Lactate-responsive enzyme electrode

Abstract

To provide L-lactate oxidase with suppressed activity loss at acidic pH as well as applications therefor.SOLUTION: An enzyme electrode includes: a polypeptide that is composed of a specific amino acid sequence; or a polypeptide that is composed of an amino acid sequence having at least 80% identity with an amino acid sequence represented by any of the specific amino acid sequences and exhibits L-lactate oxidation activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to lactate oxidase which is useful for an enzyme electrode and the use thereof. [Background technology]

[0002] L-lactate contained in human blood is known as a biomarker for fatigue, etc. However, measurement of blood L-lactate requires the collection of blood samples from the body, which is highly invasive.

[0003] Since then, L-lactate contained in human sweat has been found to have biomarker functions such as fatigue state, similar to L-lactate in blood, and L-lactate in sweat has attracted attention as a measurement target because it can be measured non-invasively (Non-Patent Document 1). On the other hand, although the pH of sweat is usually neutral, it varies from person to person and is known to shift to the acidic side due to intense exercise (Non-Patent Document 2). However, commercially available L-lactate oxidase (LOX), which is used in lactate sensors and electrodes of biofuel cells that use L-lactate as fuel and is essential for L-lactate sensing, has the problem that its activity is optimal near neutral pH, but significantly decreases at acidic pH. Therefore, there is a demand for a method of L-lactate sensing that reduces the effects of acidic pH. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Van Hoovels et al. ACS sensors 2021, 6, 10, 3496-3508 [Non-Patent Document 2] Anastasova et al. Biosensors and Bioelectronics, 2017, 93, 139-145 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention relates to providing an L-lactate oxidase in which the decrease in activity at acidic pH is suppressed, and uses thereof. [Means for solving the problem]

[0006] The present inventors conducted intensive research in light of the above problems and found that a specific L-lactate oxidase has superior acid resistance compared to existing L-lactate oxidases, and that when used as an electrode catalyst for an enzyme electrode, the current density of the enzyme electrode is improved under acidic conditions.

[0007] That is, the present invention relates to the following 1) to 9). 1) An enzyme electrode comprising a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidation activity. 2) A method for producing an enzyme electrode, comprising the step of immobilizing on an electrode a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity. 3) A lactate sensor comprising the enzyme electrode described in 1). 4) An L-lactate measurement system comprising the lactate sensor according to 3), a means for applying voltage to electrodes in the electrode system of the lactate sensor, and a means for measuring the current value in the electrode system of the lactate sensor. 5) A method for measuring L-lactate using the lactate sensor according to 3) or the lactate measurement system according to 4). 6) A biofuel cell comprising the enzyme electrode described in 1). 7) A method for generating electricity using the biofuel cell described in 6). 8) A method for measuring L-lactate using a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity. 9) An L-lactate measurement kit comprising a polypeptide having an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 or a polypeptide having an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity, peroxidase, a new Trinder reagent and a coupler compound. Effect of the Invention

[0008] According to the present invention, an enzyme electrode, a lactate sensor, and a biofuel cell using L-lactate oxidase that has acid resistance and shows activity over a wide pH range can be provided. This contributes to expanding the usage environment of devices for L-lactate sensing, stabilizing them, increasing their sensitivity, increasing their output, and making them smaller. In addition, the L-lactate oxidase is also useful as a reagent for measuring L-lactate. [Brief description of the drawings]

[0009] [Figure 1] Specific activity of L-lactate oxidase. [Diagram 2] Effect of pH on L-lactate oxidase activity. (A) Specific activity, (B) Relative activity. [Diagram 3] Current density evaluation using an L-lactate responsive enzyme electrode. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In the present specification, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing an analysis using the search homology program of the genetic information processing software GENETYX Ver. 12 with the unit size to compare (ktup) set to 2.

[0011] In the present specification, the "corresponding position" on an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence shown in SEQ ID NO: 3) so as to give maximum homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. D. et al., 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can be used. Clustal W, Clustal W2 and Clustal omega are available, for example, on the Clustal website operated by University College Dublin [www.clustal.org], the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), and the website of the DNA Data Bank of Japan operated by the National Institute of Genetics (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]). The position of the target sequence aligned to any position of the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" the any position.

[0012] Those skilled in the art can further fine-tune the alignment of the amino acid sequences obtained above to optimize it. Such an optimal alignment is preferably determined taking into consideration the similarity of the amino acid sequences, the frequency of gaps to be inserted, and the like. Here, the similarity of the amino acid sequences refers to the ratio (%) of the number of positions at which identical or similar amino acid residues exist in both sequences when the two amino acid sequences are aligned to the total number of amino acid residues. The similar amino acid residues refer to amino acid residues that have similar properties in terms of polarity and charge among the 20 types of amino acids that constitute proteins, and that cause so-called conservative substitution. Such groups of similar amino acid residues are well known to those skilled in the art, and examples thereof include, but are not limited to, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; leucine and isoleucine.

[0013] As used herein, L-lactate oxidase (LOX, EC 1.1.3.2) refers to an enzyme having L-lactate oxidizing activity that oxidizes L-lactate to produce pyruvic acid and hydrogen peroxide. L-lactate oxidation activity can be evaluated using a method well known in the art. For example, L-lactate oxidation activity can be determined by contacting a sample with L-lactate, peroxidase, a new Trinder reagent and a coupler compound, measuring the amount of hydrogen peroxide produced by oxidation of L-lactate by the L-lactate oxidase in the sample, and the amount of a dye compound produced by oxidative condensation of the new Trinder reagent and the coupler compound by the action of peroxidase, using the absorbance as an index. In this specification, one unit (U) of L-lactate oxidase is defined as the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute under the conditions described in the Examples below. Alternatively, for example, L-lactate oxidation activity can be determined by contacting a sample with L-lactate and 2,4-dinitrophenylhydrazine, measuring the amount of pyruvic acid produced by oxidation of L-lactate by L-lactate oxidase in the sample, and the amount of a pigment compound produced by dehydration and condensation of 2,4-dinitrophenylhydrazine by measuring the absorbance, and using this amount as an index. Hereinafter, when simply referring to "lactate oxidase," "lactic acid," and "lactate oxidation activity," these refer to "L-lactate oxidase," "L-lactic acid," and "L-lactate oxidation activity," respectively.

[0014] As used herein, lactate oxidase "has acid resistance" means that the relative activity at an acidic pH (e.g., pH 4 or 5) of lactate oxidase relative to the lactate oxidizing activity at the optimal pH is higher than that of a reference sequence. The optimal pH and relative activity of lactate oxidase can be determined by measuring the lactate oxidizing activity under the conditions described in the Examples below. As the reference sequence, EfLOX (SEQ ID NO: 2), a known lactate oxidase derived from Enterococcus bacteria, is preferably used.

[0015] <1. L-Lactate oxidase> The L-lactate oxidase of the present invention is a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity.

[0016] The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 is lactate oxidase (LsLOX) derived from Ligilactobacillus salitolerans, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is lactate oxidase (EaLOX) derived from Enterococcus asini, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 is lactate oxidase (CkLOX) derived from Companilactobacillus kimchiensis, and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 is lactate oxidase (LcLOX) derived from Lactobacillus crispatus. The lactate oxidase consisting of any of the amino acid sequences shown in SEQ ID NO: 3 to 6 has acid resistance as shown in the examples described later.

[0017] Examples of polypeptides having lactate oxidizing activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 include polypeptides having lactate oxidizing activity consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, specifically, 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. The amino acid sequence having at least 80% identity includes an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added. The amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added includes an amino acid sequence in which 1 to 60, preferably 30 or less, more preferably 20 or less, even more preferably 10 or less, and even more preferably 5 or less amino acids are deleted, inserted, substituted or added.

[0018] An example of a polypeptide having lactate oxidizing activity and consisting of an amino acid sequence having at least 80% identity with any of the amino acid sequences shown in SEQ ID NOs: 3 to 6 is an artificially produced mutant of lactate oxidase consisting of any of the amino acid sequences shown in SEQ ID NOs: 3 to 6. The mutant can be produced, for example, by introducing a mutation into a gene encoding the amino acid sequence shown in any of SEQ ID NOs: 3 to 6 by a known mutagenesis method such as ultraviolet irradiation or site-directed mutagenesis, expressing the gene having the mutation, and selecting a protein having the desired lactate oxidizing activity. Procedures for producing such mutants are well known to those skilled in the art.

[0019] A polypeptide having acid resistance is preferred as a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having lactate oxidizing activity. Examples of such a polypeptide include a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having a relative activity at pH 5 of 40% or more, preferably 50% or more, remaining relative activity at pH 4 of the lactate oxidizing activity at the optimal pH.

[0020] Of the lactate oxidases of the present invention described above, from the viewpoint of acid resistance, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 3 and having lactate oxidizing activity is preferred, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 is more preferred.

[0021] The lactate oxidase of the present invention can be produced, for example, by expressing a polynucleotide encoding the lactate oxidase of the present invention. Preferably, the lactate oxidase of the present invention can be produced from a transformant into which a polynucleotide encoding the lactate oxidase of the present invention has been introduced. For example, a polynucleotide encoding the lactate oxidase of the present invention or a vector containing the polynucleotide is introduced into a host to obtain a transformant, which is then cultured in an appropriate medium, whereby the lactate oxidase of the present invention is produced from the polynucleotide encoding the lactate oxidase of the present invention introduced into the transformant. The lactate oxidase of the present invention can be obtained by isolating or purifying the produced lactate oxidase from the culture.

[0022] The polynucleotide encoding the lactate oxidase of the present invention may be a polynucleotide encoding a lactate oxidase consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6. The polynucleotide encoding the lactate oxidase of the present invention may be in the form of single-stranded or double-stranded DNA, RNA, or an artificial nucleic acid, or may be cDNA or a chemically synthesized DNA containing no introns.

[0023] The polynucleotide encoding the lactate oxidase of the present invention can be synthesized chemically or genetically based on the amino acid sequence of the lactate oxidase. For example, the polynucleotide can be chemically synthesized based on the amino acid sequence of the lactate oxidase of the present invention described above. For chemical synthesis of the polynucleotide, a nucleic acid synthesis contract service (for example, provided by Medical and Biological Laboratories, Inc., Genscript, Inc., etc.) can be used. Furthermore, the synthesized polynucleotide can be amplified by PCR, cloning, etc.

[0024] Alternatively, the polynucleotide encoding the lactate oxidase of the present invention can be prepared by introducing a mutation into the polynucleotide synthesized by the above procedure using a known mutagenesis method such as the above-mentioned UV irradiation or site-directed mutagenesis. For example, the polynucleotide encoding the lactate oxidase of the present invention can be obtained by introducing a mutation into a polynucleotide consisting of any of the nucleotide sequences shown in SEQ ID NOs: 9 to 12 using a known method, expressing the obtained polynucleotide, examining the lactate oxidase activity, and selecting a polynucleotide encoding a protein having the desired lactate oxidase activity.

[0025] Site-specific mutagenesis into a polynucleotide can be carried out by any method, such as the inverse PCR method or the annealing method (Muramatsu et al., eds., "New Genetic Engineering Handbook, 4th Revised Edition," Yodosha, pp. 82-88). If necessary, various commercially available site-specific mutagenesis kits, such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit, can also be used.

[0026] The polynucleotide encoding the lactate oxidase of the present invention can be incorporated into a vector. The type of vector containing the polynucleotide is not particularly limited, and may be any vector, such as a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC vector, or a shuttle vector. The vector is preferably, but not limited to, a vector that can be amplified in bacteria, preferably in Bacillus bacteria (e.g., Bacillus subtilis or a mutant thereof), and more preferably an expression vector that can induce expression of an introduced gene in Bacillus bacteria. Among them, a shuttle vector, which is a vector that can be replicated in both Bacillus bacteria and other organisms, can be suitably used for recombinantly producing the lactate oxidase of the present invention. Preferred examples of the vector include, but are not limited to, shuttle vectors such as pHA3040SP64, pHSP64R or pASP64 (Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), and pAC3 (Nucleic Acids Res, 1988, 16:8732); and plasmid vectors that can be used for transformation of bacteria of the genus Bacillus, such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0027] The above vector may contain a DNA replication origin region or a DNA region containing a replication origin. Alternatively, in the above vector, a control sequence such as a promoter region for initiating transcription of the gene, a terminator region, or a secretion signal region for secreting the expressed protein outside the cell may be operably linked upstream of the polynucleotide encoding the lactate oxidase of the present invention (i.e., the lactate oxidase gene of the present invention). As used herein, the phrase "operably linked" between a gene and a control sequence means that the gene and the control region are arranged so that the gene can be expressed under the control of the control region.

[0028] The types of the control sequences such as the promoter region, terminator region, and secretion signal region are not particularly limited, and promoters and secretion signal sequences that are commonly used can be appropriately selected and used depending on the host to be introduced. For example, suitable examples of the control sequences that can be incorporated into the vector include the promoter and secretion signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain.

[0029] Alternatively, the vector of the present invention may further incorporate a marker gene (e.g., a resistance gene to a drug such as ampicillin, neomycin, kanamycin, or chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Alternatively, when an auxotrophic strain is used as a host, a gene encoding an enzyme for synthesizing the required nutrient may be incorporated as a marker gene into the vector. Furthermore, when a selective medium requiring a specific metabolism for growth is used, a gene related to the metabolism may be incorporated as a marker gene into the vector. An example of such a metabolism-related gene is the acetamidase gene for utilizing acetamide as a nitrogen source.

[0030] The polynucleotide encoding the lactate oxidase of the present invention can be ligated to a regulatory sequence and a marker gene by a method known in the art, such as splicing by overlap extension (SOE)-PCR (Gene, 1989, 77:61-68). The procedure for introducing the ligated fragment into a vector is well known in the art.

[0031] The transformed cell of the present invention can be obtained by introducing a vector containing a polynucleotide encoding the lactate oxidase of the present invention into a host, or by introducing a DNA fragment containing a polynucleotide encoding the lactate oxidase of the present invention into the genome of a host.

[0032] Examples of host cells include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include bacteria belonging to the genera Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus, among which Escherichia coli and Bacillus bacteria (e.g., Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) or mutants thereof) are preferred. Examples of Bacillus subtilis mutants include the KA8AX protease 9-fold deletion strain described in J. Biosci. Bioeng., 2007, 104(2): 135-143, and the D8PA strain, which is an 8-fold deletion strain of protease with improved protein folding efficiency described in Biotechnol. Lett., 2011, 33(9): 1847-1852. Examples of filamentous fungi include the genera Trichoderma, Aspergillus, and Rhizopus.

[0033] The vector can be introduced into the host by a method commonly used in the art, such as the protoplast method, electroporation, etc. A strain into which the vector has been appropriately introduced can be selected based on the expression of a marker gene, nutritional requirements, etc., to obtain a desired transformant into which the vector has been introduced.

[0034] Alternatively, a fragment in which the polynucleotide encoding the lactate oxidase of the present invention, a control sequence, and a marker gene are linked can be directly introduced into the genome of the host. For example, a DNA fragment in which sequences complementary to the genome of the host are added to both ends of the linked fragment is constructed by SOE-PCR or the like, and this is introduced into the host to cause homologous recombination between the host genome and the DNA fragment, thereby introducing the polynucleotide encoding the lactate oxidase of the present invention into the genome of the host.

[0035] When the thus obtained transformant into which the polynucleotide encoding the lactate oxidase of the present invention or a vector containing the polynucleotide is introduced is cultured in an appropriate medium, the gene encoding the protein on the vector is expressed to produce the lactate oxidase of the present invention. The medium used for culturing the transformant can be appropriately selected by those skilled in the art depending on the type of the microorganism of the transformant.

[0036] Alternatively, the lactate oxidase of the present invention may be expressed from a polynucleotide encoding the lactate oxidase of the present invention or a transcription product thereof using a cell-free translation system. The "cell-free translation system" refers to an in vitro transcription / translation system or an in vitro translation system that is constructed by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0037] The lactate oxidase of the present invention produced in the above culture or cell-free translation system can be isolated or purified by a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination. The protein recovered from the culture may be further purified by known means.

[0038] As shown in the Examples below, the lactate oxidase of the present invention has acid resistance compared to existing lactate oxidases, and therefore exhibits good activity even in samples that may have an acidic pH range (for example, preferably pH 6 or less, more preferably pH 5.5 or less) where the activity of existing lactate oxidases is significantly reduced. Thus, the lactate oxidase of the present invention can be used as an electrode catalyst for lactate sensors or enzyme electrodes of biofuel cells that use lactate as fuel, and can contribute to the expansion, stabilization, high sensitivity, and miniaturization of the use environment of lactate sensors, and the expansion, stabilization, high sensitivity, high output, and miniaturization of the use environment of biofuel cells. The lactate oxidase of the present invention is also useful as a reagent for measuring lactate.

[0039] <2. Enzyme electrode> The enzyme electrode of the present invention contains the lactate oxidase of the present invention. The enzyme electrode may have a structure in which the lactate oxidase of the present invention is immobilized on an electrode. The enzyme electrode of the present invention is a lactate-responsive enzyme electrode that converts an enzymatic reaction of lactate oxidase occurring in the presence of lactic acid into an electric signal.

[0040] Examples of the electrode material include carbon materials such as glassy carbon, carbon paste, carbon fiber, graphite, hard carbon, and conductive diamond, metal materials such as Pt, Au, and Ag, conductive polymers, and metal oxides. Of these, carbon materials are preferable. The electrode is manufactured, for example, by applying an electrode material to a substrate such as paper, ceramic, synthetic resin, glass, etc., by coating or printing such as screen printing. In manufacturing the electrode, a binder such as a fluorine-based resin (e.g., polyvinylidene fluoride, polytetrafluoroethylene) may be used.

[0041] The amount of lactate oxidase in the electrode can be appropriately selected depending on the activity of lactate oxidase and the use of the electrode. For example, from the viewpoint of measurement sensitivity and cost, the amount of lactate oxidase is preferably 6.35 μg or more, more preferably 10 μg or more, even more preferably 100 μg or more, and preferably 500 μg or less, more preferably 400 μg or less, even more preferably 300 μg or less per electrode. Also, it is preferably 6.35 to 500 μg, more preferably 10 to 400 μg, even more preferably 100 to 300 μg.

[0042] The enzyme electrode of the present invention may further contain an electron mediator from the viewpoint of improving the measurement sensitivity. The enzyme electrode may have a configuration in which the lactate oxidase of the present invention and an electron mediator are immobilized on the electrode. The electron mediator is a redox substance that mediates electron transfer, and those that are usually used in enzyme electrodes can be used. Examples of the electron mediator include quinones, cytochromes, viologens, phenazines, phenoxazines, phenothiazines, ferredoxins, ferricyanides, ferrocene and its derivatives, metal complexes, etc. Any one of these electron mediators may be used alone, or two or more may be used in combination.

[0043] The amount of the electron mediator in the electrode can be appropriately selected depending on the type of lactate oxidase to be combined, the type of the electron mediator, and the use of the electrode. For example, the amount of the electron mediator is preferably 0.001 μmol or more, more preferably 0.01 μmol or more, and even more preferably 0.1 μmol or more per electrode, from the viewpoints of measurement sensitivity and cost, and is preferably 100 μmol or less, more preferably 10 μmol or less, and even more preferably 1 μmol or less. Also, it is preferably 0.001 to 100 μmol, more preferably 0.01 to 10 μmol, and even more preferably 0.1 to 1 μmol.

[0044] The enzyme electrode of the present invention can be obtained by a production method including a step of immobilizing the lactate oxidase of the present invention on an electrode. The immobilization of lactate oxidase can be carried out by a conventional method such as an adsorption method, an ionic bond method, a covalent bond method, a crosslinking method, or an entrapment method using a polymer matrix. It is preferable to use lactate oxidase as a solution dissolved in water, an aqueous alkali solution such as sodium carbonate or sodium hydroxide, a buffer solution such as a phosphate buffer or a Tris buffer, or an alcohol such as methanol or ethanol, or as a dispersion solution dispersed therein. When the enzyme electrode of the present invention further comprises an electron mediator, it can be obtained by a production method including a step of immobilizing the lactate oxidase of the present invention and the electron mediator on the electrode. The electron mediator can be immobilized on the electrode in the same manner as lactate oxidase. The order of immobilization of lactate oxidase and the electron mediator on the electrode may be either first or simultaneously, but from the viewpoint of electron transfer efficiency, it is preferable to immobilize lactate oxidase after immobilizing the electron mediator, or to immobilize both simultaneously. From the viewpoint of storage stability, it is preferable that the lactate oxidase and the electron mediator on the electrode are present in a dry state.

[0045] <3. Lactic acid sensor> The lactate sensor of the present invention includes the enzyme electrode of the present invention. The lactate sensor includes an electrode system including a working electrode and a counter electrode, and the working electrode may be configured to include the enzyme electrode of the present invention.

[0046] The electrode system of the lactate sensor of the present invention may include at least a working electrode and a counter electrode, and may further include a reference electrode indicating a reference potential, if necessary. The electrode material and manufacturing method thereof may be the same as those described for the enzyme electrode above. The electrode system is preferably disposed on a substrate. Thus, the lactate sensor of the present invention may be configured to include a substrate and an electrode system including a working electrode and a counter electrode disposed on the substrate, with the enzyme electrode of the present invention being disposed on the working electrode. Examples of materials for the substrate include silicon, glass, glass epoxy, ceramic, polyethylene, polypropylene, polyester, polyvinyl chloride, polyimide, etc.

[0047] The lactate sensor of the present invention can be used to measure the lactate concentration in a sample. In one embodiment, the lactate measurement method of the present invention includes a step of contacting the lactate sensor of the present invention with a sample, and a step of electrochemically measuring the reaction between lactate in the sample and the lactate oxidase of the present invention. The means for contacting the lactate sensor with the sample may be any means known in the art, and includes adding, dropping, and applying the sample to the lactate sensor. The electrochemical measurement method is not particularly limited, and includes chronoamperometry, cyclic voltammetry, potentiometry, and the like. For example, when the lactate sensor of the present invention is contacted with a sample and a certain voltage is applied to the electrode system of the lactate sensor, a current flows through the electrode system based on the reaction between lactate in the sample and the lactate oxidase of the present invention. Since the lactate concentration and the current value are correlated, the lactate concentration in the sample can be calculated from the measured current value and a calibration curve created in advance from the current value when a lactate standard solution of known concentration is used. Therefore, in a preferred embodiment, the lactate measurement method of the present invention includes the steps of contacting the lactate sensor of the present invention with a sample, applying a voltage to the electrode system of the lactate sensor, and measuring the current value in the electrode system. The method may further include a step of calculating the lactate concentration in the sample based on the measured current value. The applied voltage may be appropriately set, and is preferably 10 to 700 mV, more preferably 100 to 600 mV, for example.

[0048] The sample to be measured is not particularly limited as long as it contains or may contain lactic acid, and examples of the sample include body fluids such as sweat, blood, urine, saliva, and tissue exudate, serum and plasma prepared from blood, cells, tissues, organs, beverages, foods, and microbial cultures, and is preferably body fluid, and more preferably sweat.

[0049] The lactate measurement method of the present invention can be preferably carried out using a lactate measurement system comprising the lactate sensor of the present invention, a means for applying a voltage to the electrode system of the lactate sensor, and a means for measuring a current value in the electrode system of the lactate sensor. The means for applying a voltage to the electrode system and the means for measuring a current value in the electrode system can be any means commonly used in the art.

[0050] <4. Biofuel Cell> The biofuel cell of the present invention includes the enzyme electrode of the present invention. The biofuel cell includes an anode (negative electrode) and a cathode (positive electrode), and the anode can be configured to include the enzyme electrode of the present invention.

[0051] The anode of the biofuel cell of the present invention is provided with the enzyme electrode of the present invention. The cathode is preferably provided with an electrode using a reductase capable of transferring electrons to oxygen as an electrode catalyst, an electrode using a metal such as Pt as an electrode catalyst, or a metal electrode such as Pt, and more preferably with an enzyme electrode containing a reductase. The enzyme electrode as the cathode can be configured to have a reductase immobilized on the electrode. Examples of the reductase include laccase, bilirubin oxidase, and multicopper oxidase such as ascorbate oxidase. Among them, bilirubin oxidase is preferable. The reductase may be a natural enzyme molecule or an enzyme fragment containing an active site. Such an enzyme molecule or enzyme fragment may be extracted from animals, plants, or microorganisms, chemically synthesized, or derived from a transformant. The enzyme electrode as the cathode can be manufactured in the same manner as the enzyme electrode of the present invention, except for the enzyme used. The anode and the cathode are preferably connected by an external circuit.

[0052] Lactic acid, which is the fuel for the biofuel cell of the present invention, may or may not contain a solvent, but is preferably supplied to the anode in the form of a fuel solution or fuel gel dissolved in a suitable solvent. Examples of the solvent include water and buffer solutions such as phosphate buffer and Tris buffer. The amount of lactic acid in the fuel solution or fuel gel can be appropriately changed depending on the form of the fuel, the types of the anode and cathode, the power generation method, etc.

[0053] The biofuel cell of the present invention may further include components that can be used in a biofuel cell, such as a fuel tank containing a fuel, an electrolyte as a proton conductor, etc. The biofuel cell of the present invention may further include a separator that separates the anode and the cathode, but since a biofuel cell does not require a separator in principle, not including a separator can improve the freedom of cell design.

[0054] When the fuel lactic acid is supplied to the biofuel cell, the lactic acid is oxidized by the lactate oxidase of the present invention on the anode side to generate electrons and protons. The electrons are transferred to the anode directly or via an electron mediator, and the electrons then travel from the anode through an external circuit to the cathode, generating an electric current. On the cathode side, oxygen receives the electrons that have traveled from the anode through the external circuit to the cathode and the protons generated on the anode side, generating water.

[0055] Therefore, the biofuel cell of the present invention can be used to generate electricity. In one embodiment, the power generation method of the present invention includes a step of supplying lactic acid as a fuel to the biofuel cell of the present invention (specifically, the anode). The means for supplying the fuel is not particularly limited as long as the fuel can come into contact with the lactate oxidase of the anode.

[0056] <5. L-lactate measurement method using lactate oxidase> The lactate oxidase of the present invention can be used to measure lactate contained in a sample. A method for measuring lactate using lactate oxidase has been established in the art. Thus, the lactate concentration in various samples can be measured using the lactate oxidase of the present invention according to a known method. This method is a method for measuring the lactate concentration using hydrogen peroxide or pyruvic acid produced by the oxidation of lactate by the action of lactate oxidase as an indicator. Here, the means for measuring hydrogen peroxide or pyruvic acid is not particularly limited, and known methods can be used. Hydrogen peroxide can be quantified, for example, by measuring a pigment compound formed by the oxidative condensation of hydrogen peroxide, new Trinder's reagent, and coupler compound by the action of peroxidase in the presence of peroxidase, new Trinder's reagent, and coupler compound. In addition, pyruvic acid can be quantified, for example, by measuring a pigment compound formed by dehydration condensation of pyruvic acid and 2,4-dinitrophenylhydrazine in the presence of 2,4-dinitrophenylhydrazine, and by measuring the pigment compound.

[0057] The lactate measurement method of the present invention uses the lactate oxidase of the present invention. In one embodiment, the lactate measurement method using the lactate oxidase of the present invention comprises the following steps (1) and (2): (1) A step of contacting the lactate oxidase, peroxidase, new Trinder reagent and coupler compound of the present invention with a sample. (2) A step of measuring the dye compound obtained in (1)

[0058] The sample to be measured is not particularly limited as long as it is a sample that contains or may contain lactic acid. Examples of the sample include body fluids such as sweat, blood, urine, saliva, and tissue exudate, serum and plasma prepared from blood, as well as cells, tissues, organs, beverages, foods, and microbial culture solutions, and preferably body fluids, and more preferably sweat.

[0059] The means for contacting the lactate oxidase, peroxidase, new Trinder's reagent and coupler compound of the present invention with a sample may be any means known in the art, and examples of such means include adding, dropping, etc. the lactate oxidase, peroxidase, new Trinder's reagent and coupler compound of the present invention to a sample. The order of contacting the lactate oxidase, peroxidase, new Trinder's reagent and coupler compound of the present invention with the sample is not particularly limited, and the lactate oxidase, peroxidase, new Trinder's reagent and coupler compound of the present invention may be contacted with the sample in any order, or the lactate oxidase, peroxidase, new Trinder's reagent and coupler compound of the present invention may be mixed in advance, and the resulting mixture may be contacted with the sample.

[0060] The amount of lactate oxidase of the present invention used, in terms of concentration in the measurement reaction solution, is preferably 0.00001 U / mL or more, more preferably 0.0001 U / mL or more, even more preferably 0.001 U / mL or more, and preferably 0.02 U / mL or less, more preferably 0.015 U / mL or less, even more preferably 0.01 U / mL or less, and preferably 0.00001 to 0.02 U / mL, more preferably 0.0001 to 0.015 U / mL, even more preferably 0.001 to 0.01 U / mL.

[0061] Any known peroxidase can be used, and may be an enzyme derived from a living organism or a recombinant enzyme. Examples of the peroxidase include horseradish peroxidase (HRP).

[0062] The amount of peroxidase used, as a concentration in the measurement reaction solution, is preferably 0.001 U / mL or more, more preferably 0.01 U / mL or more, even more preferably 0.1 U / mL or more, and is preferably 500 U / mL or less, more preferably 100 U / mL or less, even more preferably 10 U / mL or less, and is preferably 0.001 to 500 U / mL, more preferably 0.01 to 100 U / mL, even more preferably 0.1 to 10 U / mL.

[0063] As the new Trinder reagent, known ones can be used, and examples thereof include N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS). The Trinder reagent may be used alone or in combination of two or more kinds. Among them, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) is preferably used.

[0064] The amount of the new Trinder reagent used is, as a concentration in the measurement reaction solution, preferably 0.001 μmol / mL or more, more preferably 0.01 μmol / mL or more, even more preferably 0.1 μmol / mL or more, and preferably 50 μmol / mL or less, more preferably 10 μmol / mL or less, even more preferably 5 μmol / mL or less. Also, it is preferably 0.001 to 50 μmol / mL, more preferably 0.01 to 10 μmol / mL, even more preferably 0.1 to 5 μmol / mL.

[0065] The coupler compound may be any compound that generates a dye compound by oxidative condensation with the new Trinder reagent, such as 4-aminoantipyrine (4-AA), vanillindiaminesulfonic acid, methylbenzthiazolinonehydrazone (MBTH), sulfonated methylbenzthiazolinonehydrazone (SMBTH), aminodiphenylamine, or derivatives thereof. Among these, 4-aminoantipyrine (4-AA) is preferably used.

[0066] The amount of the coupler compound used is preferably 0.001 μmol / mL or more, more preferably 0.01 μmol / mL or more, even more preferably 0.1 μmol / mL or more, and is preferably 100 μmol / mL or less, more preferably 10 μmol / mL or less, even more preferably 5 μmol / mL or less, as a concentration in the measurement reaction solution. Also, it is preferably 0.001 to 100 μmol / mL, more preferably 0.01 to 10 μmol / mL, even more preferably 0.1 to 5 μmol / mL.

[0067] Step (1) is a step of reacting lactic acid that may be contained in a sample with the lactate oxidase of the present invention to generate hydrogen peroxide, and then reacting the hydrogen peroxide with the new Trinder reagent, a coupler compound, and peroxidase to generate a dye compound. The temperature, pH, time, and the like for carrying out step (1) can be appropriately set in consideration of the optimal reaction conditions of the enzyme used and the stability of the compound. The temperature is, for example, preferably 20 to 50°C, more preferably 25 to 45°C, and even more preferably 30 to 40°C. The pH is, for example, preferably pH 4.0 to 10.0, and more preferably pH 5.0 to 9.0. The reaction time is, for example, preferably 0.5 minutes to 1 hour, and more preferably 1 minute to 30 minutes.

[0068] Next, in step (2), the dye compound obtained in step (1) is measured. An example of a measurement method is measurement of absorbance change using a spectrophotometer. The measurement wavelength in the absorbance measurement can be appropriately set according to the type of dye compound. For example, when N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) is used as the new Trinder reagent and 4-aminoantipyrine (4-AA) is used as the coupler compound, the quinone imine of the dye compound generated can be measured at a measurement wavelength of about 555 nm. Since the lactic acid concentration in a sample and the measured value (e.g., absorbance) of the dye compound generated are correlated, the lactic acid concentration in the sample can be calculated from a calibration curve previously prepared from the measured value of the dye compound and the measured value of the dye compound generated when a lactic acid standard solution of known concentration is used. Therefore, the lactic acid measurement method using the lactate oxidase of the present invention may further include a step (3) of calculating the lactic acid concentration in the sample based on the measured value.

[0069] <6. L-Lactic Acid Measurement Kit> The above-mentioned method for measuring lactate using the lactate oxidase of the present invention can be preferably carried out using a lactate measurement kit containing the above-mentioned lactate oxidase of the present invention, peroxidase, the new Trinder reagent and a coupler compound.

[0070] The peroxidase, new Trinder's reagent and coupler compound used in the kit of the present invention can be the same as those described in relation to the lactate measurement method using the lactate oxidase of the present invention. The kit of the present invention is preferably in the form of a composition dissolved in a solution suitable for storage or measurement of lactate (e.g., a buffer solution), or in the form of a freeze-dried solution (e.g., powder). The kit of the present invention may be in the form of a single agent containing the lactate oxidase, peroxidase, new Trinder's reagent and coupler compound of the present invention, or in the form of two or more agents containing one or more of these. Of these, from the viewpoint of suppressing oxidative condensation between the new Trinder's reagent and the coupler compound, it is preferable to use a two-agent form in which the new Trinder's reagent and the coupler compound are separate agents. The lactate oxidase and peroxidase of the present invention may be contained in either agent.

[0071] The kit of the present invention may contain the lactate oxidase, peroxidase, new Trinder's reagent, and coupler compound of the present invention in amounts sufficient for at least one measurement. For example, the kit may contain preferably 0.02 to 3 mU, more preferably 0.2 to 2 mU of the lactate oxidase of the present invention, preferably 0.002 to 20 U, more preferably 0.02 to 2 U of the peroxidase, preferably 0.002 to 2 μmol, more preferably 0.02 to 1 μmol of the new Trinder's reagent, and preferably 0.002 to 2 μmol, more preferably 0.02 to 1 μmol of the coupler compound.

[0072] In addition to the enzyme and compound, the kit of the present invention may appropriately contain stabilizing components known to those skilled in the art, such as stabilizers or antioxidants, in order to enhance the storage stability of the enzyme and compound. In addition to the enzyme and compound, the kit of the present invention may also contain a lactic acid standard, a sample dilution solution, instruments required for measurement, a tool for collecting samples, a storage reagent for the collected samples, a storage container, etc.

[0073] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed in this specification, but the present invention is not limited to these embodiments.

[0074] [1] An enzyme electrode comprising a polypeptide having an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidation activity. [2] The enzyme electrode described in [1], further comprising an electron mediator. [3] The enzyme electrode according to [1] or [2], wherein the polypeptide is immobilized on an electrode. [4] The enzyme electrode according to [1] or [2], wherein the polypeptide and an electron mediator are immobilized on the electrode. [5] The enzyme electrode according to any one of [1] to [4], wherein the polypeptide has an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6. [6] A method for producing an enzyme electrode, comprising a step of immobilizing on an electrode a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity. [7] The method for producing an enzyme electrode according to [6], further comprising the step of immobilizing an electron mediator on the electrode. [8] The method for producing an enzyme electrode according to [6] or [7], wherein the polypeptide is a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6.

[0075] [9] A lactate sensor comprising the enzyme electrode according to any one of [1] to [5].

[10] The lactate sensor according to [9], comprising an electrode system including a working electrode and a counter electrode, the working electrode being provided with the enzyme electrode.

[11] An L-lactate measurement system comprising the lactate sensor according to

[10] , a means for applying voltage to electrodes in the electrode system of the lactate sensor, and a means for measuring the current value in the electrode system of the lactate sensor.

[12] A method for measuring L-lactate using the lactate sensor according to [9] or

[10] or the L-lactate measurement system according to

[11] .

[0076]

[13] A biofuel cell comprising the enzyme electrode according to any one of [1] to [5].

[14] The biofuel cell according to

[13] , comprising an anode and a cathode, the anode being provided with the enzyme electrode.

[15] A method for generating electricity using the biofuel cell according to

[13] or

[14] .

[0077]

[16] A method for measuring L-lactate using a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity.

[17] The method for measuring L-lactic acid according to

[16] , comprising the steps of (1) and (2) below: (1) contacting a sample with a polypeptide having an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 or a polypeptide having an amino acid sequence having at least 80% identity with an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity, peroxidase, a new Trinder reagent and a coupler compound. (2) A step of measuring the dye compound obtained in (1)

[18] The method for measuring L-lactate according to

[16] or

[17] , wherein the polypeptide has an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6.

[19] A kit for measuring L-lactate, comprising a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity, peroxidase, a new Trinder reagent, and a coupler compound.

[20] The L-lactate measurement kit according to

[19] , wherein the new Trinder reagent is at least one selected from the group consisting of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS).

[21] The L-lactic acid measurement kit according to

[19] or

[20] , wherein the coupler compound is 4-aminoantipyrine.

[0078]

[22] Use of a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidation activity, as an electrode catalyst for an enzyme electrode.

[23] Use of a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity, as an enzyme for measuring L-lactate.

[24] A polypeptide having an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 for use as an electrode catalyst of an enzyme electrode, or a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidation activity.

[25] A polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, for use as an enzyme for measuring L-lactate, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 3 to 6 and having L-lactate oxidizing activity. EXAMPLES

[0079] Example 1 Screening for acid-tolerant L-lactate oxidase sequences (1) Preparation of expression vector for candidate L-lactate oxidase Public databases were used to obtain candidate amino acid sequences for acid-resistant L-lactate oxidase. A heterologous recombinant expression system using Escherichia coli was used to prepare a crude enzyme solution for evaluating the activity of L-lactate oxidase. A DNA fragment containing a gene encoding L-lactate oxidase and a recombinant vector carrying the DNA fragment were obtained from Eurofins Genomics using an artificial gene synthesis method, and the recombinant vector was obtained with a genetic structure in which the DNA fragment was inserted into the 216 bp site from the initiation codon (ATG) to the termination codon (TAA) immediately before the T7 terminator in the restriction enzyme NdeI recognition sequence of the pET22b(+) plasmid.

[0080] (2) Expression of candidate L-lactate oxidase Next, we attempted to prepare transformed cells and express L-lactate oxidase. The recombinant vector was introduced into Escherichia coli ECOS Competent E. coli BL21 (DE3) (Nippon Gene) by the heat shock method to obtain transformed cells. The transformed cells were then cultured overnight at 30°C with 2 mL of LB medium (sterilized by autoclaving at 121°C for 20 minutes) and 2 μL of 100 mg / mL ampicillin in a round-bottomed spitzer (Eiken Chemical) to prepare grown cells. Next, 2 μL of the grown bacteria solution was inoculated into 2 mL of Overnight Express Instant TB medium (Novagen) (sterilized by filtration with a polyethersulfone (PES) membrane) and 2 μL of 100 mg / mL ampicillin in a liquid medium, and L-lactate oxidase was expressed by culturing the cells in a round-bottomed spitzer at 37°C for 18 hours with shaking.

[0081] (3) Activity evaluation of candidate L-lactate oxidase The cells in which the enzyme was expressed in (2) were collected by centrifugation and then washed twice with 20 mM phosphate buffer (pH 7). The cells were then resuspended in 2 mL of a buffer solution containing the same buffer, 10% BugBuster Master Mix (Merck) at a final concentration, and 1% protease inhibitor cocktail VII at a final concentration, and allowed to stand at room temperature for 30 minutes. After standing, the supernatant was collected by centrifugation to prepare a crude enzyme solution for screening. Next, the L-lactate oxidation activity of the crude enzyme solution in acidic artificial sweat was measured. The solution used for activity measurement was a composition of 0.5 g / L L-histidine hydrochloride monohydrate, 5 g / L sodium chloride, and 2.2 g / L sodium dihydrogen phosphate dihydrate, based on the composition of acidic artificial sweat (pH 5.5) disclosed in Japanese Industrial Standard JIS L0848. The activity measurement method was based on the academic paper by Hiraka et al. (Hiraka et al. “Minimizing the effects of oxygen interference on L-lactate sensors by a single amino acid mutation in Aerococcus viridans L-lactate oxidase.” Biosensors and Bioelectronics 103 (2018): 163-170.), and a reaction solution was prepared by dissolving 1.5 mM 4-aminoantipyrine, 1.5 mM TOOS (Dojindo Laboratories), 2 U / mL peroxidase, and 5 mM L-lactic acid in the above solution. Next, 10 μL of the crude enzyme solution was added to a 96-well multi-well plate, and 190 μL of the reaction solution, which had been preheated to 37°C, was mixed. The quinone imine (absorbance 555 nm) generated in an atmosphere of 37°C was measured over a 5-minute time course using a microplate reader Infinite 200 PRO (TECAN). (The molar extinction coefficient of TOOS was set to 39.2 mM(-1) cm(-1).) The enzyme activity (U / mL) in this method is defined by the following arithmetic formula:

[0082]

number

[0083] Here, ΔOD555 is the change in absorbance at 555 nm per minute at 37°C, A is the reaction solution volume (mL), B is the enzyme solution volume (mL), and C is the absorbance cell path length (cm). The weight of L-lactate oxidase protein in the crude enzyme solution was estimated by comparing the band intensity around 50 kDa obtained by electrophoresis (SDS-PAGE) with a calibration curve obtained from the band intensity of a bovine serum albumin standard solution (TaKaRa) of known concentration. Mini-PROTEAN TGX Stain-Free precast gel (BIO-RAD) was used for electrophoresis, and band detection was performed using a Chemi-Doc XRS System (BIO-RAD). Image analysis software ImageJ was used to quantify the band intensity after detection. Specific activity (U / mg) was calculated from the enzyme activity and protein weight, and the results are shown in Figure 1. The control was a crude enzyme solution prepared using transformed cells prepared by introducing pET22b(+) (Merck) into E. coli in (2).

[0084] (4) Results Existing L-lactate oxidases are known to be derived from Aerococcus viridans (Yorita et al. "On the interpretation of quantitative structure-function activity relationship data for lactate oxidase." Proceedings of the National Academy of Sciences 97.6 (2000): 2480-2485.) and Lactococcus lactis (JP Patent Publication 10-248574). The former sequence was prepared as AvLOX (SEQ ID NO: 1), and the latter sequence was prepared as EfLOX (SEQ ID NO: 2), which has an amino acid sequence homology of 98%, for comparison. As a result of intensive investigation using the above-mentioned method, it was determined that the following L-lactate oxidases were newly derived from Lisylactobacillus salitolatorans (LsLOX, SEQ ID NO: 3), Enterococcus asini (EaLOX, SEQ ID NO: 4), Companilactobacillus kimchiensis (CkLOX, SEQ ID NO: 5), and Lactobacillus crispatus (LcLOX, SEQ ID NO: 6) as acid-resistant L-lactate oxidases (Figure 1). The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOs: 1 to 6 are shown in SEQ ID NOs: 7 to 12, respectively.

[0085] Example 2: Evaluation of the effect of buffer pH on the activity of candidate acid-resistant L-lactate oxidases (1) Evaluation of L-lactate oxidase activity Britton-Robinson buffer (5.42 g / L phosphoric acid, 2.47 g / L boric acid, 2.42 g / L acetic acid. pH adjusted with 1M sodium hydroxide) was used to evaluate the activity of L-lactate oxidase. 10 μL of L-lactate oxidase solution was added to a 96-well multi-well plate, and 100 μL of each pH buffer solution from pH 2 to 11 containing 5 mM L-lactate was added and left at 30°C for 30 minutes to start the reaction in which L-lactate is converted to pyruvate. After the reaction, 40 μL of 1 mM 2,4-dinitrophenylhydrazine (dissolved in 1M hydrochloric acid) was added and left at 37°C for 10 minutes to stop the reaction. 150 μL of 1M sodium hydroxide was added and left at room temperature for 5 minutes, and the absorbance at 445 nm was measured using a microplate reader. On the other hand, a calibration curve of 445 nm absorbance and pyruvic acid concentration was created by using a pyruvic acid standard solution with a known concentration instead of the enzyme solution, and the lactate oxidation activity of the enzyme solution at each pH was evaluated as the amount of pyruvic acid produced by comparing this calibration curve with the measured values ​​of the enzyme solution.

[0086] (2) Results FIG. 2 shows the evaluation results, in which (A) shows the specific activity (U / mg) at which 1 mg of the enzyme produces 1 mM pyruvic acid per minute, and (B) shows the relative activity (%) when the activity at the optimal pH is taken as 100. Compared with the existing enzyme EfLOX, the newly discovered LsLOX was highly active at neutral pH (e.g., pH 6, 7, and 8) and acidic pH (e.g., pH 4 and pH 5), especially at acidic pH. Furthermore, compared with the relative activity of EfLOX, the relative activity of LsLOX at acidic pH (e.g., pH 4 and pH 5) was significantly improved. From these results, it was determined that LsLOX is an acid-resistant L-lactate oxidase.

[0087] Example 3 Current density evaluation of an L-lactate responsive electrode using acid-resistant L-lactate oxidase in an acidic solution (1) Purification of L-lactate oxidase In order to analyze the properties of the acid resistance candidate sequence (LsLOX: SEQ ID NO: 3) found in Example 1 and the sequence (EfLOX: SEQ ID NO: 2) that was highly active among the known sequences, purified samples were prepared. First, the enzyme expression method of Example 1 (2) was scaled up to 10 500 mL baffled flasks (100 mL culture volume per flask), and the inoculation port was aerated and blocked with a breathable seal (corning), followed by shaking culture at 37 ° C for 18 hours to prepare enzyme-expressing bacteria. The bacteria were collected by centrifugation, washed twice with 20 mM phosphate buffer (pH 7), resuspended in 250 mL of the same buffer containing lysozyme at a final concentration of 1 mg / mL, and then shaken at 37 ° C for 30 minutes. This treated solution was subjected to ultrasonic treatment to disrupt the bacteria, and after centrifugation, the supernatant was filtered through a PES membrane to prepare a crude enzyme solution. The crude enzyme solution was further purified by anion exchange chromatography fractionation using TOYOPEARL SuperQ-650 (Tosoh) and gel filtration of the active fraction using HiLoad 26 / 600 Superdex 200pg (Cytiva) connected to AKTA pure (GE Healthcare). The purified L-lactate oxidase was concentrated by centrifugal ultrafiltration and stored at 4°C.

[0088] (2) Preparation of L-lactate-responsive electrode The concentration of purified L-lactate oxidase (EfLOX or LsLOX) was adjusted with 20 mM phosphate buffer (pH 7) so that the protein concentration was 8.9 mg / mL. Gasenshi Shoun (Hanshiya e-shop) was used as the electrode substrate, and carbon ink JELCON CH-8 (Jujo Chemical) was applied by screen printing and baked at 120 °C for 15 minutes to form a single layer, forming a total of six layers to produce a lead part. Next, 1 g of porous carbon Knobel (Toyo Tanso), 10.1 mL of N-methyl-2-pyrrolidone solution containing 5 wt% KF polymer W # 9300 (Kureha), and 3.5 mL of N-methyl-2-pyrrolidone were injected, and then mixed and degassed with a rotation and revolution mixer to prepare a porous carbon ink. This porous carbon ink was applied on the above-mentioned lead part, and baked at 60 °C for 24 hours to form a single layer, forming a total of three layers to produce an anode electrode. After UV-ozone treatment of the prepared anode electrode, 10 μL of a 50 mM thionine acetate (Tokyo Chemical Industry) methanol solution was applied twice as a mediator to the top of the porous carbon. After drying under reduced pressure for 90 minutes, 10 μL of purified L-lactate oxidase was applied twice in the same manner to prepare an L-lactate responsive electrode.

[0089] (3) Evaluation of L-lactate-responsive electrodes Chronoamperometry was performed using the L-lactate responsive electrode prepared in (2) as the anode. At this time, an Ag / AgCl electrode was used as the reference electrode, a platinum electrode was used as the counter electrode, and the artificial sweat solution described in Example 1 (3) containing various concentrations of L-lactate was used as the substrate solution. The measurement was performed in a thermostatic bath at 37°C. After holding at the natural potential for 30 seconds, five measurements were performed under the conditions of a potential of 0.2 V and a holding time of 300 seconds, and the average current density after the measurement was calculated. The results are shown in Figure 3, and it was confirmed that the lactate responsive electrode using LsLOX, an acid-resistant L-lactate oxidase, generates a higher current density in an acidic solution than an electrode using an existing enzyme.

Claims

1. An enzyme electrode comprising a polypeptide consisting of an amino acid sequence represented by any of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence represented by any of SEQ ID NOs: 3 to 6 and having L-lactic acid oxidation activity.

2. The enzyme electrode according to claim 1, further comprising an electron mediator.

3. The enzyme electrode according to claim 1 or 2, wherein the polypeptide is immobilized on the electrode.

4. A method for producing an enzyme electrode, comprising the step of immobilizing a polypeptide consisting of an amino acid sequence shown in any of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in any of SEQ ID NOs: 3 to 6 and having L-lactic acid oxidation activity, onto an electrode.

5. A lactic acid sensor comprising the enzyme electrode described in claim 1.

6. The lactic acid sensor according to claim 5, comprising an electrode system including a working electrode and a counter electrode, wherein the working electrode is provided with the enzyme electrode.

7. An L-lactic acid measurement system comprising a lactic acid sensor according to claim 6, means for applying current to electrodes in the electrode system of the lactic acid sensor, and means for measuring the current value in the electrode system of the lactic acid sensor.

8. A method for measuring L-lactic acid using the lactic acid sensor according to claim 5 or 6, or the L-lactic acid measurement system according to claim 7.

9. A biofuel cell comprising the enzyme electrode according to claim 1.

10. The biofuel cell according to claim 9, comprising an anode and a cathode, wherein the anode is provided with the enzyme electrode.

11. A method for generating electricity using a biofuel cell according to claim 9 or 10.

12. A method for measuring L-lactic acid using a polypeptide consisting of an amino acid sequence represented by any of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence represented by any of SEQ ID NOs: 3 to 6 and having L-lactic acid oxidation activity.

13. A method for measuring L-lactic acid according to claim 12, comprising the steps (1) and (2) below. (1) A step of bringing a sample into contact with a polypeptide consisting of an amino acid sequence represented by any of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence represented by any of SEQ ID NOs: 3 to 6 and having L-lactic acid oxidation activity, peroxidase, novel Trinder reagent, and coupler compound. (2) Step of measuring the dye compound obtained in (1)

14. An L-lactic acid measurement kit comprising a polypeptide consisting of an amino acid sequence represented by any of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence represented by any of SEQ ID NOs: 3 to 6 and having L-lactic acid oxidation activity, a peroxidase, a novel Trinder reagent, and a coupler compound.

15. The L-lactic acid measurement kit according to claim 14, wherein the new Trinder reagent is at least one selected from the group consisting of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS).

16. The L-lactic acid measurement kit according to claim 14 or 15, wherein the coupler compound is 4-aminoantipyrine.

Images