Ethanolamine oxidoreductase variant, quantification composition, quantification kits, quantification electrode, quantification sensor chip, and quantification sensor for ethanolamine phosphate, and method for quantifying ethanolamine phosphate

Abstract

To provide a highly activated ethanolamine oxidoreductase variant, a composition, kit, electrode, sensor chip, and quantification sensor for quantifying ethanolamine phosphate comprising the same, and a method for quantifying ethanolamine phosphate using the same.SOLUTION: An ethanolamine oxidoreductase variant contains a copper ion in which the amino acid at position 338 of a specific amino acid sequence is substituted with alanine, glutamic acid, or lysine. A composition for quantifying ethanolamine phosphate contains the variant, a mediator that is reduced by adding the variant, and a reagent that reacts with the reduced mediator.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] One embodiment of the present invention relates to a highly activated ethanolamine oxidoreductase variant. Alternatively, one embodiment of the present invention relates to a composition for quantifying ethanolamine phosphate. Alternatively, one embodiment of the present invention relates to an electrode for quantifying ethanolamine phosphate. Alternatively, one embodiment of the present invention relates to a sensor chip for quantifying ethanolamine phosphate. Alternatively, one embodiment of the present invention relates to a kit for quantifying ethanolamine phosphate. Alternatively, one embodiment of the present invention relates to a sensor for quantifying ethanolamine phosphate. Alternatively, one embodiment of the present invention relates to a method for quantifying ethanolamine phosphate. [Background technology]

[0002] Human blood contains ethanolamine phosphate (EAP). Patent Document 1 describes that EAP is a biomarker for diagnosing depression. Non-Patent Document 1 describes that depression can be diagnosed with an EAP concentration of 1.5 μM or less as a reference value. Patent Document 2 describes a method for measuring EAP concentration using EAP phosphorylase and acetaldehyde dehydrogenase.

[0003] The present inventors have disclosed an oxidoreductase acting on EAP in Patent Document 3. Furthermore, they have disclosed a method capable of measuring trace amounts of EAP using an oxidoreductase acting on EAP in Patent Document 4. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2011 / 019072 [Patent Document 2] International Publication No. 2013 / 069645 [Patent Document 3] International Publication No. 2020 / 122231 [Patent Document 4] International Publication No. 2021 / 256503 [Non-patent literature]

[0005] [Non-Patent Document 1] Kawamura N, “Plasma metabolome analysis of patients with major depressive disorder.” Psychiatry Clin Neurosci. 2018 May;72(5):349-361 [Non-Patent Document 2] Hirano Y et al., “Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination”Appl Microbiol Biotechnol (2016) 100:3999-4013 Summary of the Invention [Problem to be solved by the invention]

[0006] One embodiment of the present invention provides a highly activated variant of ethanolamine oxidoreductase. Alternatively, one embodiment of the present invention provides a composition for quantifying ethanolamine phosphate, comprising a highly activated variant of ethanolamine oxidoreductase. Alternatively, one embodiment of the present invention provides a kit for quantifying ethanolamine phosphate, comprising a highly activated variant of ethanolamine oxidoreductase. Alternatively, one embodiment of the present invention provides an electrode for quantifying ethanolamine phosphate, comprising a highly activated variant of ethanolamine oxidoreductase. Alternatively, one embodiment of the present invention provides a sensor chip for quantifying ethanolamine phosphate, comprising a highly activated variant of ethanolamine oxidoreductase. Alternatively, one embodiment of the present invention provides a sensor for quantifying ethanolamine phosphate, comprising a highly activated variant of ethanolamine oxidoreductase. Alternatively, one embodiment of the present invention provides a method for quantifying ethanolamine phosphate using a highly activated variant of ethanolamine oxidoreductase. [Means for solving the problem]

[0007] According to one embodiment of the present invention, there is provided a modified ethanolamine oxidoreductase which contains a copper ion and in which the amino acid at position 338 of the amino acid sequence of SEQ ID NO:1 has been substituted with alanine, glutamic acid, or lysine.

[0008] In the modified ethanolamine oxidoreductase, the amino acid at position 331 of the amino acid sequence of SEQ ID NO:1 may be substituted with alanine, glutamic acid, or lysine.

[0009] It may further include topaquinone.

[0010] According to one embodiment of the present invention, there is provided a composition for quantifying ethanolamine phosphate, the composition comprising the above-mentioned ethanolamine oxidoreductase variant, a mediator that is reduced by adding the ethanolamine oxidoreductase variant, and a reagent that reacts with the reduced mediator.

[0011] According to one embodiment of the present invention, there is provided a composition for quantifying ethanolamine phosphate, comprising the above-mentioned ethanolamine oxidoreductase variant and a reagent that reacts with hydrogen peroxide generated by adding the ethanolamine oxidoreductase variant.

[0012] The composition for quantifying ethanolamine phosphate may further comprise a phosphatase.

[0013] According to one embodiment of the present invention, there is provided a kit for quantifying ethanolamine phosphate, the kit comprising the above-mentioned ethanolamine oxidoreductase variant, a mediator that is reduced by adding the ethanolamine oxidoreductase variant, and a reagent that reacts with the reduced mediator.

[0014] According to one embodiment of the present invention, there is provided a kit for quantifying ethanolamine phosphate, comprising the above-mentioned ethanolamine oxidoreductase variant and a reagent that reacts with hydrogen peroxide generated by adding the ethanolamine oxidoreductase variant.

[0015] The above-mentioned kit for quantifying ethanolamine phosphate may further contain a phosphatase.

[0016] According to one embodiment of the present invention, there is provided an electrode comprising the above-described ethanolamine oxidoreductase variant.

[0017] According to one embodiment of the present invention, there is provided a sensor chip comprising a working electrode, a counter electrode, and a reference electrode, and a reaction layer disposed on the working electrode, the counter electrode, and the reference electrode, wherein the working electrode has the above-mentioned electrode, or the reaction layer contains the above-mentioned ethanolamine oxidoreductase variant.

[0018] According to one embodiment of the present invention, there is provided a sensor comprising the above sensor chip.

[0019] According to one embodiment of the present invention, there is provided a method for quantifying ethanolamine phosphate, comprising reacting a sample with a phosphatase to convert ethanolamine phosphate contained in the sample to ethanolamine, and then reacting the ethanolamine oxidoreductase variant described above with the ethanolamine.

[0020] The above-mentioned method for quantifying ethanolamine phosphate may further include reducing a mediator by allowing an ethanolamine oxidoreductase variant to act on ethanolamine, reacting the reduced mediator with a reagent, and determining the concentration of ethanolamine phosphate.

[0021] The above-mentioned method for quantifying ethanolamine phosphate may further include determining the concentration of ethanolamine phosphate by allowing an ethanolamine oxidoreductase variant to act on ethanolamine and quantifying the amount of hydrogen peroxide produced or the amount of oxygen consumed.

[0022] The above-mentioned method for quantifying ethanolamine phosphate may further include, when the sample contains at least one amine other than ethanolamine phosphate, treating the at least one amine with an amine oxidoreductase prior to treating the sample with a phosphatase. Effect of the Invention

[0023] According to one embodiment of the present invention, a highly activated ethanolamine oxidoreductase variant is provided. Alternatively, according to one embodiment of the present invention, a composition for quantifying ethanolamine phosphate, comprising a highly activated ethanolamine oxidoreductase variant, is provided. Alternatively, according to one embodiment of the present invention, a kit for quantifying ethanolamine phosphate, comprising a highly activated ethanolamine oxidoreductase variant, is provided. Alternatively, according to one embodiment of the present invention, an electrode for quantifying ethanolamine phosphate, comprising a highly activated ethanolamine oxidoreductase variant, is provided. Alternatively, according to one embodiment of the present invention, a sensor chip for quantifying ethanolamine phosphate, comprising a highly activated ethanolamine oxidoreductase variant, is provided. Alternatively, according to one embodiment of the present invention, a sensor for quantifying ethanolamine phosphate, comprising a highly activated ethanolamine oxidoreductase variant, is provided. Alternatively, according to one embodiment of the present invention, a method for quantifying ethanolamine phosphate using a highly activated ethanolamine oxidoreductase variant is provided. [Brief description of the drawings]

[0024] [Figure 1A] FIG. 1 is a top view of an electrode 1 according to one embodiment of the present invention. [Figure 1B] 1B is a cross-sectional end view of the electrode 1 taken along line AA' in FIG. 1A. [Figure 2A] FIG. 1 is a top view of a sensor chip 10 according to an embodiment of the present invention. [Figure 2B] FIG. 2 is a schematic diagram showing components constituting the sensor chip 10. [Figure 2C] FIG. 2 is a schematic diagram showing components constituting the sensor chip 10. [Figure 2D] FIG. 2 is a schematic diagram showing components constituting the sensor chip 10. [Figure 3A] FIG. 1 is a schematic diagram of a sensor 100 according to one embodiment of the present invention. [Figure 3B] FIG. 1 is a block diagram of a sensor 100 according to an embodiment of the present invention. [Figure 4]FIG. 1 shows the relationship between the length of holoconversion treatment time and the specific activity of LrEAOX after in vitro holoconversion of ethanolamine oxidase for 16, 40, and 64 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The following describes the highly activated ethanolamine oxidoreductase variant, the composition for quantifying ethanolamine phosphate, the kit for quantifying ethanolamine phosphate, the electrode for quantifying ethanolamine phosphate, the sensor chip for quantifying ethanolamine phosphate, and the method for quantifying ethanolamine phosphate according to the present invention. However, the highly activated ethanolamine oxidoreductase variant, the composition for quantifying ethanolamine phosphate, the kit for quantifying ethanolamine phosphate, the electrode for quantifying ethanolamine phosphate, the sensor chip for quantifying ethanolamine phosphate, and the method for quantifying ethanolamine phosphate according to the present invention are not to be construed as being limited to the description of the embodiments and examples shown below.

[0026] [Holoenzyme] Amine oxidase is known to be a holoenzyme containing the cofactor 2,4,5-trihydroxyphenylalanylquinone (topaquinone, TPQ), which is generated by oxidation of a specific tyrosine side chain in the enzyme (Non-Patent Document 2). Amine oxidase is also a holoenzyme that contains, for example, copper (II) ions as metal ions, and in which the copper (II) ions are involved in the oxidation reaction of amino acid residues to generate the cofactor.

[0027] [Modified ethanolamine oxidoreductase] The ethanolamine oxidoreductase variant according to the present invention is a holoenzyme containing a copper(II) ion. In one embodiment, the ethanolamine oxidoreductase variant is a holoenzyme containing TPQ in which the tyrosine side chain at position 398 of SEQ ID NO: 1 is oxidized. In this embodiment, the ethanolamine oxidoreductase variant is a holoenzyme in which the amino acid at position 338 of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (SEQ ID NO: 3), glutamic acid (SEQ ID NO: 4), or lysine (SEQ ID NO: 5). In other words, in this embodiment, the ethanolamine oxidoreductase variant has an alanine residue, a glutamic acid residue, or a lysine residue at position 338 of the amino acid sequence of SEQ ID NO: 1.

[0028] The ethanolamine oxidoreductase variant of this embodiment has the amino acid at position 338 of the amino acid sequence of SEQ ID NO: 1 replaced with alanine, glutamic acid, or lysine, and thus has higher oxidation activity or dehydrogenation activity for ethanolamine than the ethanolamine oxidoreductase of a wild-type strain having TPQ at position 398 of SEQ ID NO: 1.

[0029] In one embodiment, the variant ethanolamine oxidoreductase may be a double mutant (SEQ ID NOs: 6 to 14) in which the amino acid at position 331 is substituted with alanine, glutamic acid or lysine in addition to the above-mentioned substitution of the amino acid at position 338 in the amino acid sequence of SEQ ID NO: 1. In other words, the variant ethanolamine oxidoreductase contains a copper(II) ion, and has TPQ at position 398 of SEQ ID NO: 1, an alanine residue, a glutamic acid residue or a lysine residue at position 331, and an alanine residue, a glutamic acid residue or a lysine residue at position 338.

[0030] In one embodiment, the ethanolamine oxidoreductase variant may be a holoenzyme (SEQ ID NO: 6) in which the amino acid at position 331 and the amino acid at position 338 in the amino acid sequence of SEQ ID NO: 1 are alanine. Alternatively, the ethanolamine oxidoreductase variant may be a holoenzyme (SEQ ID NO: 8) in which the amino acid at position 331 and the amino acid at position 338 in the amino acid sequence of SEQ ID NO: 1 are alanine and lysine, respectively. Alternatively, the ethanolamine oxidoreductase variant may be a holoenzyme (SEQ ID NO: 12) in which the amino acid at position 331 and the amino acid at position 338 in the amino acid sequence of SEQ ID NO: 1 are alanine.

[0031] The variant ethanolamine oxidoreductase according to this embodiment has an alanine residue at position 331 and an alanine residue at position 338 in the amino acid sequence of SEQ ID NO: 1. Alternatively, the variant ethanolamine oxidoreductase has an alanine residue at position 331 and a lysine residue at position 338 in the amino acid sequence of SEQ ID NO: 1. Alternatively, the variant ethanolamine oxidoreductase has a lysine residue at position 331 and an alanine residue at position 338 in the amino acid sequence of SEQ ID NO: 1. These holoenzymes contain a copper(II) ion and have TPQ at position 398.

[0032] The ethanolamine oxidoreductase variant of this embodiment has the amino acid at position 331 of the amino acid sequence of SEQ ID NO: 1 substituted with alanine, glutamic acid, or lysine, in addition to the amino acid at position 338. As a result, the variant has higher oxidation activity or dehydrogenation activity for ethanolamine than the ethanolamine oxidoreductase, which is the holoenzyme of the wild-type strain.

[0033] [Ethanolamine oxidoreductase variant gene] In one embodiment, a gene encoding the above-mentioned ethanolamine oxidoreductase variant is provided. The ethanolamine oxidoreductase variant gene according to this embodiment is a gene in which the amino acid at position 338 in the amino acid sequence of SEQ ID NO: 1 in the ethanolamine oxidoreductase gene derived from Lichtheimia ramosa of SEQ ID NO: 2 is substituted with alanine (SEQ ID NO: 3), glutamic acid (SEQ ID NO: 4), or lysine (SEQ ID NO: 5).

[0034] In one embodiment, the ethanolamine oxidoreductase variant gene of this embodiment is a double mutant gene (SEQ ID NOs: 6 to 14) in which the amino acid at position 338 of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, glutamic acid, or lysine, and the amino acid at position 331 is substituted with alanine, glutamic acid, or lysine.

[0035] In one embodiment, the ethanolamine oxidoreductase variant gene according to this embodiment may be a double mutant gene (SEQ ID NO: 6) in which the amino acid at position 331 and the amino acid at position 338 in the amino acid sequence of SEQ ID NO: 1 are alanine. Alternatively, the ethanolamine oxidoreductase variant gene may be a double mutant gene (SEQ ID NO: 8) in which the amino acid at position 331 and the amino acid at position 338 in the amino acid sequence of SEQ ID NO: 1 are alanine and lysine, respectively. Alternatively, the ethanolamine oxidoreductase variant gene may be a double mutant gene (SEQ ID NO: 12) in which the amino acid at position 331 and the amino acid at position 338 in the amino acid sequence of SEQ ID NO: 1 are lysine and alanine, respectively.

[0036] It should be noted that all of the above-mentioned ethanolamine oxidoreductase variant genes according to this embodiment are genes for an apoenzyme in which position 398 of SEQ ID NO:1 is tyrosine.

[0037] [Ethanolamine oxidoreductase modified DNA construct] In one embodiment, a DNA construct containing the above-mentioned ethanolamine oxidoreductase variant gene is provided. In this specification, the term "DNA construct" refers collectively to a recombinant vector containing a DNA fragment containing the above-mentioned ethanolamine oxidoreductase variant gene. Such a vector may be a plasmid, a bacteriophage, a cosmid, or the like, containing a DNA fragment containing the ethanolamine oxidoreductase variant gene.

[0038] [Method of producing a modified ethanolamine oxidoreductase gene] The present ethanolamine oxidoreductase variant gene can be produced by a conventional method.

[0039] (Construction of expression plasmid) In one embodiment, the plasmid for expressing the ethanolamine oxidoreductase variant gene is obtained by a commonly used method. For example, DNA is extracted from Lichtheimia ramosa that produces the ethanolamine oxidoreductase variant (SEQ ID NO: 1) to prepare a DNA library. From the prepared DNA library, a DNA fragment encoding the ethanolamine oxidoreductase variant is identified and isolated. The isolated DNA fragment is amplified by polymerase chain reaction (PCR) using complementary primers with the isolated DNA fragment as a template, and the gene encoding the ethanolamine oxidoreductase variant (SEQ ID NO: 2) is cloned. The amplified DNA fragment is ligated to a vector to obtain a plasmid having a DNA fragment encoding the ethanolamine oxidoreductase variant.

[0040] In the obtained plasmid, the gene (SEQ ID NO: 2) encoding the present ethanolamine oxidoreductase variant can be substituted with alanine (SEQ ID NO: 3), glutamic acid (SEQ ID NO: 4), or lysine (SEQ ID NO: 5) for the amino acid at position 338 of the amino acid sequence of SEQ ID NO: 1 to obtain a plasmid for expressing the ethanolamine oxidoreductase variant gene according to the present embodiment. Note that mutants in which the amino acid at position 338 of the amino acid sequence of SEQ ID NO: 1 is substituted with a base sequence encoding such an amino acid can be prepared by known methods, and detailed explanations thereof will be omitted.

[0041] Alternatively, a DNA fragment is chemically synthesized in which the base sequence (SEQ ID NO: 2) of the gene encoding the present ethanolamine oxidoreductase variant is replaced with a base sequence encoding alanine (SEQ ID NO: 3), glutamic acid (SEQ ID NO: 4), or lysine (SEQ ID NO: 5) at position 338 of the amino acid sequence of SEQ ID NO: 1, and the DNA fragment is ligated to a vector to obtain a plasmid having DNA encoding the ethanolamine oxidoreductase variant of this embodiment.

[0042] A strain of E. coli or the like is transformed with the obtained plasmid to obtain a strain of E. coli or the like having a DNA encoding the modified ethanolamine oxidoreductase according to this embodiment.

[0043] Also, for example, a strain such as yeast may be transformed with the obtained plasmid to obtain a strain such as yeast having a DNA encoding an ethanolamine oxidoreductase variant. As a method for transforming yeast, a known method, for example, a method using lithium acetate or electroporation, etc., may be suitably used, but is not limited thereto, and transformation may be performed using any method including the spheroplast method and the glass bead method. Examples of microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Saccharomyces, Pichia, and Candida. The plasmid having the DNA encoding the ethanolamine oxidoreductase variant may contain a marker gene to enable the selection of transformed cells. Examples of the marker gene include genes that complement the auxotrophy of the host, such as URA3 and TRP1. Furthermore, the plasmid having the DNA encoding the variant ethanolamine oxidoreductase desirably contains a promoter or other control sequence (e.g., a secretion signal sequence, an enhancer sequence, a terminator sequence, or a polyadenylation sequence) capable of expressing the variant ethanolamine oxidoreductase gene in a host cell. Specific examples of promoters include the GAL1 promoter and the ADH1 promoter.

[0044] Other examples of host cells include filamentous fungi such as those of the genus Aspergillus and Trichoderma. The method for producing a transformant of a filamentous fungus is not particularly limited, and examples thereof include a method in which a DNA encoding an ethanolamine oxidoreductase variant is inserted into a host filamentous fungus in a manner that allows the DNA to be expressed according to a conventional method. Specifically, a DNA construct is prepared in which a gene encoding an ethanolamine oxidoreductase variant is inserted between an expression-inducing promoter and a terminator, and then a host filamentous fungus is transformed with the DNA construct containing the gene encoding the ethanolamine oxidoreductase variant, thereby obtaining a transformant that overexpresses the gene encoding the ethanolamine oxidoreductase variant.

[0045] The method for inserting a gene encoding an ethanolamine oxidoreductase variant into a host filamentous fungus in a manner such that the gene is expressed is not particularly limited, and examples of the method include a method of directly inserting the gene into a chromosome of the host organism by utilizing homologous recombination, or a method of introducing the gene into the host filamentous fungus by linking the gene onto a plasmid vector.

[0046] In the method using homologous recombination, a DNA construct can be ligated between sequences homologous to the upstream and downstream regions of the recombination site on the chromosome and inserted into the genome of the host filamentous fungus. A transformant can be obtained by self-cloning by overexpressing the gene in the host filamentous fungus under the control of the host filamentous fungus' own high expression promoter. The high expression promoter is not particularly limited, and examples thereof include the promoter region of the TEF1 gene (tef1), which is a translation elongation factor, the promoter region of the α-amylase gene (amy), and the promoter region of the alkaline protease gene (alp).

[0047] In the method using a vector, the DNA construct can be inserted into a plasmid vector used for transformation of filamentous fungi by standard methods, and the corresponding host filamentous fungus can be transformed by standard methods.

[0048] Such a suitable vector-host system is not particularly limited as long as it is a system that allows the apoenzyme to be produced in the host filamentous fungus, and examples thereof include a system of pUC19 and a filamentous fungus, and a system of pSTA14 (Unkles et al., Mol. Gen. Genet. (1989) 218, 99-104) and a filamentous fungus.

[0049] The DNA construct is preferably introduced into the chromosome of the host filamentous fungus for use, but as an alternative, the DNA construct can be incorporated into an autonomously replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. (1995) 59, 1133-1134) for use without being introduced into the chromosome.

[0050] The DNA construct may contain a marker gene that allows the selection of transformed cells. The marker gene is not particularly limited, and examples thereof include genes that complement the auxotrophy of the host, such as pyrG, niaD, and adeA; and drug resistance genes against drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably contains a promoter, terminator, and other control sequences (e.g., enhancer, polyadenylation sequence, etc.) that allow the gene encoding the ethanolamine oxidoreductase variant to be overexpressed in the host cell. The promoter is not particularly limited, and examples thereof include appropriate expression-inducing promoters and constitutive promoters, such as tef1 promoter, alp promoter, and amy promoter. The terminator is also not particularly limited, and examples thereof include alp terminator, amy terminator, and tef1 terminator.

[0051] In the DNA construct, the expression control sequence of the gene encoding the ethanolamine oxidoreductase variant is not necessarily required when the DNA fragment containing the gene encoding the ethanolamine oxidoreductase variant to be inserted contains a sequence having an expression control function. In addition, when transformation is performed by co-transformation, the DNA construct may not need to have a marker gene.

[0052] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, a gene encoding a variant ethanolamine oxidoreductase, the alp gene terminator, and the pyrG marker gene are linked to the In-Fusion Cloning Site in the multiple cloning site of pUC19.

[0053] As a method for transforming a filamentous fungus, a method known to those skilled in the art can be appropriately selected. For example, a protoplast PEG method using polyethylene glycol and calcium chloride after preparing a protoplast of a host filamentous fungus (see, for example, Unkles et al., Mol. Gen. Genet. 218, 99-104, 1989, JP 2007-222055 A) can be used. A medium for regenerating a transformed filamentous fungus is used that is appropriate depending on the host filamentous fungus and the transformation marker gene used. For example, when Aspergillus sojae is used as a host filamentous fungus and the pyrG gene is used as a transformation marker gene, the regeneration of the transformed filamentous fungus can be performed, for example, in Czapek-Dox minimal medium (manufactured by Difco) containing 0.5% agar and 1.2 M sorbitol.

[0054] (Recombinant expression of modified ethanolamine oxidoreductase apoenzyme) A strain such as Escherichia coli having a DNA encoding the ethanolamine oxidoreductase variant according to the present embodiment is cultured in a medium. The microbial host cells may be cultured at a culture temperature of 10°C to 42°C, preferably at a culture temperature of around 25°C for several hours to several days, more preferably at a culture temperature of around 25°C for 1 to 7 days, by aeration and stirring submerged culture, shaking culture, stationary culture, or the like. The medium for culturing the microbial host cells may be, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran leachate, to which one or more inorganic salts such as sodium chloride, potassium monophosphate, potassium diphosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and further, if necessary, carbohydrate raw materials, vitamins, and the like are appropriately added. The cells are separated from the culture solution obtained by the culture by centrifugation. The separated cells are subjected to ultrasonic pulverization, grinding, etc., or treated with a lytic enzyme such as lysozyme or ytalase to prepare a suspension, and the suspension is centrifuged to obtain a fraction from which a crude enzyme solution is obtained.

[0055] (Purification of modified ethanolamine oxidoreductase apoenzyme) The method for purifying the modified ethanolamine oxidoreductase apoenzyme may be any method capable of purifying the enzyme from a crude enzyme solution, for example, a commonly used method such as ion exchange chromatography or gel filtration chromatography.

[0056] (Production of modified ethanolamine oxidoreductase holoenzyme) The tyrosine residue at position 398 of the prepared apoenzyme is oxidized by oxidation reaction to convert it to TPQ, thereby producing the ethanolamine oxidoreductase modified holoenzyme of the present invention. The oxidation reaction for producing the holoenzyme can be carried out, for example, by adding copper (II) ions to oxidize the tyrosine residue at position 398 of the apoenzyme and convert it to TPQ, thereby producing the holoenzyme of the present invention.

[0057] The ethanolamine oxidoreductase modified holoenzyme can be produced by oxidizing the apoenzyme in an appropriate solution. The amount of the apoenzyme to be added to the solution may be, for example, 0.01 mg / mL to 50 mg / mL, preferably 0.05 mg / mL to 10 mg / mL. The amount of copper (II) ions to be added to the solution may be, for example, 0.001 mM to 100 mM, preferably 0.01 mM to 10 mM. The pH of the solution is preferably adjusted using a buffer so that the holoenzyme is not inactivated. For example, the pH of the solution is pH 3 to 11, preferably pH 4 to 10, more preferably pH 5 to 9. Examples of buffers used to adjust the pH of the solution include, but are not limited to, N-[tris(hydroxymethyl)methyl]glycine, phosphate, acetate, carbonate, tris(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutamate, tricine, HEPES, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, phthalic acid, and tartaric acid.

[0058] [Identification of topaquinone] The topaquinone formed in the ethanolamine oxidoreductase variant holoenzyme can be identified, for example, by the following method. The topaquinone formed in the ethanolamine oxidoreductase variant holoenzyme is derivatized with, for example, p-nitrophenylhydrazine (pNPH), and then released from the polypeptide chain by protease treatment and purified. The released topaquinone can be identified by analysis using NMR, ultraviolet-visible spectroscopy, and electrospray tandem mass spectrometry (ESI-MS / MS) (for example, Steinebach V. et al., Analytical Biochemistry (1995) 230 (1), 159-166).

[0059] [Measurement of activity of modified ethanolamine oxidoreductase] The activity of an ethanolamine oxidoreductase variant (holoenzyme) can be measured by catalyzing an oxidation-reduction reaction to generate a reduced product, and measuring the current value generated when the reduced product transfers electrons to an electrode. Preferably, the enzyme activity can be measured by reacting the reduced product produced by the oxidation-reduction reaction catalyzed by the holoenzyme with a reagent containing a light-absorbing substance that reacts with the reduced product (hereinafter referred to as "light-absorbing reagent") and measuring the absorbance.

[0060] In one embodiment, the activity of an ethanolamine oxidoreductase variant (holoenzyme) can be measured by reacting a product of an oxidation reaction catalyzed by the ethanolamine oxidoreductase variant with a reagent that reacts with the product (hereinafter, "product reaction reagent"), and measuring the light-absorbing substance produced by the reaction, by measuring absorbance.

[0061] [EAP quantitative composition and EAP quantitative kit] The method for quantifying EAP using the present ethanolamine oxidoreductase variant (holoenzyme) may be carried out by providing a composition containing the present ethanolamine oxidoreductase variant (holoenzyme), or by combining the present ethanolamine oxidoreductase variant (holoenzyme) with a commercially available product reaction reagent. In one embodiment, a composition for quantifying EAP can be provided that contains the present ethanolamine oxidoreductase variant (holoenzyme), a mediator that is reduced by adding ethanolamine oxidoreductase, and a reagent that reacts with the reduced mediator.

[0062] Alternatively, in one embodiment, a composition for quantifying EAP can be provided, comprising the present ethanolamine oxidoreductase variant (holoenzyme) and a reagent that reacts with hydrogen peroxide generated by adding ethanolamine oxidase.

[0063] In one embodiment, a composition containing the present ethanolamine oxidoreductase variant (holoenzyme) and a phosphatase can be provided. In one embodiment, a composition for quantifying EAP can be provided, the composition containing a phosphatase, the present ethanolamine oxidoreductase variant (holoenzyme), a mediator that is reduced by adding the ethanolamine oxidoreductase, and a reagent that reacts with the reduced mediator.

[0064] Alternatively, in one embodiment, a composition for quantifying EAP can be provided, comprising a phosphatase, the present ethanolamine oxidoreductase variant (holoenzyme), and a reagent that reacts with hydrogen peroxide generated by adding ethanolamine oxidase.

[0065] In one embodiment, a kit for quantifying EAP can be provided, comprising the present ethanolamine oxidoreductase variant (holoenzyme), a mediator that is reduced by adding the ethanolamine oxidoreductase, and a reagent that reacts with the reduced mediator. For example, the kit for quantifying EAP may comprise a first reagent comprising the ethanolamine oxidoreductase variant (holoenzyme) and a second reagent comprising the mediator that is reduced by adding the ethanolamine oxidoreductase variant (holoenzyme). The kit may further comprise a third reagent comprising an amine oxidoreductase or amine oxidase that acts on at least one amine other than EAP, and may be configured to add the third reagent to the sample before adding the first and second reagents.

[0066] The mediator (also called artificial electron mediator, artificial electron acceptor or electron mediator) used in the quantification method or quantification kit of this embodiment is not particularly limited as long as it can accept electrons from oxidoreductase. Mediators include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides such as potassium ferricyanide, ferredoxins, ferrocene, osmium complexes and derivatives thereof, and phenazine compounds include, but are not limited to, 5-methylphenazinium methosulfate (PMS) and methoxy PMS.

[0067] Alternatively, in one embodiment, a kit for quantifying EAP can be provided, comprising the present ethanolamine oxidoreductase variant (holoenzyme) and a reagent that reacts with hydrogen peroxide generated by adding ethanolamine oxidase. For example, the kit for quantifying EAP may comprise a first reagent comprising the ethanolamine oxidoreductase variant (holoenzyme) and a second reagent that reacts with hydrogen peroxide generated by adding the ethanolamine oxidase variant.

[0068] Alternatively, the kit may further include a third reagent containing an amine oxidoreductase or amine oxidase acting on at least one amine other than EAP, and the third reagent may be added to the sample before the first and second reagents are added. Generally, clinical specimens may contain amines such as EA, β-alanine, and taurine as impurities. In the kit for quantifying EAP according to the present invention, since a modified ethanolamine oxidoreductase is used, if these amines are contained in the sample, the modified ethanolamine oxidoreductase will also act on these amines, making it difficult to accurately quantify the EA derived from the EAP that should be measured. For this reason, it is preferable to decompose or eliminate amines other than EAP contained in the sample before quantifying EAP.

[0069] The reagent used in the quantitative determination method or quantitative determination kit of this embodiment, which reacts with hydrogen peroxide generated by adding ethanolamine oxidase, may be a color-developing substrate. Examples of the color-developing substrate include, in addition to 4-aminoantipyrine, ADOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), ALOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline), TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine), and DA-64 (N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine). ADOS, ALOS, and TOOS develop color when condensed with 4-aminoantipyrine. DA-64 and DA-67 do not require 4-aminoantipyrine and will develop color when formulated alone. In either case, the color reaction is catalyzed by peroxidase. The measured consumables include dissolved oxygen, and the amount of dissolved oxygen in the reaction solution can be measured using a dissolved oxygen meter or the like. For example, the degree of color development (change in absorbance) of the above-mentioned measurement reagent can be measured using a spectrophotometer or an automatic biochemical analyzer, and compared with the absorbance of a standard sample to measure the EAP contained in the sample.

[0070] [Electrodes, sensor chips and electrodes] Fig. 1A is a top view of an electrode 1 according to one embodiment of the present invention, and Fig. 1B is a cross-sectional end view of the electrode 1 taken along line AA' in Fig. 1A. The electrode 1 includes a wiring 1a and an upper electrode 1b disposed on the wiring 1a. The upper electrode 1b contains the above-mentioned ethanolamine oxidoreductase variant. Figs. 1A and 1B show the electrode 1 disposed on a substrate 11. In one embodiment, the upper electrode 1b may be disposed at one end of the wiring 1a, but is not limited thereto.

[0071] In one embodiment, the upper electrode 1b may be formed by coating, adsorbing, or immobilizing the present ethanolamine oxidoreductase variant on the wiring 1a. The upper electrode 1b may be formed by immobilizing the present ethanolamine oxidoreductase variant on the wiring 1a by crosslinking, coating with a dialysis membrane, encapsulation in a polymer matrix, use of a photocrosslinkable polymer, use of an electrically conductive polymer, use of an oxidation / reduction polymer, or the like. In another embodiment, a phosphatase may be coated, adsorbed, or immobilized on the wiring 1a together with the present ethanolamine oxidoreductase variant. The ethanolamine oxidoreductase variant and the phosphatase may be immobilized in a polymer or adsorbed and immobilized on the wiring 1a together with a mediator, or these methods may be combined.

[0072] The wiring 1a can be a carbon electrode, or a metal electrode such as platinum, gold, silver, nickel, or palladium. In the case of a carbon electrode, the material can be pyrolytic graphite carbon (PG), glassy carbon (GC), carbon paste, or plastic formed carbon (PFC).

[0073] FIG. 2A is a top view of a sensor chip 10 according to an embodiment of the present invention, and FIGS. 2B to 2D are schematic diagrams showing components constituting the sensor chip 10. The sensor chip 10 includes two or more electrodes arranged on a substrate 11. The substrate 11 is made of an insulating material. In FIGS. 2A and 2B, as an example, a working electrode 2, a counter electrode 3, and a reference electrode 5 are arranged on the substrate 11. Each electrode is electrically connected to a wiring portion 7, and the wiring portion 7 is electrically connected to a terminal 9 located on the opposite side of the wiring direction from each electrode. The working electrode 2, the counter electrode 3, and the reference electrode 5 are arranged apart from each other. In addition, the working electrode 2, the counter electrode 3, and the reference electrode 5 are preferably integrally formed with the wiring portion 7 and the terminal 9. In addition, the counter electrode 3 and the reference electrode 5 may be integral.

[0074] As shown in Figs. 2A and 2C, a spacer 13 is disposed at the end of the substrate 11 parallel to the wiring portion 7, and a cover 15 is disposed to cover the working electrode 2, the counter electrode 3, the reference electrode 5, and the spacer 13. The spacer 13 and the cover 15 are made of an insulating material. The spacer 13 has a thickness approximately equal to that of the working electrode 2, the counter electrode 3, and the reference electrode 5, and preferably adheres closely to the working electrode 2, the counter electrode 3, and the reference electrode 5. The spacer 13 and the cover 15 may be integrally formed. The cover 15 is a protective layer that prevents the wiring portion 7 from being deteriorated due to exposure to the outside air and from being short-circuited due to seepage of a measurement sample.

[0075] In one embodiment, the modified ethanolamine oxidoreductase is applied to, adsorbed on, or immobilized on the working electrode 2. In another embodiment, a phosphatase may be applied to, adsorbed on, or immobilized on the working electrode 2 together with the modified ethanolamine oxidoreductase. In another embodiment, a mediator may be applied to, adsorbed on, or immobilized on the working electrode 2 together with the modified ethanolamine oxidoreductase and the phosphatase. The working electrode 2 may have the configuration described for the electrode 1 above.

[0076] The ethanolamine oxidoreductase variant, or the ethanolamine oxidoreductase variant and phosphatase, or the ethanolamine oxidoreductase variant, phosphatase, and mediator may be included in a reaction layer 19 arranged on the working electrode 2, counter electrode 3, and reference electrode 5. The counter electrode 3 and reference electrode 5 may be a carbon electrode, or a metal electrode such as platinum, gold, silver, nickel, or palladium. In the case of a carbon electrode, examples of the material include pyrolytic graphite carbon (PG), glassy carbon (GC), carbon paste, and plastic formed carbon (PFC). The measurement system may be a two-electrode system or a three-electrode system, and for example, the ethanolamine oxidoreductase variant may be immobilized on the working electrode 2. Examples of the reference electrode 5 include a standard hydrogen electrode, a reversible hydrogen electrode, a silver-silver chloride electrode (Ag / AgCl), a palladium hydrogen electrode, and a saturated calomel electrode. From the viewpoint of stability and reproducibility, it is preferable to use Ag / AgCl.

[0077] The mediator (also referred to as artificial electron mediator, artificial electron acceptor or electron mediator) used in the composition, kit, electrode or sensor chip according to the present embodiment is not particularly limited as long as it can accept electrons from the modified ethanolamine oxidoreductase. Examples of mediators include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides such as potassium ferricyanide, ferredoxins, ferrocene, osmium complexes and derivatives thereof, and examples of phenazine compounds include, but are not limited to, PMS and methoxy PMS.

[0078] The present ethanolamine oxidoreductase variant can be applied to various electrochemical measurement methods by using a potentiostat, a galvanostat, or the like. Examples of electrochemical measurement methods include various methods such as amperometry, potentiometry, and coulometry. For example, by the amperometry method, hydrogen peroxide generated when the ethanolamine oxidoreductase variant reacts with EAP is measured by applying +600 mV to +1000 mV (vs. Ag / AgCl) to a hydrogen peroxide electrode, and the concentration of EAP in a sample can be calculated by measuring the current value. For example, a calibration curve can be created by measuring the current value for known EAP concentrations (0 μM, 0.1 μM, 0.15 μM, 0.3 μM, 0.5 μM, 50 μM, 100 μM, 150 μM, 200 μM) and plotting it against the EAP concentration. The EAP concentration can be obtained from the calibration curve by measuring the current value of unknown EAP. For example, a carbon electrode or a platinum electrode can be used as the hydrogen peroxide electrode. Alternatively, instead of the hydrogen peroxide electrode, an electrode with immobilized reductase such as peroxidase or catalase can be used, and the amount of hydrogen peroxide can be quantified by measuring the reduction current value generated by applying -400mV to +100mV (vs. Ag / AgCl), thereby measuring the EAP value.

[0079] In addition, the concentration of EAP in the sample can be calculated by mixing a mediator in the reaction solution, transferring the electrons generated when the ethanolamine oxidoreductase variant reacts with EAP to the oxidized mediator by, for example, amperometry to generate a reduced mediator, and then measuring the current value generated by applying -1000mV to +500mV (vs. Ag / AgCl). A carbon electrode or a platinum electrode is preferable as the counter electrode. For example, a calibration curve can be created by measuring the current value for known EAP concentrations (0μM, 0.1μM, 0.15μM, 0.3μM, 0.5μM, 50μM, 100μM, 150μM, 200μM) and plotting it against the EAP concentration. The EAP concentration can be obtained from the calibration curve by measuring the current value of unknown EAP.

[0080] Furthermore, in order to reduce the amount of solution required for measurement, a printed electrode (sensor chip) can also be used. In this case, the electrodes are preferably formed on a base material made of an insulating substrate. Specifically, it is desirable to form the electrodes on the base material by photolithography technology or printing technology such as screen printing, gravure printing, and flexographic printing. In addition, examples of materials for the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, and it is more preferable to use one that is highly resistant to various solvents and chemicals.

[0081] [EAP measurement sensor] In one embodiment, an EAP measurement sensor using the present ethanolamine oxidoreductase variant is provided. FIG. 3A is a schematic diagram of a sensor 100 according to one embodiment of the present invention. The sensor is an EAP measurement device using the oxidoreductase of the present invention, and includes a sensor chip 10 containing the ethanolamine oxidoreductase variant, and a measurement unit 30. The measurement unit 30 may include, for example, a switch 31 as an input unit, and a display 33 as a display unit. The switch 31 may be used, for example, to control the ON / OFF of the power supply of the measurement unit 30, and may be used to control the start and interruption of EAP measurement in the sensor 100. The display 33 may display, for example, the measured EAP value, and may include a touch panel as an input unit for controlling the measurement unit 30.

[0082] 3B is a block diagram of the sensor 100 according to an embodiment of the present invention. The sensor 100 may include, for example, a control unit 110, a display unit 120, an input unit 130, a storage unit 140, a communication unit 150, and a power supply 160 in the measurement unit 30, which may be electrically connected to each other by wiring 190. A terminal of the sensor chip 10 described below and a terminal of the measurement unit 30 are electrically connected, and a current generated in the sensor chip 10 is detected by the control unit 110. The control unit 110 is a control device that controls the sensor 100, and is composed of, for example, a known central processing unit (CPU) and an operation program that controls the sensor 100. Alternatively, the control unit 110 may include a central processing unit and an operating system (OS), and may include an application program or module for performing EAP measurement.

[0083] The display unit 120 may include, for example, a known display 33, and may display the EAP measurement value, the state of the measurement unit 30, and operation requests to the person taking the measurement. The input unit 130 is an input device that enables the person to operate the sensor 100, and may be, for example, a switch 31 or a touch panel arranged on the display 33. The measurement unit 30 may be provided with a plurality of switches 31.

[0084] The storage unit 140 is composed of a main storage device (memory), and a secondary storage device (hard disk) may be arranged externally. The main storage device (memory) may be composed of a read-only memory (ROM) and / or a random access memory (RAM). Operation programs, operating systems, application programs or modules are stored in the storage unit 140 and executed by the central processing unit to configure the control unit 110. In addition, measured values ​​and current values ​​can be stored in the storage unit 140.

[0085] The communication unit 150 is a known communication device that connects the sensor 100 or the measurement unit 30 to an external device (a computer, a printer, or a network). The communication unit 150 and the external device are connected by wired or wireless communication. The power supply 160 is a known power supply device that supplies power to the sensor 100 or the measurement unit 30.

[0086] [Quantification of EAP using modified ethanolamine oxidoreductase] Ethanolamine phosphate can be quantified by allowing the above-mentioned modified ethanolamine oxidoreductase to act on a sample containing ethanolamine phosphate.

[0087] (Quantification of EAP using reductase activity of modified ethanolamine oxidoreductase) A sample is treated with phosphatase to convert EAP contained in the sample into ethanolamine (EA) (step S101). The ethanolamine oxidoreductase variant is then treated with EA (step S103). The reaction product generated by this reaction is detected (step S105). Using a calibration curve prepared in advance, EAP is quantified from the detected value of the reaction product (step S107).

[0088] In step S101, the EAP concentration in the sample is not particularly limited, but may be, for example, 0.01 μM to 10,000 μM. The pH when the sample is subjected to phosphatase treatment conditions may be unadjusted, or may be adjusted to pH 2 to 13, preferably pH 3 to 12, using an appropriate pH buffer so as to be a suitable pH for the action of the phosphatase used. Examples of buffers that can be used include N-[tris(hydroxymethyl)methyl]glycine, phosphate, acetate, carbonate, tris(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutamate, tricine, HEPES, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, phthalic acid, and tartaric acid. The treatment temperature may be, for example, 20 to 50° C., or, depending on the phosphatase used, may be higher, at 45 to 70° C. The treatment time may be a time sufficient for liberating EA, and may be 5 seconds to 180 minutes, preferably 0.5 minutes to 60 minutes, more preferably 1 minute to 30 minutes, and even more preferably 1 minute to 10 minutes. The resulting treatment solution may be used as is, or may be appropriately heated, centrifuged, concentrated, diluted, etc., as necessary.

[0089] In step S103, the reaction time of the modified ethanolamine oxidoreductase can be, for example, 5 seconds to 180 minutes, preferably 0.5 minutes to 60 minutes, more preferably 1 minute to 30 minutes, and even more preferably 1 minute to 10 minutes. The reaction temperature varies depending on the optimum temperature of the modified ethanolamine oxidoreductase used, but is, for example, 20° C. to 45° C., and can be appropriately selected from temperatures used in ordinary enzyme reactions.

[0090] The preferred amount of the present ethanolamine oxidoreductase variant to be used is, for example, 0.001 U / ml to 500 U / ml, preferably 0.01 U / ml to 100 U / ml in final concentration. In general, the lower the concentration of the substrate contained in the sample solution, the higher the final concentration of the ethanolamine oxidoreductase variant to be added. The pH during the reaction is preferably adjusted to a suitable pH for the reaction by using a buffer, taking into consideration the optimal pH of the ethanolamine oxidoreductase variant, but is not limited thereto as long as the pH is operable. For example, the pH is 3 to 11, preferably 5 to 9. Examples of buffers that can be used include N-[tris(hydroxymethyl)methyl]glycine, phosphate, acetate, carbonate, tris(hydroxymethyl)-aminomethane, borate, citrate, dimethylglutamate, tricine, HEPES, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, phthalic acid, and tartaric acid.

[0091] In step S105 of detecting the reaction product, the reaction product can be detected by selecting from two detection methods having different measurement principles. In step S105A, the mediator is reduced by an ethanolamine oxidoreductase variant, and the reduced mediator is reacted with a color-developing or color-fading reagent. In addition to DCIP (2,6-dichlorophenolindophenol), examples of the color-developing or color-fading substrate used in this embodiment include tetrazolium compounds (Tetrazolium blue, Nitro-tetrazolium blue, Water soluble tetrazolium (WST)-1, WST-3, WST-4, WST-5, WST-8, WST-9), etc.

[0092] In step S105B using a different measurement principle, hydrogen peroxide is generated by a modified ethanolamine oxidoreductase, and the hydrogen peroxide generated is reacted with a color-developing reagent by utilizing a catalytic reaction of peroxidase. The color-developing or color-fading substrates used in the present invention include color-developing agents such as ADPS (N-ethyl-N-sulfopropyl-3-methoxyaniline), ALPS (N-ethyl-N-sulfopropylaniline), TOPS (N-ethyl-N-sulfopropyl-3-methylaniline), ADOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), DAOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline), and ALOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline). , HDAOS (N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline), MAOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline), TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine), DA-64 (N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine), etc. ADOS, ALOS, and TOOS develop color when condensed with 4-aminoantipyrine. DA-64 and DA-67 do not require 4-aminoantipyrine and will develop color when formulated alone.Further, color-developing agents such as methylene blue, 10-(acetylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, 10-(phenylcarbonyl)-3,7-bis(dimethylamino)phenothiazine, 10-(3-(methylcarboxyamino)-hexamethylamino)-phenothiazine, 10-(3-(methylcarboxyamino)-4-methylphenyl)- ... Examples of the color-developing agent include, but are not limited to, 0-((3-(methylcarboxyaminomethyl)-phenyl)-methylamino)-phenothiazine, 10-(1-naphthaleneamino)-phenothiazine, 10-(methyl)-phenothiazine, 10-(phenylamino)-phenothiazine, 10-(methylamino)-phenothiazine, Azure A, Azure B, Azure C, Toluidine Blue O, 1,9-dimethyl-3,7-bis(dimethylamino)phenothiazine salt, methylene green or a salt thereof, or a leuco form thereof. The color-developing agent may be added so that the final concentration in the reaction solution is 0.001 mM to 10 mM, for example, 0.005 mM to 2 mM.

[0093] In step S107, the amount of EAP in the sample is determined from the detected value of the reaction product using a calibration curve prepared in advance. In this manner, the amount of EAP in the sample can be quantified.

[0094] The sample used in the method for quantifying EAP according to the present invention may be any biological sample that may contain EAP, such as a sample derived from blood, plasma, interstitial fluid, urine, tears, sweat, saliva, etc. The sample may be appropriately processed. For example, the sample may be concentrated using a centrifugal concentrator.

[0095] In such a quantitative determination method, if a sample containing EAP is contaminated with other substances other than EAP that can be acted on by the ethanolamine oxidoreductase modified to quantify EAP, there is a risk of a positive error in the measured value. In particular, the higher the rate at which the ethanolamine oxidoreductase modified acts on such contaminating substances, the less accurately the EAP, which is the intended quantitative target substance, can be quantified.

[0096] In one embodiment, the sample may contain at least one amine other than EAP. In general, clinical specimens may contain amines such as EA, β-alanine, and taurine as impurities. In the method for quantifying EAP according to the present invention, since a modified ethanolamine oxidoreductase is used, if these amines are contained in the sample, the modified ethanolamine oxidoreductase will also act on these amines, making it difficult to accurately quantify the EA derived from the EAP that should be measured. For this reason, it is preferable to decompose or eliminate amines other than EAP contained in the sample before quantifying EAP.

[0097] In one embodiment, it is preferable to allow at least an oxidoreductase acting on amines to act on amines contained in the sample before allowing phosphatase to act on the sample. In this embodiment, a modified ethanolamine oxidoreductase can be used as at least an oxidoreductase acting on amines. First, the modified ethanolamine oxidoreductase is added to the sample as an oxidoreductase acting on amines, and amines contained in the sample are decomposed or eliminated (step S201). Note that the modified ethanolamine oxidoreductase does not act on EAP, but acts only on amines. Therefore, in step S201, EAP contained in the sample is not decomposed or eliminated. A phosphatase is added to the sample, and allowed to act on EAP to convert it into EA (step S203). The modified ethanolamine oxidoreductase added in step S201 acts on the converted EA. A reaction product generated by this reaction is detected (step S205). Using a previously prepared calibration curve, the EAP is quantified from the detected value of the reaction product (step S207).

[0098] In step S201, an ethanolamine oxidoreductase variant is first added to the sample as an oxidoreductase that acts on at least amines, and the amines contained in the sample are decomposed or eliminated by the ethanolamine oxidoreductase variant. The degree of influence of error-causing substances in the sample on the measured value in the EAP quantification method according to the present embodiment depends on the amount of amines such as free EA contained in the sample. When measuring a sample containing a small amount of amines such as free EA, step S201 for eliminating amines such as free EA in advance is not necessary, and phosphatase can be directly applied to the sample. In this case, it can be assumed that all EA generated in the sample is cleaved from EAP in the sample by the action of phosphatase.

[0099] On the other hand, when a sample containing free amines such as EA is measured, it becomes impossible to distinguish between EA liberated from the EAP, which is the target substance to be quantified, by the action of phosphatase, resulting in measurement errors. In order to avoid such measurement errors, it is preferable to eliminate free amines such as EA from the sample in advance.

[0100] When an ethanolamine oxidoreductase variant is selected that has high specificity for EA and has substantially no effect on EAP, EAP, which is the target substance to be quantified, will not be eliminated in step S201. However, if the nature of the ethanolamine oxidoreductase variant used is such that EAP, which is the target substance to be quantified, may be eliminated in step S201, erroneous elimination of EAP can be avoided by performing step S201 under conditions of a concentration at which EAP is not eliminated and only amines such as EA are eliminated.

[0101] In step S203, phosphatase is added to the sample to act on the EAP and convert it into EA. Step S203 is the same as step S101 described above, and therefore a detailed description thereof will be omitted.

[0102] In step S205, the modified ethanolamine oxidoreductase added in step S201 acts on the EA converted in step S203. The reaction product produced by this reaction can be detected. Step S205 is similar to step S103 except that the modified ethanolamine oxidoreductase already added acts on EA, and is similar to step S105 in that the reaction product produced in the redox reaction is detected, so a detailed description will be omitted.

[0103] In step S207, the EAP is quantified from the detection value of the reaction product using a calibration curve prepared in advance. Step S207 is the same as step S107, and therefore a detailed description thereof will be omitted.

[0104] In the above embodiment, the action of the ethanolamine oxidoreductase variant added in the amine elimination step is directly utilized, but the EAP quantification method according to the present invention is not limited thereto. For example, a separate oxidoreductase may be added in the amine elimination step, and the ethanolamine oxidoreductase variant may be further added in the EAP quantification step.

[0105] First, an oxidoreductase different from the modified ethanolamine oxidoreductase is added to a sample to decompose or eliminate amines contained in the sample (step S301). A phosphatase is added to the sample to act on EAP and convert it to EA (step S303). The modified ethanolamine oxidoreductase is added to the sample to act on the converted EA (step S305). The reaction product generated by this reaction is detected (step S307). Using a calibration curve created in advance, EAP is quantified from the detected value of the reaction product (step S309).

[0106] Step S303 may be the same as step S101 described above. Step S305 may be the same as step S103 described above. Step S307 may be the same as step S105 described above. Step S309 may be the same as step S107 described above, and detailed description thereof will be omitted.

[0107] <Phosphatase> The phosphatase that can be used in the EAP quantification method of the present invention may be any phosphatase that can be used in clinical tests and can effectively cleave EA from EAP in the sample to be treated. Examples of such phosphatases include acid phosphatase, alkaline phosphatase, and protein phosphatase. The above phosphatases may be used alone or in combination of two or more kinds.

[0108] In the present invention, when selecting a phosphatase, an enzyme can be selected from the viewpoint of efficiently cleaving EA from EAP in a sample while suppressing the generation of contaminants as much as possible. That is, various compounds with added phosphate other than the object to be measured contained in the sample are predicted as contaminants, and the generation of contaminants can be suppressed by selecting a type of phosphatase that is unlikely to generate them. A phosphatase can be selected based on knowledge regarding what types of bonds between structures are easily cleaved by various known phosphatases.

[0109] In fact, the EAP quantification method of the present invention can also be controlled by selecting the substrate specificity of the modified ethanolamine oxidoreductase in the step of action by the modified ethanolamine oxidoreductase following cleavage by phosphatase, and such selection is expected to reduce the influence of erroneous reactions.

[0110] The conditions for treating the sample with phosphatase may be any conditions as long as the phosphatase used acts on EAP and efficiently releases EA in a short time. The amount of phosphatase used is appropriately selected depending on the content of EAP in the sample or the treatment conditions, and as an example, phosphatase can be added to a final concentration of 0.001 U / ml to 10000 U / ml, preferably 0.01 U / ml to 1000 U / ml. If necessary, other enzymes may be added appropriately.

[0111] The activity of the phosphatase usable in the present invention is calculated by measuring the amount of 4-nitrophenol (maximum absorption wavelength 405 nm) generated by hydrolysis of 4-nitrophenyl phosphate using a spectrophotometer. As an example, when alkaline phosphatase (ALP) is used, the amount of enzyme that hydrolyzes 1 μmole of 4-nitrophenyl phosphate per minute at 37° C. and pH 9.8 (diethanolamine buffer) is defined as 1 U.

[0112] <Oxidoreductase> The oxidoreductase different from the present ethanolamine oxidoreductase variant is preferably an oxidoreductase having high specificity for EA and substantially no effect on EAP. For example, examples of oxidoreductases belonging to EC number 1.4 or EC number 1.5 include primary amine dehydrogenase, monoamine dehydrogenase, diamine dehydrogenase, polyamine dehydrogenase, ethanolamine dehydrogenase, tyramine dehydrogenase, phenylethylamine dehydrogenase, benzylamine dehydrogenase, histamine dehydrogenase, serotonin dehydrogenase, spermine dehydrogenase, spermidine dehydrogenase, β-alanine dehydrogenase, γ-aminobutyric acid (GABA) dehydrogenase, taurine dehydrogenase, cadaverine dehydrogenase, and agmatine dehydrogenase.

[0113] In one embodiment, oxidases belonging to EC number 1.4.3 or EC number 1.5.3 can be used, such as primary amine oxidase, monoamine oxidase, diamine oxidase, polyamine oxidase, tyramine oxidase, phenylethylamine oxidase, benzylamine oxidase, histamine oxidase, serotonin oxidase, spermine oxidase, spermidine oxidase, β-alanine oxidase, γ-aminobutyric acid (GABA) oxidase, taurine oxidase, cadaverine oxidase, and agmatine oxidase. In particular, phenylethylamine oxidase (PEAOX) can be preferably used.

[0114] The reaction conditions of the oxidoreductase according to the present embodiment may be any conditions as long as it acts on amines and efficiently catalyzes the oxidation reaction. However, enzymes generally have optimum temperature and optimum pH at which they exhibit the highest activity. Therefore, the reaction conditions are preferably near the optimum temperature and optimum pH. For example, the reaction conditions of the oxidoreductase may be preferably, but are not limited to, a temperature of 30°C and a pH of 8.5. For example, the reaction conditions of the PEAOX may be preferably, but are not limited to, a temperature of 37°C and a pH of 8.5, which will be described later.

[0115] (Quantification of EAP using the oxidase activity of modified ethanolamine oxidoreductase) In one embodiment, EAP in a sample can be measured using the oxidase activity of the ethanolamine oxidoreductase variant. More specifically, the concentration of EAP can be determined by allowing the ethanolamine oxidoreductase variant to act on EA and quantifying the amount of hydrogen peroxide produced or the amount of oxygen consumed.

[0116] First, phosphatase is added to a sample, and EAP is reacted with the phosphatase to convert it to EA (step S101). The present modified ethanolamine oxidoreductase is added to the sample, and the modified ethanolamine oxidoreductase is reacted with the converted EA (step S103). Hydrogen peroxide produced by this reaction is detected (step S105). Using a calibration curve prepared in advance, EAP is quantified from the detected value of hydrogen peroxide (step S107).

[0117] Step S101 has been described above, and detailed description thereof will be omitted. In step S103, the EAP concentration in the sample is not particularly limited, but may be, for example, 0.01 μM to 10,000 μM. The reaction time may be, for example, 5 seconds to 180 minutes, preferably 0.5 minutes to 60 minutes, more preferably 1 minute to 30 minutes, and even more preferably 1 minute to 10 minutes. The reaction temperature varies depending on the optimum temperature of the enzyme used, but is, for example, 20° C. to 45° C., and may be appropriately selected from temperatures used in ordinary enzyme reactions.

[0118] The preferred amount of the ethanolamine oxidoreductase variant to be used is, for example, 0.001 U / ml to 500 U / ml, preferably 0.01 U / ml to 100 U / ml in final concentration. In general, the lower the concentration of the substrate contained in the sample solution, the higher the final concentration of the ethanolamine oxidoreductase variant to be added. The pH when the ethanolamine oxidoreductase variant is allowed to act is preferably adjusted using a buffer to a pH suitable for the reaction, taking into consideration the optimal pH of the ethanolamine oxidoreductase variant, but is not limited thereto as long as the pH is operable. The pH when the ethanolamine oxidoreductase variant is allowed to act is, for example, pH 3 to pH 11, preferably pH 5 to pH 9. The buffer that can be used is the same as that when EAP is quantified based on the reductase activity of the ethanolamine oxidoreductase variant.

[0119] In this embodiment, a method for measuring EAP is provided by measuring products or products consumed by the oxidase activity of an ethanolamine oxidoreductase variant acting on EA. Hydrogen peroxide is an easy-to-measure product and is a preferred measurement target. Hydrogen peroxide produced by the action of an ethanolamine oxidoreductase variant may be detected using a chromogenic substrate, etc. Examples of the chromogenic substrate used in the present invention include, in addition to 4-aminoantipyrine, ADOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), ALOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline), TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine), DA-64 (N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine), etc. ADOS, ALOS, and TOOS develop color when condensed with 4-aminoantipyrine. DA-64 and DA-67 do not require 4-aminoantipyrine and will develop color when formulated alone. In either case, the color reaction is catalyzed by peroxidase. Dissolved oxygen can be measured as a consumable, and the amount of dissolved oxygen in the reaction solution can be measured using a dissolved oxygen meter or the like. For example, the degree of color development (change in absorbance) of the above-mentioned measurement reagent can be measured using a spectrophotometer or an automatic biochemical analyzer, and compared with the absorbance of a standard sample to measure the EAP contained in the sample.

[0120] In step S107, the amount of EAP in the sample is determined from the detected value of the reaction product using a calibration curve prepared in advance. In this manner, the amount of EAP in the sample can be quantified.

[0121] Examples of samples used in the EAP quantification method according to the present invention include any biological sample that may contain EAP, such as samples derived from blood, plasma, etc. The sample may be appropriately processed. For example, the sample may be concentrated using a centrifugal concentrator.

[0122] However, in such a quantitative determination method, if a sample containing EAP is contaminated with other substances other than EAP that can be acted on by the ethanolamine oxidoreductase modified to quantify EAP, there is a risk of a positive error in the measured value. In particular, the higher the rate at which the ethanolamine oxidoreductase modified acts on such contaminating substances, the more difficult it becomes to accurately quantify EAP, which is the substance to be quantified.

[0123] In one embodiment, the sample may contain at least one amine other than EAP. Generally, clinical specimens may contain amines such as EA, β-alanine, and taurine as impurities. In the method for quantifying EAP according to the present invention, a modified ethanolamine oxidoreductase acting on EA is used, and if these amines are contained in the sample, the modified ethanolamine oxidoreductase also acts on these amines, making it difficult to accurately quantify the EA derived from the EAP that should be measured. For this reason, it is preferable to decompose or eliminate amines other than EAP contained in the sample before quantifying EAP.

[0124] In one embodiment, it is preferable to allow at least an oxidase acting on amines to act on amines contained in the sample before allowing the sample to act with phosphatase. In this embodiment, a modified ethanolamine oxidoreductase can be used as at least an oxidase acting on amines. First, the modified ethanolamine oxidoreductase is added to the sample as an oxidase acting on amines to decompose or eliminate amines contained in the sample (step S201). Note that the modified ethanolamine oxidoreductase does not act on EAP, but acts only on amines. Therefore, in step S201, the EAP contained in the sample is not decomposed or eliminated. A phosphatase is added to the sample to act on the EAP to convert it into EA (step S203). The modified ethanolamine oxidoreductase added in step S201 acts on the converted EA. Hydrogen peroxide produced by this reaction is detected (step S205). Using a calibration curve created in advance, EAP is quantified from the detected value of hydrogen peroxide (step S207).

[0125] In step S201, an ethanolamine oxidoreductase variant is first added to the sample as an oxidase that acts on at least amines, and the amines contained in the sample are decomposed or eliminated by the ethanolamine oxidoreductase variant. The degree of influence of error-causing substances in the sample on the measured value in the EAP quantification method according to the present embodiment depends on the amount of free EA contained in the sample. When measuring a sample containing a small amount of free EA, step S201 of eliminating free EA in advance is not necessary, and phosphatase can be directly applied to the sample. In that case, it can be assumed that all EA generated in the sample is cleaved from the EAP in the sample by the action of phosphatase.

[0126] On the other hand, when measuring a sample containing free EA, it becomes impossible to distinguish between the EA released from the EAP, which is the target substance to be quantified, by the action of phosphatase, resulting in measurement errors. In order to avoid such measurement errors, it is preferable to eliminate the free EA in the sample in advance.

[0127] When an ethanolamine oxidoreductase variant is selected that has high specificity for EA and has substantially no effect on EAP, EAP, which is the target substance to be quantified, will not be eliminated in step S201. However, if the nature of the ethanolamine oxidoreductase variant used is such that EAP, which is the target substance to be quantified, may be eliminated in step S201, erroneous elimination of EAP can be avoided by performing step S201 by setting a condition concentration at which only EA is eliminated but not EAP.

[0128] In step S203, phosphatase is added to the sample to act on the EAP and convert it into EA. Step S203 is the same as step S101 described above, and therefore a detailed description thereof will be omitted.

[0129] In step S205, the modified ethanolamine oxidoreductase added in step S201 acts on the EA converted in step S203. The reaction product produced by this reaction can be detected. Step S205 is similar to step S103 except that the modified ethanolamine oxidoreductase already added acts on EA, and is similar to step S105 in that the reaction product produced in the redox reaction is detected, so a detailed description will be omitted.

[0130] In step S207, the EAP is quantified from the detection value of the reaction product using a calibration curve prepared in advance. Step S207 is the same as step S107, and therefore a detailed description thereof will be omitted.

[0131] In the above embodiment, the action of the modified ethanolamine oxidoreductase added in the amine elimination step is directly utilized, but the EAP quantification method according to the present invention is not limited thereto. For example, a separate oxidase may be added in the amine elimination step, and the modified ethanolamine oxidoreductase may be further added in the EAP quantification step.

[0132] First, an oxidase different from the modified ethanolamine oxidoreductase is added to the sample to decompose or eliminate amines contained in the sample (step S301). A phosphatase is added to the sample to act on EAP and convert it to EA (step S303). The modified ethanolamine oxidoreductase is added to the sample to act on the converted EA (S305). Hydrogen peroxide produced by this reaction is detected (step S307). Using a calibration curve created in advance, EAP is quantified from the detected value of hydrogen peroxide (step S309).

[0133] Step S303 may be the same as step S101 described above. Step S305 may be the same as step S103 described above. Step S307 may be the same as step S105 described above. Step S309 may be the same as step S107 described above, and detailed description thereof will be omitted.

[0134] <Oxidase> The oxidase different from the present ethanolamine oxidoreductase variant is preferably an oxidase that has high specificity for EA and has substantially no effect on EAP, such as phenylethylamine oxidase (PEAOX), ethanolamine oxidase, tyramine oxidase, benzylamine oxidase, histamine oxidase, serotonin oxidase, spermine oxidase, spermidine oxidase, β-alanine oxidase, γ-aminobutyric acid (GABA) oxidase, taurine oxidase, cadaverine oxidase, agmatine oxidase, etc.

[0135] The reaction conditions of the oxidase used in this embodiment may be any conditions that act on amines and efficiently catalyze the oxidation reaction. However, enzymes generally have optimal temperatures and optimal pHs at which they exhibit the highest activity. Therefore, the reaction conditions are preferably near the optimal temperature and optimal pH. For example, the reaction conditions of PEAOX can be preferably, but are not limited to, a temperature of 37°C and a pH of 8.5. EXAMPLES

[0136] (Construction of plasmids for expression of LrEAOX mutants) The gene (SEQ ID NO: 2) encoding Lichtheimia ramosa-derived ethanolamine oxidase (LrEAOX) (SEQ ID NO: 1) was cloned into the plasmid vector pKK223-3, and the LrEAOX expression plasmid was named pKK223-3-LrHP (see Patent Document 4). The expression plasmids of the LrEAOX mutants were prepared by PCR using pKK223-3-LrHP as a template and two types of primers. The combinations of primers used to prepare each mutant are shown in Table 1.

[0137] [Table 1]

[0138] For PCR, KOD One PCR Master Mix (Toyobo Co., Ltd.) was used, and PCR conditions were determined according to the three-step cycle in the attached manual. The number of PCR cycles was 7. 1.0 μl of DpnI (New England Biolab) was added to the solution after PCR and treated at 37°C for 1 hour. 2.5 μl of Ligation high Ver.2 (Toyobo Co., Ltd.), 0.5 μl of T4 polynucleotide Kinase (5 U / μl) (Toyobo Co., Ltd.), and 3.5 μl of ultrapure water were added to 1.0 μl of the reaction solution treated with DpnI, and treated at 16°C for 1 hour. E. coli JM109 strain was transformed with the obtained plasmid and plated on LB agar medium containing ampicillin. The grown colony was inoculated into LB liquid medium (LB-amp medium) containing 50 μg / ml of ampicillin, cultured with shaking, and the plasmid was isolated using FastGene Plasmid Mini Kit (Nihon Genetics Co., Ltd.). The nucleotide sequence of the region encoding the LrEAOX mutant in the isolated plasmid was analyzed by FASMAC Co., Ltd., and it was confirmed that the plasmid for expressing the desired mutant had been correctly constructed.

[0139] (Recombinant production of wild-type LrEAOX and LrEAOX mutants) The LrEAOX mutant recombinant production strain, which was prepared by transforming E. coli BL21 strain with the wild-type LrEAOX or LrEAOX mutant expression plasmid, was inoculated into 2.5 ml of LB-amp medium (ampicillin concentration 50 μg / ml) and cultured overnight at 37°C and 180 rpm in a test tube. 0.5 ml of the seed culture was inoculated into 50 ml of LB-amp medium containing 0.02 mM CuSO4 and 0.1 mM IPTG in a Sakaguchi flask and cultured at 25°C and 130 rpm for 20 hours.

[0140] (Preparation of crude enzyme solutions of wild-type LrEAOX and LrEAOX mutants) 50 ml of the culture medium was centrifuged at 6,500 × g for 10 minutes, and the pellet was resuspended in 4 ml of Tris-HCl buffer (pH 7.5). The bacterial suspension was ultrasonically disrupted, and then centrifuged at 20,400 × g for 15 minutes to recover the supernatant, which was used as a crude enzyme solution.

[0141] (Anion exchange chromatography of wild-type LrEAOX and LrEAOX mutants) A crude enzyme solution of wild-type LrEAOX or an LrEAOX mutant was applied to HiScreen (registered trademark) Q-FF (Cytiva, resin amount 4.7 ml) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) to bind to the anion exchange resin.

[0142] The resin was then washed with 47 ml (10 CV) of 20 mM Tris-HCl buffer (pH 7.5), and the NaCl concentration in 20 mM Tris-HCl buffer (pH 7.5) was linearly increased from 0 mM to 130 mM while pumping 92 ml (19.5 CV) to elute the LrEAOX mutant bound to the resin.

[0143] (Size exclusion chromatography of wild-type LrEAOX and LrEAOX mutants) The eluted fraction was concentrated using Amicon (registered trademark) Ultra Ultracel-30K and purified using a HiLoad (registered trademark) 26 / 60 Superdex 200 column. The resin was equilibrated and eluted with 20 mM Bis-Tris-HCl buffer (pH 7.0) containing 150 mM NaCl.

[0144] The purity of each eluted fraction was evaluated by SDS-PAGE, and fractions free of contaminating proteins were collected to prepare purified preparations of wild-type LrEAOX or LrEAOX mutants.

[0145] (In vitro holoconjugation of EAOX) It is known that the specific activity of copper amine oxidase including EAOX is increased by incubating it with copper ions in vitro, whereby the tyrosine in the active site is converted to TPQ (holo conversion) (Matsuzaki et al., Biochemistry (1995) 34, 4524-4530). LrEAOX was mixed with CuSO4 and a buffer solution to the composition shown in Table 2 to prepare a holo conversion treatment mixture. The mixture was incubated at 20°C for 0, 16, 40, and 64 hours. The specific activity of LrEAOX treated for each time was calculated according to the following activity measurement method in accordance with the description in Patent Document 3.

[0146] [Table 2]

[0147] (Activity evaluation of LrEAOX mutants) The following reagents were used: Reagents: 4-aminoantipyrine (4-AA) (Fujifilm Wako Pure Chemical Industries, Ltd.), horseradish peroxidase (POD) (Toyobo Co., Ltd.), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) (Dojindo Laboratories, Ltd.), ethanolamine (EA) (Tokyo Chemical Industry Co., Ltd.)

[0148] 22 μl of LrEAOX solution was added to 616 μl of activity measurement reagent (161 mM bicine-NaOH (pH 8.07.5), 0.48 mM 4-AA, 4.8 U / ml POD, 0.54 mM TOOS) and incubated at 37°C for 2 minutes, after which 22 μl of substrate solution (150 mM ethanolamine EA) was added and mixed, and the A per 5 minutes at 37°C was measured using a spectrophotometer (U-3900, Hitachi High-Tech Science Corporation). 555 Amount of change (ΔA S ) was measured.

[0149] The specific activity of LrEAOX was calculated using the following formula. U / mg = (ΔA S×df×1.53) / LrEAOX concentration (mg / ml) df; dilution factor The concentration of LrEAOX is A 280 was measured and calculated as follows: mg / mL=A 280 ×df / 1.339 df: dilution rate 1.339: Absorbance of EAOX 1 g / l at 280 nm wavelength

[0150] (Test results: Evaluation of the effect of improving specific activity by holoprocessing of LrEAOX) Figure 4 shows the relationship between the length of holoconversion time and the specific activity of LrEAOX after in vitro holoconversion for 16, 40, and 64 hours. The specific activity of LrEAOX was improved by holoconversion, and compared with 0.35 U / mg without holoconversion, the specific activity was 1.5 times higher after 16 hours, 1.9 times higher after 40 hours, and 2.1 times higher after 64 hours. This confirmed that the inactivation of LrEAOX was improved by holoconversion, and that the degree of improvement in specific activity changed gradually over time with the duration of holoconversion, with only a slight improvement after 40 hours.

[0151] Based on these results, in the subsequent performance evaluation of various LrEAOX mutants, the holoconversion mixture prepared using various LrEAOX mutants instead of unmutated LrEAOX was incubated at 20°C for 40 hours to perform holoconversion, and then the specific activity was calculated using the activity measurement method and specific activity calculation method described above.

[0152] (Test results: Evaluation of the specific activity improvement effect of various LrEAOX mutants) The specific activities of the various LrEAOX mutants listed in Table 1 were calculated using the above-mentioned holoconversion conditions, activity measurement method, and specific activity calculation method, and the results are shown in Table 3.

[0153] [Table 3]

[0154] The specific activity of wild-type LrEAOX before holoconversion was 0.35, but after holoconversion at 20°C for 40 hours, the specific activity of wild-type LrEAOX was 0.66, which was a 1.9-fold increase in specific activity. In addition to the above-mentioned holoconversion treatment, mutants in which the amino acid at position 331 was substituted with an amino acid other than cysteine ​​showed a significant increase in specific activity compared to the wild-type. In mutants in which cysteine ​​at position 338 was substituted, the specific activity was increased by 1.3 to 2.4 times. Furthermore, a further increase in specific activity was confirmed in mutants in which a mutation was introduced into cysteine ​​at position 331 in addition to cysteine ​​at position 338. Among these, the effect was remarkable in Cys331Ala / Cys338Ala, Cys331Lys / Cys338Ala and Cys331Ala / Cys338Lys, with specific activity improved by 2.6-fold, 2.9-fold and 4.2-fold, respectively, compared to the wild type. [Explanation of symbols]

[0155] 1 electrode, 1a wiring, 1b upper electrode, 2 working electrode, 3 counter electrode, 5 reference electrode, 7 wiring section, 9 terminal, 10 sensor chip, 11 substrate, 13 spacer, 15 cover, 19 reaction layer, 30 measuring section, 31 switch, 33 display, 100 sensor, 110 control section, 120 display section, 130 input section, 140 memory section, 150 communication section, 160 power supply, 190 wiring

Claims

1. Contains copper ions, A modified ethanolamine oxidoreductase in which the amino acid at position 338 of the amino acid sequence of SEQ ID NO:1 is substituted with alanine, glutamic acid or lysine.

2. The ethanolamine oxidoreductase variant according to claim 1 , wherein the amino acid at position 331 of the amino acid sequence of SEQ ID NO:1 is substituted with alanine, glutamic acid, or lysine.

3. The ethanolamine oxidoreductase variant according to claim 1 or 2, further comprising topaquinone.

4. The ethanolamine oxidoreductase variant according to claim 1 ; a mediator that is reduced by adding the ethanolamine oxidoreductase variant; and a reagent which reacts with the reduced mediator.

5. The ethanolamine oxidoreductase variant according to claim 1 ; and a reagent that reacts with hydrogen peroxide generated by adding the modified ethanolamine oxidoreductase.

6. The composition for quantifying ethanolamine phosphate according to claim 4 or 5, further comprising a phosphatase.

7. The ethanolamine oxidoreductase variant according to claim 1 ; a mediator that is reduced by adding the ethanolamine oxidoreductase variant; and a reagent that reacts with the reduced mediator.

8. The ethanolamine oxidoreductase variant according to claim 1 ; and a reagent that reacts with hydrogen peroxide generated by adding the modified ethanolamine oxidoreductase.

9. The kit for quantifying ethanolamine phosphate according to claim 7 or 8, further comprising a phosphatase.

10. An electrode comprising the ethanolamine oxidoreductase variant of claim 1.

11. A working electrode, a counter electrode, and a reference electrode; A reaction layer disposed on the working electrode, the counter electrode, and the reference electrode, The working electrode comprises the electrode according to claim 10 , or A sensor chip, wherein the reaction layer comprises the ethanolamine oxidoreductase variant according to claim 1.

12. A sensor comprising the sensor chip according to claim 11.

13. A phosphatase is allowed to act on the sample to convert ethanolamine phosphate contained in the sample into ethanolamine; A method for quantifying ethanolamine phosphate, comprising reacting the modified ethanolamine oxidoreductase of claim 1 with ethanolamine.

14. the modified ethanolamine oxidoreductase is allowed to act on the ethanolamine to reduce a mediator; The method for quantifying ethanolamine phosphate according to claim 13, further comprising reacting the reduced mediator with a reagent to determine the concentration of ethanolamine phosphate.

15. allowing the modified ethanolamine oxidoreductase to act on the ethanolamine; 14. The method of claim 13, further comprising determining the concentration of ethanolamine phosphate by quantifying hydrogen peroxide produced or oxygen consumed.

16. the sample comprises at least one amine other than the ethanolamine phosphate; The method for quantifying ethanolamine phosphate according to claim 13, further comprising the step of reacting the at least one amine with an amine oxidoreductase prior to reacting the sample with the phosphatase.

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