction, and is capable of transferring electrons to and from the substrate in the catalytic reaction and is also capable of directly releasing the electrons to the outside of the enzyme or allowing the electrons to directly flow in from the outside; and the measurement target substance detection site is a detection site which is connected to the direct electron transfer site and is capable of changing the amount of release or inflow of the electrons when the detection site comes into contact with the measurement target substance.

Description

Direct electron transfer enzymes and their uses

[0001] The present disclosure relates to a direct electron transfer enzyme modified with a modifier. More specifically, the present disclosure relates to an electron transfer enzyme comprising a direct electron transfer site and an analyte sensing site. The present disclosure also relates to a kit comprising the enzyme, an enzyme electrode comprising the enzyme and an electrode substrate, an enzyme sensor comprising the electrode, and an electrochemical measurement method using these.

[0002] Known methods for monitoring the presence or absence and concentration of in vivo substances such as biomarkers include the ELISA method and the redox probe method (Non-Patent Document 1). However, these monitoring methods have difficulty in monitoring the concentration of in vivo substances, which changes from moment to moment, in real time.

[0003] Biosensors capable of real-time monitoring have been developed to date, including those using electrochemical measurement methods. However, a drawback is that they are limited in the types of substances they can measure. For example, a sensor using a direct electron transfer enzyme is an example of such a biosensor, but the substance to be measured in such a sensor is limited to the substrate of the direct electron transfer enzyme. Specifically, when the substrate to be measured comes into contact with the direct electron transfer enzyme, a reaction occurs in which the substrate is converted into a product by the activity of the enzyme. Furthermore, based on the principles of substrate-enzyme reactions, the concentration of the substrate in a reaction between a substrate and an enzyme correlates with the enzyme's reaction rate. In particular, with direct electron transfer enzymes, electrons are transferred between the substrate and the direct electron transfer enzyme during the reaction. Therefore, in a direct electron transfer enzyme reaction, when the amount of substrate is large, the reaction rate increases, which in turn increases the amount of electron transfer (in other words, the amount of electrons transferred increases), resulting in a larger current flowing through the direct electron transfer enzyme at that point. Thus, in a biosensor using a direct electron transfer enzyme, the concentration of the substrate as the substance to be measured is converted into a current value that flows through the direct electron transfer enzyme, making it possible to monitor it in real time.

[0004] Furthermore, existing electrochemical biosensors are sometimes limited to measuring redox substances, and there is a need for the development of technology that can monitor non-redox substances in real time.

[0005] S.Takamatsu et al.,Continuous electrochemical monitoring of L-glutamine using redox-probe-modified L-glutamine-binding protein based on intermittent pulse amperometry,Sensors and Actuators B:Chemical,346,130554(2021)F.Schachinger et al., Amperometric Biosensors Based on Direct Electron Transfer Enzymes, Molecules 26 (2021) 4525.K.Kano,Redox Potentials of Proteins and Other Compounds of Bioelectrochemical Interest in Aqueous Solutions,Review of Polarography,Vol.48,No.1,(2002)29-46.S.Kawai et al.,Heterologous overexpression and characterization of a flavoprotein-cytochrome c complex fructose dehydrogenase of Gluconobacter japonicus NBRC3260,Appl.Environ.Microbiol.79(2013)1654-1660.K.Kataoka et al.,Structure and function of the engineered multicopper oxidase CueO from Escherichia coli-Deletion of the methionine-rich helical region covering the substrate-binding site,J.Mol.Biol.373(2007)141-152.

[0006] An object of the present disclosure is to provide a technology for monitoring not only redox substances but also non-redox substances in real time. In particular, with conventional biosensors using the above-mentioned direct electron transfer enzymes, the analyte is limited to the substrate of the direct electron transfer enzyme, making it impossible to monitor in real time substances other than the substrate of the direct electron transfer enzyme. Therefore, an object of the present disclosure is to provide a technology that can monitor the analyte in real time, even when the analyte is a substance other than the substrate of the direct electron transfer enzyme.

[0007] The present inventors have found that modifying a direct electron transfer enzyme with a substance that specifically binds to the analyte enables the analyte to be measured electrochemically, and have made further improvements.

[0008] The present disclosure encompasses, for example, the subject matter described in the following paragraphs. Item 1. An electron transfer enzyme comprising a direct electron transfer site and an analyte sensing site, wherein the direct electron transfer site has catalytic reaction activity for converting a substrate into a product and direct electron transfer activity associated with the catalytic reaction, and is a site that receives and gives off electrons to and from the substrate in the catalytic reaction and is capable of directly releasing or influxing the electrons to and from the outside of the enzyme, and the analyte sensing site is linked to the direct electron transfer site and is a sensing site that is capable of changing the amount of electron release or influx when it comes into contact with the analyte. Item 2. An electron transfer enzyme comprising an analyte sensing site selected from the group consisting of fructose dehydrogenase, multicopper oxidase, glucose dehydrogenase, glucose oxidase, pyranose dehydrogenase, and ferredoxin-NADP. ＋Item 1. The enzyme according to Item 1, wherein the direct electron transfer site has a protein comprising the entire amino acid sequence encoding sulfite oxidase, sulfite dehydrogenase, nitrate reductase, carbon monoxide dehydrogenase, hydrogenase, galactose oxidase, or nitrous oxide reductase, or a protein domain comprising a part of said amino acid sequence. Item 3. The enzyme according to Item 1 or 2, wherein the direct electron transfer site has a protein comprising the entire amino acid sequence encoding sulfite oxidase, sulfite dehydrogenase, nitrate reductase, carbon monoxide dehydrogenase, hydrogenase, nitrous oxide reductase, alcohol dehydrogenase, or aldehyde dehydrogenase, or a protein domain comprising a part of said amino acid sequence. Item 4. The enzyme according to any one of Items 1 to 3, wherein the direct electron transfer site has a protein containing the entire amino acid sequence encoding fructose dehydrogenase or multicopper oxidase, or a protein domain containing a part of the amino acid sequence. Item 5. The enzyme according to any one of Items 1 to 4, wherein the analyte detection site contains at least one species selected from the group consisting of biotin, an antigen, an antibody, RNA, DNA, ethylenediaminetetraacetic acid, and nitrilotriacetic acid. Item 6. A kit comprising the enzyme according to any one of Items 1 to 5. Item 7. An enzyme electrode comprising the enzyme according to any one of Items 1 to 5 and an electrode substrate, wherein the electrode substrate is an electrode substrate that comes into contact with the enzyme to receive electrons released from the enzyme or to allow electrons to flow into the enzyme. Item 8. An enzyme sensor comprising the enzyme electrode according to Item 7 as a working electrode. Item 9. An electrochemical measurement method using the enzyme according to any one of Items 1 to 5, the kit according to Item 6, the enzyme electrode according to Item 7, or the enzyme sensor according to Item 8.Item 10. An electrochemical measurement method comprising the following steps (I) and (II): (I) a step of contacting the enzyme according to any one of Items 1 to 5, the kit according to Item 6, the enzyme electrode according to Item 7, or the enzyme sensor according to Item 8 with a substrate for the enzyme, and (II) a step of contacting the enzyme according to any one of Items 1 to 5, the kit according to Item 6, the enzyme electrode according to Item 7, or the enzyme sensor according to Item 8 with a substrate for the enzyme and a substance to be measured. Item 11. The method according to Item 10, wherein step (I) is a step of contacting the enzyme according to any one of Items 1 to 5, the kit according to Item 6, the enzyme electrode according to Item 7, or the enzyme sensor according to Item 8 with a substrate for the enzyme to measure an electrochemical signal, and step (II) is a step of contacting the enzyme according to any one of Items 1 to 5, the kit according to Item 6, the enzyme electrode according to Item 7, or the enzyme sensor according to Item 8 with a substrate for the enzyme and a substance to be measured, and measuring an electrochemical signal. Item 12. Item 12. The method according to Item 11, wherein the electrochemical signal measured in step (I) is compared with the electrochemical signal measured in step (II), and the presence / absence or concentration of the substance to be measured is determined based on the amount of change in the electrochemical signal. Item 13. The method according to Item 11 or 12, wherein the electrochemical signal is a current value.

[0009] A direct electron transfer enzyme suitable for real-time monitoring of not only redox substances but also non-redox substances is provided. An electron transfer enzyme including a direct electron transfer site and an analyte detection site is provided. Also provided are a kit and enzyme electrode including the enzyme, and an enzyme sensor including the enzyme electrode. Also provided is an electrochemical measurement method using the enzyme, kit, enzyme electrode, or enzyme sensor. According to the technology disclosed herein, even when the analyte is a substance other than a substrate of a direct electron transfer enzyme, the analyte can be monitored in real time.

[0010] 1 shows the current-time curves when a fructose dehydrogenase (FDH)-modified electrode was used, and a copper efflux oxidase (CueO, an oxygen reductase)-modified electrode was used.

[0011] Each embodiment of the present disclosure will be described in more detail below. The present disclosure includes a direct electron transfer enzyme modified with a modifying substance. In this specification, such an enzyme may be referred to as the "enzyme of the present disclosure."

[0012] As used herein, the term "direct electron transfer enzyme" refers to an enzyme that can directly donate and receive electrons to and from an electrode via a redox substance present within the enzyme or on its surface, and that catalyzes a redox reaction.

[0013] Direct electron transfer enzymes are not particularly limited, but include, for example, fructose dehydrogenase, multicopper oxidase (e.g., copper efflux oxidase, laccase, bilirubin oxidase, ascorbate oxidase, etc.), glucose dehydrogenase, glucose oxidase, pyranose dehydrogenase, and ferredoxin-NADP. ＋ Examples of suitable oxidases include reductase, cellobiose dehydrogenase, gluconate dehydrogenase, succinate dehydrogenase, histamine dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, cytochrome P450, peroxidase, xanthine dehydrogenase, xanthine oxidase, sulfite oxidase, sulfite dehydrogenase, formate dehydrogenase, nitrogenase, nitrate reductase, carbon monoxide dehydrogenase, hydrogenase, galactose oxidase, and nitrous oxide reductase (Non-Patent Document 2). Among these, fructose dehydrogenase or multicopper oxidase (especially copper efflux oxidase) is preferred. Also preferred are sulfite oxidase, sulfite dehydrogenase, nitrate reductase, carbon monoxide dehydrogenase, hydrogenase, nitrous oxide reductase, alcohol dehydrogenase, and aldehyde dehydrogenase. The direct electron transfer enzyme may be a wild-type enzyme, or may contain mutations such as amino acid deletions, substitutions, or additions, as long as it has the activity of a direct electron transfer enzyme. For example, fructose dehydrogenase is a heterotrimer composed of three subunits (I to III), and it is known that it functions as a direct electron transfer enzyme even when unnecessary subunits are removed. Therefore, the direct electron transfer enzyme also includes, for example, mutants in which the unnecessary subunits have been removed.

[0014] Thus, the direct electron transfer enzyme modified with a modifier may be an enzyme (protein) containing the entire amino acid sequence of the enzyme (the amino acid sequence encoding each enzyme), or an enzyme (which may be referred to as an oligomer containing a protein domain and / or subunit) containing a part of the amino acid sequence (which may be referred to as a protein domain) thereof, as long as it has the activity of a direct electron transfer enzyme, i.e., as long as it has catalytic reaction activity for converting a substrate to a product and direct electron transfer activity associated with the catalytic reaction. In the present disclosure, catalytic reaction activity means the ability to convert a substrate to a product accompanied by electron transfer, and direct electron transfer activity means the ability to directly release electrons to the outside of the enzyme (the direct electron transfer site) or to directly inject electrons from the outside into the enzyme (the direct electron transfer site). In other words, the direct electron transfer enzyme at least comprises a direct electron transfer site that is responsible for the catalytic reaction activity of converting a substrate into a product and the direct electron transfer activity associated with the catalytic reaction, and electrons are transferred at this site due to electron transfer with the substrate in the catalytic reaction, making it possible for the electrons to be directly released outside the enzyme or directly flow into the enzyme from the outside.

[0015] The amino acid sequence of each enzyme can be easily found based on databases such as UniProt (https: / / www.uniprot.org / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ). As described above, the enzyme (particularly the direct electron transfer site) may be one in which unnecessary subunits have been removed, as long as it has catalytic reaction activity and direct electron transfer activity. Furthermore, one or more amino acids in the amino acid sequence of the enzyme (particularly the direct electron transfer site) may be mutated (deleted, substituted, or added). For example, one to ten amino acids are exemplified as one or more, more preferably one, two, three, four, or five. While not limiting the present disclosure, the mutation is preferably one that, when an electrode is prepared using the mutated enzyme in the same manner as in the test examples described below and electrochemical measurements are performed, an electrochemical signal resulting from the direct electron transfer activity associated with the catalytic activity is obtained when the enzyme is reacted with its substrate.

[0016] Furthermore, a modifying substance can be said to be a substance that detects the analyte, and therefore, in the present disclosure, a direct electron transfer enzyme modified with a modifying substance can be said to be, in other words, an electron transfer enzyme that includes a direct electron transfer site and an analyte detection site linked to the direct electron transfer site.

[0017] While not limiting the present disclosure, to more specifically describe one embodiment of the enzyme of the present disclosure, the direct electron transfer site has catalytic activity for converting a substrate into a product and direct electron transfer activity associated with the catalytic reaction, and is a site that receives and gives off electrons to and from the substrate in the catalytic reaction and is capable of directly releasing or influxing the electrons to or from the outside of the enzyme (for example, when the electron transfer enzyme is in contact with an electrode, the site can directly release or influx the electrons to or from the electrode). In particular, when the electron transfer enzyme is in contact with an electrode, direct release or influx of electrons becomes possible between the electron transfer enzyme and the electrode in contact with it.

[0018] In an electron transfer enzyme having such a direct electron transfer site, for example, electrons obtained in a catalytic reaction (oxidation reaction) are transferred without delay at the site and transmitted directly from the enzyme to the outside (e.g., an electrode), or electrons are lost in a catalytic reaction (reduction reaction) and electrons flow directly into the enzyme from the outside (e.g., an electrode) without delay, resulting in electron transfer, and this series of electron transfers and donations occurs continuously. Therefore, the enzyme of the present disclosure can donate and accept electrons to and from a substrate in the catalytic reaction, and simultaneously release the electrons directly to the outside of the enzyme or allow them to flow in directly from the outside.

[0019] The enzyme can be produced according to genetic engineering techniques such as conventionally known protein expression procedures. For example, the enzyme can be obtained by transforming a vector, host cell, or the like with a base sequence encoding the enzyme and expressing the protein (enzyme). The enzyme is not limited as long as it contains a direct electron transfer site. Furthermore, any sequence such as a promoter, linker, or restriction enzyme cleavage site may be further inserted into the vector as needed, and the enzyme may also contain any sequence other than the direct electron transfer site as long as the expressed protein has the above activity.

[0020] Examples of catalytic reactions targeted by the enzyme (particularly the direct electron transfer site) include the following.

[0021] Fructose dehydrogenase is an enzyme that oxidizes the substrate D-fructose to the product 5-keto-D-fructose and receives electrons from the substrate. SEQ ID NOS: 1 to 3 show the amino acid sequences of subunits I, II, and III constituting fructose dehydrogenase derived from the acetic acid bacterium Gluconobacter japonicus NBRC3260. In fructose dehydrogenase, the portion primarily responsible for the catalytic reaction (catalytically active portion) is exemplified by a flavin adenine dinucleotide (FAD)-containing domain, and the portion primarily responsible for direct electron transfer with the outside (direct electron transfer active portion) is exemplified by a heme C-containing domain (preferably a domain containing at least one selected from the group consisting of heme 1c, heme 2c, and heme 3c) and a 3Fe-4S iron-sulfur cluster-containing domain. For example, subunit I contains a FAD-containing domain and a 3Fe-4S iron-sulfur cluster-containing domain, and subunit II contains a heme C-containing domain. Without limiting the present disclosure, in SEQ ID NO: 2 representing subunit II, the amino acid sequence from the N-terminus at positions 27 to 169 corresponds to the heme 1c-containing domain, the amino acid sequence from positions 170 to 325 corresponds to the heme 2c-containing domain, and the amino acid sequence from positions 326 to 440 corresponds to the heme 3c-containing domain. A preferred example of a direct electron transfer site is a protein comprising an FAD-containing domain and at least one domain selected from the group consisting of a heme 1c-containing domain, a heme 2c-containing domain, a heme 3c-containing domain, and a 3Fe-4S iron-sulfur cluster-containing domain. When two or three domains selected from the group consisting of a heme 1c-containing domain, a heme 2c-containing domain, a heme 3c-containing domain, and a 3Fe-4S iron-sulfur cluster-containing domain are contained, any combination thereof may be used, and all four domains may also be contained. A preferred example is a protein comprising a heme 2c-containing domain and / or a 3Fe-4S iron-sulfur cluster-containing domain. Preferred examples of the direct electron transfer site include a protein containing subunits I and II, a protein containing subunits I and III, and a protein containing subunits I, II, and III.Copper efflux oxidase (CueO), a type of multicopper oxidase, is an enzyme that reduces the substrate oxygen to the product water and donates electrons to the substrate. SEQ ID NO: 4 shows the amino acid sequence of copper efflux oxidase (CueO) derived from Escherichia coli. In copper efflux oxidases, the portion primarily responsible for catalytic reactions (catalytically active portion) is exemplified by type II and III copper cluster-containing domains, and the portion primarily responsible for direct electron transfer with the outside (direct electron transfer active portion) is exemplified by type I copper. For example, copper efflux oxidases contain type II and III copper cluster-containing domains and a type I copper-containing domain as monomers. Therefore, a preferred example is one that contains type II and III copper cluster-containing domains and a type I copper-containing domain as direct electron transfer sites. Laccase, a type of multicopper oxidase, is an enzyme that reduces the substrate oxygen to the product water and donates electrons to the substrate. Bilirubin oxidase, a type of multicopper oxidase, is an enzyme that reduces the substrate oxygen to the product water and donates electrons to the substrate. Ascorbate oxidase, another type of multicopper oxidase, is an enzyme that reduces the substrate oxygen to the product water and donates electrons to the substrate. Glucose dehydrogenase is an enzyme that oxidizes the substrate D-glucose to the product D-glucono-1,5-lactone and accepts electrons from the substrate, or oxidizes the substrate D-maltose to the product D-maltono-1,5-lactone and accepts electrons from the substrate. Glucose oxidase is an enzyme that oxidizes the substrate D-glucose to the product D-glucono-1,5-lactone and accepts electrons from the substrate. Pyranose dehydrogenase is an enzyme that oxidizes the substrate pyranose to the product pyranolactone or ketopyranose and accepts electrons from the substrate. Ferredoxin-NADP. ＋ Reductase converts the substrate NADPH into the product NADP ＋ to accept electrons from the substrate, or to ＋to the product NADPH and donates electrons to the substrate. Cellobiose dehydrogenase is an enzyme that oxidizes the substrate cellobiose to the product cellobiono-1,5-lactone and accepts electrons from the substrate, oxidizes the substrate D-glucose to the product D-glucono-1,5-lactone and accepts electrons from the substrate, or oxidizes the substrate lactose to the product lactono-1,5-lactone and accepts electrons from the substrate. Gluconate dehydrogenase is an enzyme that oxidizes the substrate D-gluconic acid to the product 2-keto-D-gluconic acid and accepts electrons from the substrate. Succinate dehydrogenase is an enzyme that oxidizes the substrate succinic acid to the product fumaric acid and accepts electrons from the substrate. Histamine dehydrogenase is an enzyme that oxidizes the substrate histamine to the product imidazole-4-acetaldehyde and accepts electrons from the substrate. Lactate dehydrogenase is an enzyme that oxidizes the substrate lactate to the product pyruvate and receives electrons from the substrate. Alcohol dehydrogenase is an enzyme that oxidizes the substrate ethanol to the product acetaldehyde and receives electrons from the substrate. Aldehyde dehydrogenase is an enzyme that oxidizes the substrate acetaldehyde to the product acetate and receives electrons from the substrate. Cytochrome P450 is an enzyme that oxidizes various substrates (e.g., testosterone, long-chain fatty acids) and receives electrons from the substrate. Peroxidase is an enzyme that reduces the substrate hydrogen peroxide to the product water and donates electrons to the substrate. Xanthine dehydrogenase is an enzyme that oxidizes the substrate hypoxanthine to the product xanthine and receives electrons from the substrate, or oxidizes the substrate xanthine to the product urate and receives electrons from the substrate. Xanthine oxidase is an enzyme that oxidizes the substrate hypoxanthine to the product xanthine and accepts electrons from the substrate, or oxidizes the substrate xanthine to the product urate and accepts electrons from the substrate. Sulfite oxidase is an enzyme that oxidizes the substrate sulfite to the product sulfate and accepts electrons from the substrate. Sulfite dehydrogenase is an enzyme that oxidizes the substrate sulfite to the product sulfate and accepts electrons from the substrate.Formate dehydrogenase oxidizes the substrate formate to the product carbon dioxide and receives electrons from the substrate, or converts the substrate NADH to the product NAD. ＋ It is an enzyme that oxidizes the substrate carbon dioxide to the product formic acid and gives electrons to the substrate, or it oxidizes the substrate NAD ＋ Nitrogenase is an enzyme that reduces the substrate nitrogen to the product ammonia and donates electrons to the substrate. Nitrate reductase is an enzyme that reduces the substrate nitrate to the product nitrite and donates electrons to the substrate. Carbon monoxide dehydrogenase is an enzyme that oxidizes the substrate carbon monoxide to the product carbon dioxide and accepts electrons from the substrate. Hydrogenase oxidizes the substrate hydrogen to the product hydrogen ions and accepts electrons from the substrate, or oxidizes the substrate NADH to the product NAD ＋ It is an enzyme that oxidizes the substrate hydrogen ion to the product hydrogen and donates electrons to the substrate, or it oxidizes the substrate NAD ＋ Galactose oxidase is an enzyme that oxidizes the substrate D-galactose to the product D-galactohexodialdose and receives electrons from the substrate. Nitrous oxide reductase is an enzyme that reduces the substrate nitrous oxide to the product nitrogen and receives electrons from the substrate.

[0022] In the electron transfer between a direct electron transfer enzyme (electron transfer enzyme, particularly a direct electron transfer site) and a substrate, when the enzyme (site) receives electrons from the substrate, the enzyme (site) can directly release the electrons to the outside, and when the enzyme (site) donates electrons to the substrate, the enzyme (site) can directly inject the electrons from the outside.

[0023] The modifying substance (analyte detection site) is not particularly limited as long as it specifically interacts with (binds to, etc.) the analyte. Examples include biotin, antigens, antibodies, RNA, DNA, ethylenediaminetetraacetic acid, nitrilotriacetic acid, etc. These can be used alone or in combination of two or more.

[0024] Therefore, without limiting the present disclosure, the enzyme (preferably the direct electron transfer site) may be fructose dehydrogenase, multicopper oxidase, glucose dehydrogenase, glucose oxidase, pyranose dehydrogenase, ferredoxin-NADP ＋ reductase, cellobiose dehydrogenase, gluconate dehydrogenase, succinate dehydrogenase, histamine dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, cytochrome P450, peroxidase, xanthine dehydrogenase, xanthine oxidase, sulfite oxidase, sulfite dehydrogenase, formate dehydrogenase, nitrogenase, nitrate reductase, carbon monoxide dehydrogenase, hydrogenase, galactose oxidase or nitrous oxide reductase (preferably fructose dehydrogenase, maize Preferred examples of such enzymes include those selected from the group consisting of multicopper oxidase, sulfite oxidase, sulfite dehydrogenase, nitrate reductase, carbon monoxide dehydrogenase, hydrogenase, nitrous oxide reductase, alcohol dehydrogenase, or aldehyde dehydrogenase, and more preferably fructose dehydrogenase or multicopper oxidase, or protein domains derived from such enzymes, and the modifying substance is at least one selected from the group consisting of biotin, antigens, antibodies, RNA, DNA, ethylenediaminetetraacetic acid, and nitrilotriacetic acid.

[0025] As described below, according to the present disclosure, the presence / absence and / or concentration of an analyte can be easily determined based on a change in the electrochemical signal that occurs when a modifier contacts the analyte. Therefore, without limiting the present disclosure, to more specifically explain a preferred embodiment of the enzyme of the present disclosure, the analyte sensing site is linked to the direct electron transfer site, and has the effect of changing the amount of electrons released or flowing out of the direct electron transfer site (to the electrode when the electron transfer enzyme is in contact with the electrode) when the sensing site contacts the analyte.

[0026] Examples of substances to be measured include avidin, streptavidin, neutravidin, antigens, antibodies, RNA, DNA, metal ions, etc. These may be used alone or in combination of two or more.

[0027] Examples of combinations of modifiers (sites for detecting analytes) and analytes include the following.

[0028] When biotin is used as the modifier (analyte detection site, the same applies below), examples of the analyte include avidin, streptavidin, neutravidin, etc. When an antigen is used as the modifier, examples of the analyte include antibodies, etc. When an antibody is used as the modifier, examples of the analyte include antigens, etc. When RNA is used as the modifier, examples of the analyte include RNA, DNA, etc. When DNA is used as the modifier, examples of the analyte include RNA, DNA, etc. When ethylenediaminetetraacetic acid is used as the modifier, examples of the analyte include metal ions, etc. When nitrilotriacetic acid is used as the modifier, examples of the analyte include metal ions, etc. The analyte may be a redox substance or a non-redox substance. Of these, a non-redox substance is preferable.

[0029] The method for modifying a direct electron transfer enzyme with a modifier is not particularly limited and can be appropriately selected depending on the type of modifier or the type of direct electron transfer enzyme. The modification method can be any method known in the technical field and can be any condition. The modifier can be applied to any position of the direct electron transfer enzyme as long as the direct electron transfer enzyme has the above-mentioned activity.

[0030] Therefore, the analyte sensing site may be linked to any location of the direct electron transfer site within the range in which the direct electron transfer site has the above-mentioned activity. For example, it may be linked to a domain or subunit in the direct electron transfer site that is responsible for the catalytic reaction, or to a domain or subunit that is responsible for direct electron transfer. Taking fructose dehydrogenase as an example, for example, the analyte sensing site may be linked to an FAD-containing domain that is responsible for the catalytic reaction or a subunit containing said domain, or the analyte sensing site may be linked to a heme C-containing domain that is responsible for direct electron transfer or a subunit containing said domain. Furthermore, when both the catalytic reaction and direct electron transfer are possible using a single domain or subunit, the analyte sensing site may be linked to such a domain or subunit.

[0031] The modifying substance can modify the direct electron transfer enzyme (electron transfer enzyme, preferably the direct electron transfer site, the same applies hereinafter) directly or indirectly via a linker or the like.

[0032] The linker can be appropriately selected depending on the type of modifying substance or the type of direct electron transfer enzyme, and examples thereof include a maleimide group, an amino group, a carboxy group, a silane group, an epoxy group, a cysteine ​​group, a hydroxy group, an azide group, an alkyl group, an aldehyde group, and an affinity tag (e.g., a HIS tag, a GST tag, a FLAG tag, etc.).

[0033] A spacer may be included between the modifier and the direct electron transfer enzyme. The spacer can be appropriately selected depending on the type of modifier or the type of direct electron transfer enzyme. The length of the spacer is not particularly limited, and can be appropriately selected depending on the type of modifier or the type of direct electron transfer enzyme.

[0034] When biotin is used as the modifying substance, methods for directly modifying an electron transfer enzyme include, for example, a chemical modification method using a biotinylation reagent such as biotin maleimide, and an enzymatic modification method using an enzyme.

[0035] The amino acid residues that serve as a scaffold for modification by the modifying substance are preferably, for example, amino acid residues that are exposed on the enzyme surface.

[0036] Furthermore, when a direct electron transfer enzyme does not have an amino acid residue that can serve as a scaffold for modification with a modifier, a scaffold may be created by introducing a mutation (more specifically, a substitution, etc.) into the direct electron transfer enzyme within a range that retains the activity of the direct electron transfer enzyme. From the viewpoint of ease of introducing a mutation and from the viewpoint of serving as a scaffold for a modifier, the amino acid residue to be mutated is preferably, for example, an amino acid residue exposed on the enzyme surface.

[0037] Techniques for introducing amino acid mutations into a specific amino acid sequence are known in the art and can be performed using any method, such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis, or random mutagenesis.

[0038] The number of amino acids to be mutated (more specifically, substituted, etc.) is not particularly limited as long as the enzyme has the activity of a direct electron transfer enzyme, and may be, for example, 1 to 10, 1 to 5, 1 to 3, or 1 to 2.

[0039] When fructose dehydrogenase is used as the direct electron transfer enzyme, for example, the lysine residue at position 89 in the amino acid sequence shown in SEQ ID NO: 1 can be substituted with a cysteine ​​residue. When copper efflux oxidase (CueO) is used as the direct electron transfer enzyme, for example, at least one selected from the group consisting of the aspartic acid residue at position 38, the aspartic acid residue at position 132, and the lysine residue at position 401 in the amino acid sequence shown in SEQ ID NO: 4 can be substituted with a cysteine ​​residue. For example, the aspartic acid residues at positions 38 and 132 may be substituted with cysteine ​​residues, the lysine residue at position 401 may be substituted with a cysteine ​​residue, or all of the aspartic acid residues at positions 38, 132, and 401 may be substituted with cysteine ​​residues.

[0040] The number of modifiers that modify the direct electron transfer enzyme is not particularly limited as long as the enzyme has the activity of a direct electron transfer enzyme, and can be appropriately selected depending on the type of modifier or the type of direct electron transfer enzyme. For example, the number may be 1 to 10, 1 to 5, 1 to 3, or 1 to 2.

[0041] In this way, a method for directly linking an analyte sensing site to an electron transfer site is similarly described, i.e., the modifier is replaced with an analyte sensing site, and the direct electron transfer enzyme is replaced with the direct electron transfer site, and the linking procedure, linker, spacer, scaffold, mutation, etc. are similarly described, thereby obtaining an electron transfer enzyme in which an analyte sensing site is directly linked to an electron transfer site.

[0042] Although not limiting the present disclosure, when biotin is used as the detection site for the substance to be measured, methods for directly linking it to the electron transfer site include, for example, a chemical modification method using a biotinylation reagent such as biotin maleimide, or an enzymatic modification method using an enzyme.

[0043] Although not limiting the present disclosure, when fructose dehydrogenase is used as the direct electron transfer site, for example, the lysine residue at position 89 in the amino acid sequence shown in SEQ ID NO: 1 can be substituted with a cysteine ​​residue. When copper efflux oxidase (CueO) is used as the direct electron transfer site, for example, at least one selected from the group consisting of the aspartic acid residue at position 38, the aspartic acid residue at position 132, and the lysine residue at position 401 in the amino acid sequence shown in SEQ ID NO: 4 can be substituted with a cysteine ​​residue. For example, the aspartic acid residues at positions 38 and 132 may be substituted with cysteine ​​residues, the lysine residue at position 401 may be substituted with a cysteine ​​residue, or all of the aspartic acid residues at positions 38, 132, and 401 may be substituted with cysteine ​​residues.

[0044] Although not limiting the present disclosure, the number of analyte detection sites linked to a direct electron transfer site is not particularly limited as long as the enzyme has direct electron transfer activity, and may be, for example, 1 to 10, 1 to 5, 1 to 3, or 1 to 2.

[0045] The present disclosure also encompasses a kit containing the enzyme of the present disclosure. In this specification, such a kit may be referred to as a "kit of the present disclosure."

[0046] The enzymes contained in the kit of the present disclosure (enzymes of the present disclosure) can be the same as those described above. The enzymes contained in the kit of the present disclosure may be one type alone or a combination of two or more types.

[0047] The content of the enzyme of the present disclosure contained in the kit of the present disclosure is not particularly limited, but can be, for example, about 1 to 99% by mass.

[0048] The kit of the present disclosure includes the enzyme of the present disclosure and may further include other components. Such other components include, for example, water, buffer solutions, surfactants, salts, and / or other components that can be used in bioassays. These components may be used alone or in combination of two or more.

[0049] The present disclosure also encompasses an enzyme electrode comprising the enzyme and electrode substrate of the present disclosure. In this specification, the enzyme electrode may be referred to as the "enzyme electrode of the present disclosure."

[0050] The enzyme contained in the enzyme electrode of the present disclosure can be the same as that described above. The enzyme contained in the enzyme electrode of the present disclosure can be one kind of enzyme or a combination of two or more kinds of enzymes.

[0051] The electrode substrate is not particularly limited as long as it has conductivity, and examples thereof include carbon substrates such as graphite, carbon black, activated carbon, glassy carbon, and plastic-formed carbon; metal substrates such as gold, platinum, silver, palladium, and indium tin oxide; and conductive inks such as carbon paste and silver paste. Of these, carbon substrates are preferred, and glassy carbon is more preferred.

[0052] The content of the electrode substrate contained in the enzyme electrode of the present disclosure is not particularly limited.

[0053] The enzyme electrode of the present disclosure may further contain a conductive agent. The conductive agent is not particularly limited, and examples thereof include porous carbon materials such as ketjen black, carbon nanotubes, carbon nanoparticles, carbon black, and acetylene black; carbon whiskers; carbon fibers; natural graphite; artificial graphite; gold; silver; copper; nickel; vanadium; platinum; palladium; iron; aluminum; titanium; zinc; tin; tungsten; and chromium. These may be used alone or in combination of two or more. Among these, porous carbon materials are preferred, and ketjen black and carbon nanotubes are more preferred.

[0054] The content of the enzyme of the present disclosure contained in the enzyme electrode of the present disclosure is not particularly limited. For example, when the enzyme electrode of the present disclosure contains a conductive agent, the content mass ratio of the enzyme of the present disclosure to 1 part by mass of the conductive agent can be, for example, about 0.001 to 1 part by mass.

[0055] The conductive agent is preferably applied to, for example, the surface of the electrode substrate, etc. The application method is not particularly limited, and any method known in the technical field and any conditions can be adopted.

[0056] When the electrode substrate and / or the enzyme electrode of the present disclosure contains a conductive agent, the enzyme of the present disclosure is preferably immobilized on the conductive agent. The method for immobilizing the direct electron transfer enzyme (electron transfer enzyme) is not particularly limited, and any method known in the art and any conditions can be used. Examples include physical methods such as adsorption and chemical methods such as crosslinking.

[0057] In this way, an electrode can be produced in which the enzyme of the present disclosure is in contact with the electrode substrate. The enzyme electrode of the present disclosure in which the enzyme is in contact with the electrode substrate can easily receive electrons released from the enzyme or allow electrons to flow into the enzyme in response to electron transfer associated with the catalytic reaction that occurs when the enzyme comes into contact with the substrate.

[0058] The enzyme electrode of the present disclosure may or may not contain an electron mediator. Since the enzyme electrode of the present disclosure contains a direct electron transfer enzyme (electron transfer enzyme, particularly a direct electron transfer site) that can directly donate and receive electrons to and from the electrode, it does not need to contain an electron mediator.

[0059] Examples of electron mediators include compounds described in Non-Patent Document 3. Specifically, water-soluble iron porphyrin ([5,10,15,20-tetrakis(2,6-dichloro-3-sulfonate)-popirinate]Fe(H ２ O) (OH －)), ABTS (2,2'-azinobis-(3-ethylbenzthiozoline-6-sulfonic acid), promazine, chloramine T, TMPDA (N,N,N',N'-tetramethylphenylenediamine), porphyrexide, ABTS, syringaldazine, o-tolidine, bacteriochlorophyll a, dopamine, 2,5-dihydroxy-1,4-benzoquinone, p-amino-dimethylaniline, o-quinone / 1,2-hydroxybenzene (catechol), p-aminophenol, tetrahydroxybenzoyl C-p-benzoquinone, 2,5-dichloro-p-benzoquinone, 1,4-benzoquinone, TMPDA, diaminodurene, 2,5-dihydroxyphenylacetic acid, 2,6,2'-trichloroindophenol, indophenol, o-toluidine blue, DCPIP (2,6-dichlorophenol indophenol), 2,6-dibromo-indophenol, phenol blue, 3-amino-thiazine, 1,2-naphthoquinone-4-sulfonate, 2,6-dimethyl-p-benzoquinone, 2,6 -dibromo-2'-methoxy-indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone, 2,5-dimethyl-p-benzoquinone, 1,4-dihydroxy-naphthoic acid, 2,6-dimethyl-indophenol, 5-isopropyl-2-methyl-p-benzoquinone, 1,2-naphthoquinone, 1-naphthol-2-sulfonic acid indophenol, toluylene blue, TTQ (tryptophan tryptophylquinone) model compound (3-methyl-4-(3'-methylindole- 2'-yl)indole-6,7-dione), ubiquinone (coenzyme Q), PMS (N-methylphenazinium methosulfate), TPQ (topaquinone, or 6-hydroxydopaquinone), PQQ (pyrroloquinoline quinone), thionine, thionine tetrasulfonate, ascorbic acid, PES (phenazine ethosulfate), cresyl blue, 1,4-naphthoquinone, toluidine blue, thiazine blue, gallocyanine, thioindigo disulfonate, methylene blue, vitamin K ３ , (2-methyl-1,4-naphthoquinone), Pycocyanin, Indigo Tetrasulfonate, Vitamin K １(2-methyl-3-phytyl 1,4-naphthoquinone), luciferin, gallocyanin, pyocyanin, methyl capri blue, resorufin, 2-amino-3-carboxy-1,4-naphthoquinone, 2-farnesyl-3-methyl-1,4-naphthoquinone, indigo trisulfonate, 4-amino-1,2-naphthoquinone, 6,8,9-trimethyl-isoaroxazine, chlorafine, indigo disulfonate, Nile blue, indigo Carmine, 9-phenyl-isoalloxazine, 2-hydroxy-1,4-naphthoquinone, thioglycolic acid, 2-amino-N-methylphenazine methosulfate, Azure A, indigo monosulfonate, anthraquinone-1,5-disulfonate, alloxazine, brilliant alizarin blue, crystal violet, 2-methyl-3-hydroxy-1,4-naphthoquinone, patent blue, 9-methyl-isoalloxazine, ciba Examples of the antibacterial agent include Chron Blue, phenol red, anthraquinone-2,6-disulfonate, neutral blue, bromophenol blue, anthraquinone-2,7-disulfonate, quinoline yellow, riboflavin, FMN (flavin adenine mononucleotide), FAD (flavin adenine dinucleotide), anthraquinone-1-sulfonate, anthraquinone-2-sulfonate, phenosafranine, lipoamide, safranine T, lipoic acid, indulin scarlet, 4-aminoacridine, acridine, NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate), neutral red, cysteine, benzyl viologen, 3-aminoacridine, 1-aminoacridine, methyl viologen, 2-aminoacridine, water-soluble iron porphyrin, 2,8-diaminoacridine, methyl viologen, and 5-aminoacridine.

[0060] The present disclosure also encompasses an enzyme sensor that includes the enzyme electrode of the present disclosure as a working electrode. In this specification, the enzyme sensor may be referred to as the "enzyme sensor of the present disclosure."

[0061] The above description can be applied to the enzyme electrode included in the enzyme sensor of the present disclosure.

[0062] The enzyme sensor of the present disclosure includes the enzyme electrode of the present disclosure as a working electrode, and may further include a counter electrode, or may include a counter electrode and a reference electrode. In other words, the enzyme sensor of the present disclosure may be a two-electrode type or a three-electrode type. Among these, a three-electrode type including a counter electrode and a reference electrode is preferred.

[0063] The material for the counter electrode is not particularly limited, but examples thereof include platinum, gold, diamond, palladium, carbon, etc. Among these, platinum is preferred.

[0064] The material of the reference electrode is not particularly limited, but examples thereof include silver / silver chloride, silver, platinum, palladium, carbon, etc. Among these, silver / silver chloride is preferred.

[0065] The enzyme sensor of the present disclosure may or may not contain an electron mediator. Since the enzyme sensor of the present disclosure includes an enzyme electrode containing a direct electron transfer enzyme (electron transfer enzyme, particularly a direct electron transfer site) that can directly donate and receive electrons to and from the electrode, the enzyme sensor may not contain an electron mediator.

[0066] The enzyme, kit, enzyme electrode, and enzyme sensor of the present disclosure can be used in various electrochemical measurement techniques by using an electrochemical analyzer such as a potentiostat or galvanostat.

[0067] The present disclosure also encompasses an electrochemical measurement method using the enzyme of the present disclosure, the kit of the present disclosure, the enzyme electrode of the present disclosure, or the enzyme sensor of the present disclosure. In this specification, this measurement method may be referred to as the "measurement method of the present disclosure."

[0068] The measurement method of the present disclosure preferably includes, for example, a step of contacting (i) an enzyme of the present disclosure, a kit of the present disclosure, an enzyme electrode of the present disclosure, or an enzyme sensor of the present disclosure, (ii) a substrate for the enzyme, and (iii) a sample containing a substance to be measured under voltage application conditions, and measuring a current value.

[0069] More specifically, the measurement method of the present disclosure preferably includes, for example, (I) a step of contacting (i) an enzyme of the present disclosure, a kit of the present disclosure, an enzyme electrode of the present disclosure, or an enzyme sensor of the present disclosure, and (ii) a substrate of the enzyme under voltage application conditions, and (II) a further step of contacting (i) an enzyme of the present disclosure, a kit of the present disclosure, an enzyme electrode of the present disclosure, or an enzyme sensor of the present disclosure, with (ii) a substrate of the enzyme, and (iii) a sample containing a substance to be measured under voltage application conditions. Furthermore, the measurement method of the present disclosure preferably measures a current value, for example, in step (I). Furthermore, the measurement method of the present disclosure preferably also measures a current value, for example, in step (II). In other words, the measurement method of the present disclosure preferably includes, for example, (I) a step of contacting (i) an enzyme of the present disclosure, a kit of the present disclosure, an enzyme electrode of the present disclosure, or an enzyme sensor of the present disclosure, and (ii) a substrate of the enzyme under voltage application conditions, and measuring a current value; and (II) a step of further contacting (i) an enzyme of the present disclosure, a kit of the present disclosure, an enzyme electrode of the present disclosure, or an enzyme sensor of the present disclosure, and (ii) a substrate of the enzyme, and (iii) a sample containing a substance to be measured under voltage application conditions, and measuring a current value.

[0070] The voltage application conditions can be appropriately selected depending on the direct electron transfer enzyme (electron transfer enzyme, particularly the direct electron transfer site; the same applies hereinafter) and the counter electrode or reference electrode used. Furthermore, the voltage application conditions in step (I) and step (II) are preferably the same. A preferred example of the applied voltage is about −1 to +1 V. For example, when fructose dehydrogenase is used as the direct electron transfer enzyme, the applied voltage can be, for example, +0.1 to 1 V, preferably about 0.3 to 0.8 V. For example, when copper efflux oxidase (CueO) is used as the direct electron transfer enzyme, the applied voltage can be, for example, about −1.0 to +1.0 V, preferably about −0.3 to +0.3 V.

[0071] As the substrate for the direct electron transfer enzyme, a substance that can serve as a substrate for a redox reaction can be appropriately selected depending on the type of direct electron transfer enzyme. The concentration of the substrate can be appropriately selected depending on the type of direct electron transfer enzyme used, and can be, for example, about 0.1 to 10 mM, preferably about 0.5 to 5 mM. For example, when fructose dehydrogenase is used as the direct electron transfer enzyme, the concentration of fructose as the substrate can be, for example, about 0.1 to 10 mM, preferably about 0.5 to 5 mM. For example, when copper efflux oxidase (CueO) is used as the direct electron transfer enzyme, the substrate is oxygen, and therefore, there is no need to add a substrate as long as dissolved oxygen is present.

[0072] The method for contacting (i) the enzyme of the present disclosure, the kit of the present disclosure, the enzyme electrode of the present disclosure, or the enzyme sensor of the present disclosure, and (ii) the substrate of the enzyme is not particularly limited, but examples include a method of immersing (i) the enzyme of the present disclosure, the kit of the present disclosure, the enzyme electrode of the present disclosure, or the enzyme sensor of the present disclosure in an aqueous solution containing (ii) the substrate of the enzyme.

[0073] The type of aqueous solution containing the substrate is not particularly limited and can be appropriately selected depending on the type of direct electron transfer enzyme, and examples include solutions containing inorganic salts, phosphate buffer, McIlvain buffer, Tris buffer, citrate buffer, acetate buffer, etc.

[0074] Examples of electrochemical measurement methods (methods for measuring current values) include amperometry such as chronoamperometry and potential step chronoamperometry; and voltammetry such as cyclic voltammetry and differential pulse voltammetry.

[0075] The sample containing the substance to be measured is not particularly limited as long as it can contain the substance to be measured, and examples include body fluids such as whole blood, serum, plasma, saliva, cerebrospinal fluid, synovial fluid, urine, interstitial fluid, sweat, tears, and saliva; samples derived from these body fluids; production process fluids for pharmaceuticals, chemical products, fermented products, etc.; and samples derived from these production process fluids. The sample containing the substance to be measured is preferably in liquid form. The content of the substance to be measured in the sample is not particularly limited as long as the measurement is performed appropriately.

[0076] The method for contacting (i) the enzyme of the present disclosure, the kit of the present disclosure, the enzyme electrode of the present disclosure, or the enzyme sensor of the present disclosure, (ii) the substrate of the enzyme, and (iii) a sample containing the substance to be measured is not particularly limited, but examples include a method in which (iii) a sample containing the substance to be measured is added to an aqueous solution containing (ii) the substrate of the enzyme (in other words, to the aqueous solution of step (I)) in which (i) the enzyme of the present disclosure, the kit of the present disclosure, the enzyme electrode of the present disclosure, or the enzyme sensor of the present disclosure is immersed.

[0077] The conditions for measuring the current value in the measurement method of the present disclosure (contact conditions in steps (I) and (II)) can be appropriately selected depending on the type of direct electron transfer enzyme used, and examples include a temperature of 0 to 80°C, preferably about 3 to 40°C, and a pH of about 2 to 8 at that contact temperature. For example, when fructose dehydrogenase is used as the direct electron transfer enzyme, the temperature can be, for example, 0 to 80°C, preferably about 15 to 40°C. For example, when fructose dehydrogenase is used as the direct electron transfer enzyme, the pH can be, for example, 2.0 to 8.0, preferably about 4.0 to 7.5. For example, when copper efflux oxidase (CueO) is used as the direct electron transfer enzyme, the temperature can be, for example, 0 to 80°C, preferably about 3 to 40°C, more preferably about 15 to 40°C. For example, when copper efflux oxidase (CueO) is used as the direct electron transfer enzyme, the pH can be set to, for example, about 2.0 to 8.0, preferably about 4.0 to 7.5.

[0078] In the measurement method of the present disclosure, stirring or the like may be carried out as necessary.

[0079] In the measurement method of the present disclosure, in step (I), the catalytic current accompanying the oxidation or reduction reaction of the substrate by the direct electron transfer enzyme can be measured as the background current. Therefore, in the measurement method of the present disclosure, it is preferable that the sample containing the analyte is added to the measurement system after measuring the background current. In other words, it is preferable that step (II) is carried out after measuring the current value in step (I). The measurement time for the background current (contact time in step (I)) can be, for example, 10 seconds to 2 minutes, preferably about 30 seconds to 1 minute 30 seconds.

[0080] In the measurement method of the present disclosure, the contact time in step (II) is not particularly limited as long as the measurement is carried out appropriately, but can be, for example, about 30 seconds to 5 minutes, more preferably about 1 to 4 minutes.

[0081] Although not wishing to be bound by theory, according to the present disclosure, a direct electron transfer enzyme (electron transfer enzyme, particularly the direct electron transfer site) is modified (linked) with a modifier (analyte detection site) that specifically interacts with (e.g., specifically binds to) the analyte. Therefore, in the presence of the analyte, the modifier (analyte detection site) specifically interacts with the analyte, and for example, the analyte interferes with the cavity (the space that forms the active site) of the direct electron transfer enzyme (electron transfer enzyme, particularly the direct electron transfer site), thereby The enzyme-substrate reaction between the enzyme (particularly the direct electron transfer site) and the original substrate is inhibited, and / or the specific interaction between the modifying substance and the substance to be measured, for example, changes the structure of the enzyme (particularly the direct electron transfer site), thereby inhibiting the enzyme-substrate reaction between the enzyme (particularly the direct electron transfer site) and the original substrate, and / or inhibiting the electrode reaction between the enzyme (particularly the direct electron transfer site) and the electrode, thereby reducing the amount of electrons exchanged with the electrode and decreasing the catalytic current value resulting from the enzyme reaction, and it is therefore thought that the substance to be measured can be measured based on the amount of change in the current value.

[0082] That is, in the measurement method of the present disclosure, the presence of the analyte in the sample causes the current value in step (II) to be smaller than the current value in step (I), or the amount of change (decrease) in the current value in step (II) to be greater than the amount of change (decrease) in the current value in step (I), and therefore the analyte can be measured based on the amount of change in the current value. In the measurement method of the present disclosure, when the enzyme (particularly the direct electron transfer site) used catalyzes an oxidation reaction, the presence of the analyte in the sample causes the positive current value in step (II) to be smaller than the positive current value in step (I), or the amount of change (decrease) in the positive current value in step (II) to be greater than the amount of change (decrease) in the positive current value in step (I), and therefore the analyte can be measured based on the amount of change in the current value. In the measurement method of the present disclosure, when the enzyme used (particularly the direct electron transfer site) catalyzes a reduction reaction, the presence of the substance to be measured in the sample causes the negative current value in step (II) to be smaller than the negative current value in step (I), or causes the fluctuation (decrease) in the negative current value in step (II) to be larger than the fluctuation (decrease) in the negative current value in step (I), and therefore the substance to be measured can be measured based on the fluctuation in the current value.

[0083] The measurement method of the present disclosure can calculate the concentration of the analyte contained in the sample, for example, by using a solution containing a standard concentration of the analyte.

[0084] According to the present disclosure, the electrochemical signal measured in step (I) is compared with the electrochemical signal measured in step (II), and the presence or concentration of the analyte can be determined based on the amount of change in the electrochemical signal. Typically, if a change in the electrochemical signal is observed, it is determined that the analyte is present, and if no change in the electrochemical signal is observed, it is determined that the analyte is present. The measurement method of the present disclosure may further include this determination step. The measurement method of the present disclosure can also be said to be a method for determining the presence or concentration of the analyte, or a method for monitoring the presence and / or concentration of the analyte in real time.

[0085] In this way, when using the enzyme of the present disclosure, the enzyme of the present disclosure is brought into contact with a substrate and the electrochemical signal (e.g., current value) is measured in advance, and then the enzyme of the present disclosure is brought into contact with the substance to be measured in the presence of the substrate, and the amount of change in the electrochemical signal (e.g., current value) resulting from contact with the substance to be measured is confirmed. This makes it possible to easily determine the presence / absence and / or concentration of the substance, even when the substance to be measured is a substance other than the substrate of a direct electron transfer enzyme (electron transfer enzyme, particularly a direct electron transfer site).

[0086] Furthermore, according to the present disclosure, contact between the modifier (the analyte detection site) and the analyte causes a change in electron exchange between the enzyme of the present disclosure (particularly the direct electron transfer site) and the substrate, and this change can cause a change in the amount of electrons directly released or flowing in to the outside. Thus, according to the present disclosure, the presence / absence and / or concentration of the analyte can be monitored in real time.

[0087] For these reasons, according to the present disclosure, even when a substance other than the substrate of the enzyme (particularly the direct electron transfer site) of the present disclosure is used as the analyte, the presence / absence and / or concentration of the substance can be monitored in real time. Furthermore, according to the present disclosure, the presence / absence and / or concentration of the analyte can be monitored in real time based on a change in the electrochemical signal that occurs when the modifier (analyte detection site) comes into contact with the analyte.

[0088] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure encompasses any and all combinations of the constituent elements described in this specification.

[0089] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0090] The present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these examples. In the following, unless otherwise specified, the experiments were carried out under atmospheric pressure and room temperature conditions.

[0091] Preparation of a fructose dehydrogenase (FDH)-modified electrode and measurements using this electrode FDH (wild-type (rFDH), SEQ ID NOS: 1 to 3) and an FDH mutant (K89C) were purified according to Non-Patent Document 4. Structural information has confirmed that FDH (wild-type) has two cysteine ​​residues exposed on the enzyme surface that can serve as scaffolds for maleimide coupling, which will be described later. In this study, an FDH mutant was prepared by introducing the K89C mutation (substituting a cysteine ​​residue for the 89th lysine residue in the amino acid sequence shown in SEQ ID NO: 1 (the amino acid sequence of subunit I constituting fructose dehydrogenase)) to serve as a scaffold for the maleimide coupling reaction.

[0092] The FDH solution may contain 2-mercaptoethanol (a substance that interferes with the maleimide coupling reaction) as a stabilizer. Therefore, this substance was removed by buffer exchange using a centrifugal ultrafiltration filter. Subsequently, FDH (50 μM) and biotin maleimide (1 mM; 20-fold amount relative to FDH) were mixed in 0.1 M phosphate buffer (pH 7.0) and allowed to stand at 25°C for 2 hours. After standing for 2 hours, buffer exchange was performed using a centrifugal ultrafiltration filter to remove unreacted biotin maleimide, and the FDH concentration was adjusted to 20 μM. The solution obtained in this manner was designated as a biotin-modified FDH (wild-type) solution. A biotin-modified FDH mutant solution was obtained in the same manner, except that an FDH mutant was used instead of FDH.

[0093] A glassy carbon electrode (3 mm diameter; BAS, Inc. (Japan)) was used as the electrode substrate, and 30 μg of Ketjen Black (EC300J; ElectroChem, Inc. (USA)), a porous carbon material, was applied to this electrode. Then, 30 μL of the biotin-modified FDH mutant solution was dropped onto this electrode and allowed to stand at 4°C for 2 hours to prepare an FDH mutant-modified electrode. Similarly, various FDH-modified electrodes were prepared using biotin-modified FDH (wild-type), biotin-unmodified FDH (wild-type), or biotin-unmodified FDH mutant solution instead of the biotin-modified FDH mutant solution.

[0094] Electrochemical measurements were performed using an electrochemical analyzer (ALD650E; BAS, Inc. (Japan)) and a rotating ring-disk electrode system (RRDE3; BAS, Inc. (Japan)). An FDH-modified electrode, a silver / silver chloride electrode, and a platinum wire served as the working electrode, reference electrode, and counter electrode, respectively. Current-time curves were measured in 0.1 M acetate buffer (pH 5.0) containing 1 mM D-fructose at 25°C, 4000 rpm, pH 5.0, and +0.5 V. Streptavidin was added to a final concentration of 10 μg / mL one minute after the start of measurement.

[0095] The results are shown in Figure 1. As shown in Figure 1, in the case of non-biotin modification, no decrease in current value was observed after the addition of streptavidin, whether FDH (wild-type) or FDH mutant was used. On the other hand, in the case of biotin modification, a decrease in current value was observed after the addition of streptavidin when FDH (wild-type strain) was used, and an even greater decrease in current value was observed when FDH mutant was used. From these results, it is believed that while electrons are normally generated when fructose is oxidized by FDH, the biotin modification results in a decrease in the amount of electrons supplied to the electrode due to the binding of the biotin used for modification to streptavidin, inhibiting the enzyme substrate reaction or electrode reaction, etc., resulting in the decrease in current value.

[0096] Preparation of a Copper Efflux Oxidase (CueO, Oxygen Reductase)-Modified Electrode and Measurements Using This Electrode CueO (SEQ ID NO: 4) and various CueO mutants (D38C / D132C double mutant and K401C mutant) were purified according to Non-Patent Document 5. Structural information confirmed that wild-type CueO does not contain cysteine ​​residues that are exposed on the enzyme surface and could serve as a scaffold for the maleimide coupling reaction described below. Two CueO mutants were prepared, each with a D38C and D132C double mutation (in which the aspartic acid residues at positions 38 and 132 of the amino acid sequence shown in SEQ ID NO: 4 (the amino acid sequence constituting CueO) are replaced with cysteine ​​residues) or a K401C mutation (in which the lysine residue at position 401 of the amino acid sequence shown in SEQ ID NO: 4 is replaced with a cysteine ​​residue) to provide a scaffold for the maleimide coupling reaction.

[0097] CueO mutant (50 μM) and biotin maleimide (1 mM; 20-fold amount relative to CueO) were mixed in 0.1 M phosphate buffer (pH 7.0) and allowed to stand at 25°C for 2 hours. After standing for 2 hours, buffer exchange was performed using a centrifugal ultrafiltration filter to remove unreacted biotin maleimide, and the CueO concentration was adjusted to 20 μM. The solution obtained in this manner was used as a biotin-modified CueO mutant solution.

[0098] A glassy carbon electrode (diameter 3 mm; BAS, Inc. (Japan)) was used as the electrode substrate, and 5 μg of multi-walled carbon nanotubes (DRP-MWCNTNH2; Metrohm Dropsens (Spain)), a porous carbon material, was applied to this electrode. Then, 30 μL of the biotin-modified CueO mutant solution was dropped onto this electrode, and the electrode was allowed to stand at 4°C for 2 hours to prepare a CueO mutant-modified electrode. Similarly, a CueO (wild-type)-modified electrode was prepared by using a CueO (wild-type, no biotin-modified site) solution instead of the biotin-modified CueO mutant solution.

[0099] For electrochemical measurements, an electrochemical analyzer (ALS650E; BAS, Inc. (Japan)) and a rotating ring-disk electrode system (RRDE3; BAS, Inc. (Japan)) were used. A CueO-modified electrode, a silver / silver chloride electrode, and a platinum wire were used as the working electrode, reference electrode, and counter electrode, respectively. Current-time curves were measured in 0.1 M phosphate buffer (pH 7.0) at 4°C, 9000 rpm, pH 7.0, and 0 V. Streptavidin was added at a final concentration of 100 μg / mL one minute after the start of measurement.

[0100] The results are shown in Figure 2. As shown in Figure 2, when the D38C / D132C double mutant and the K401C mutant were used, an increased decrease in current value was confirmed after the addition of streptavidin, compared to when CueO (wild-type, no biotin modification site) was used. From these results, it is believed that while electrons are normally consumed when oxygen is reduced by CueO, modification with CueO causes the biotin used for modification to bind to streptavidin, inhibiting the enzyme substrate reaction or electrode reaction, thereby reducing the amount of electrons supplied from the electrode and resulting in the decrease in current value (decrease in negative current value).

[0101] From these findings, it was found that by using a direct electron transfer enzyme modified with a modifying substance, i.e., by using an electron transfer enzyme in which the detection site for the substance to be measured is linked to the direct electron transfer site, it is possible to monitor the presence / absence and / or concentration of the substance to be measured in real time, even when the substance to be measured is a substance other than the substrate of the enzyme (especially the direct electron transfer site).

Claims

1. An electron transfer enzyme comprising a direct electron transfer site and an analyte detection site, wherein the direct electron transfer site has catalytic reaction activity for converting a substrate into a product and direct electron transfer activity associated with the catalytic reaction, and is a site that can donate and receive electrons to and from the substrate in the catalytic reaction and can directly release or inject the electrons to or from the outside of the enzyme, and the analyte detection site is linked to the direct electron transfer site and is a detection site that can change the amount of electrons released or injected when it comes into contact with the analyte.

2. The direct electron transfer site is selected from the group consisting of fructose dehydrogenase, multicopper oxidase, glucose dehydrogenase, glucose oxidase, pyranose dehydrogenase, and ferredoxin-NADP. ＋ The enzyme according to claim 1, having a protein comprising the entire amino acid sequence encoding reductase, cellobiose dehydrogenase, gluconate dehydrogenase, succinate dehydrogenase, histamine dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, cytochrome P450, peroxidase, xanthine dehydrogenase, xanthine oxidase, sulfite oxidase, sulfite dehydrogenase, formate dehydrogenase, nitrogenase, nitrate reductase, carbon monoxide dehydrogenase, hydrogenase, galactose oxidase or nitrous oxide reductase, or a protein domain comprising a part of said amino acid sequence.

3. The enzyme according to claim 1, wherein the direct electron transfer site comprises a protein containing the entire amino acid sequence encoding sulfite oxidase, sulfite dehydrogenase, nitrate reductase, carbon monoxide dehydrogenase, hydrogenase, nitrous oxide reductase, alcohol dehydrogenase, or aldehyde dehydrogenase, or a protein domain containing a part of said amino acid sequence.

4. The enzyme according to claim 1, wherein the direct electron transfer site has a protein containing the entire amino acid sequence encoding fructose dehydrogenase or multicopper oxidase, or a protein domain containing a part of said amino acid sequence.

5. The enzyme according to any one of claims 1 to 4, wherein the detection site comprises at least one selected from the group consisting of biotin, an antigen, an antibody, RNA, DNA, ethylenediaminetetraacetic acid, and nitrilotriacetic acid.

6. A kit comprising the electron transfer enzyme of claim 1.

7. An enzyme electrode comprising the electron transfer enzyme according to claim 1 and an electrode substrate, wherein the electrode substrate is in contact with the enzyme and receives electrons released from the enzyme or allows electrons to flow into the enzyme.

8. An enzyme sensor comprising the enzyme electrode according to claim 7 as a working electrode.

9. An electrochemical measurement method using the electron transfer enzyme according to any one of claims 1 to 4, the kit according to claim 6, the enzyme electrode according to claim 7, or the enzyme sensor according to claim 8.

10. An electrochemical measurement method comprising the following steps (I) and (II): (I) contacting the enzyme described in claim 1 with a substrate for said enzyme; and (II) contacting the enzyme described in claim 1 with a substrate for said enzyme and a substance to be measured.

11. The method according to claim 10, wherein step (I) is a step of contacting the enzyme according to claim 1 with a substrate of the enzyme and measuring an electrochemical signal, and step (II) is a step of contacting the enzyme according to claim 1 with a substrate of the enzyme and a substance to be measured and measuring an electrochemical signal.

12. The method according to claim 11, wherein the electrochemical signal measured in step (I) is compared with the electrochemical signal measured in step (II), and the presence or concentration of the substance to be measured is determined based on the amount of change in the electrochemical signal.

13. The method of claim 11 or 12, wherein the electrochemical signal is a current value.

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