Quantification method of 25-hydroxyvitamin D3, composition for quantification, kit for quantification, electrode, sensor chip, and sensor

Abstract

Provided are a new method for quantifying vitamin D derivatives, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor. Provided is a method for quantifying vitamin D derivatives, the method including adding an oxidoreductase into a sample. The concentration of vitamin D derivatives may be determined by reducing a mediator through the addition of the oxidoreductase and then reacting the reduced mediator with a reagent. The oxidoreductase is an oxidase, and the concentration of vitamin D derivatives may be determined by quantifying the hydrogen peroxide produced through the addition of the oxidase or quantifying the consumed oxygen. In addition, the concentration of vitamin D derivatives may be determined by reacting a reagent and the hydrogen peroxide produced through the addition of the oxidase.

Description

Technical Field

[0001] The present invention relates to a method for quantifying vitamin D derivatives, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor.

Background Art

[0002] Vitamin D is a physiologically active substance that acts on various biological processes in vivo. Vitamin D is divided into vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). The sources of vitamin D are biosynthesis of vitamin D3 in the skin and ingestion of vitamin D2 and vitamin D3 from food, supplements, etc. Here, since vitamin D2 and vitamin D3 undergo the same metabolism and act, they are referred to as vitamin D when not distinguishing between vitamin D2 and vitamin D3.

[0003] Vitamin D taken into the body is hydroxylated in the liver and converted to 25-hydroxyvitamin D, which is stored in hepatocytes. 25-Hydroxyvitamin D binds to vitamin D-binding protein and is released into the blood. Since the blood half-life of 25-hydroxyvitamin D is as long as about 2 to 3 weeks, the blood concentration of 25-hydroxyvitamin D is considered to be an index reflecting the vitamin D concentration in the body.

[0004] 25-hydroxyvitamin D in the blood is taken into cells by endocytosis via megalin receptors. 25-hydroxyvitamin D transported to the renal tubules of the kidney is hydroxylated and converted to the active form, 1,25-dihydroxyvitamin D. 1,25-dihydroxyvitamin D released back into the blood binds to vitamin D receptors in target cells and acts as a transcription factor that controls the expression of various types of genes related to calcium transport and utilization. The blood half-life of 1,25-dihydroxyvitamin D is short, about 15 hours, and the blood concentration of 1,25-dihydroxyvitamin D is tightly regulated by parathyroid hormone, calcium, and phosphate. Therefore, it is considered that the blood concentration of 1,25-dihydroxyvitamin D does not change unless there is extreme vitamin D deficiency or excess.

[0005] Vitamin D plays an essential role in controlling calcium and phosphate concentrations in the body. Vitamin D mainly affects intestinal cells and osteoblasts, assisting in controlling calcium uptake in the former and controlling bone formation and maintenance in the latter. It is also known that vitamin D is involved in cell growth and differentiation and the immune system. Vitamin D deficiency or excess can have various consequences for the body. In particular, vitamin D deficiency has been pointed out as potentially leading to serious diseases such as rickets, osteomalacia, osteoporosis, chronic kidney disease, hyperparathyroidism, and psoriasis.

[0006] As criteria for determining vitamin D deficiency or insufficiency, a serum 25-hydroxyvitamin D concentration of 30 ng / ml or more is considered sufficient vitamin D status, 20 ng / ml or more but less than 30 ng / ml is considered vitamin D insufficiency, and less than 20 ng / ml is considered vitamin D deficiency (based on the guidelines for the determination of vitamin D deficiency / insufficiency by the Japanese Society for Bone and Mineral Research and the Japanese Society of Endocrinology). The number of patients with vitamin D deficiency is currently estimated to be 1 billion worldwide. In particular, early detection of vitamin D deficiency is useful in modern society where people tend to avoid direct sunlight. In Japan, electrochemical luminescence immunoassay (ECLIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), etc. for 25-hydroxyvitamin D in serum are covered by insurance. These are all immunological assays using anti-25-hydroxyvitamin D antibodies.

[0007] For example, Patent Document 1 discloses an antibody that recognizes 25-hydroxyvitamin D or its antigen-binding fragment, and a method for measuring 25-hydroxyvitamin D using the same. In the measurement of 25-hydroxyvitamin D using the anti-25-hydroxyvitamin D antibody, since the detection limit of 25-hydroxyvitamin D is less than 3.0 ng / ml and the sensitivity is high, it is considered useful for the early diagnosis of vitamin D deficiency. However, since it is an assay method using an immunological assay, there are problems such as a long measurement time and high assay costs.

[0008] Therefore, a method for measuring 25-hydroxyvitamin D itself rather than an immunological assay is desired. For example, Patent Document 2 discloses a method for measuring 25-hydroxyvitamin D by competitive binding to vitamin D-binding protein using 25-hydroxyvitamin D labeled with biotin or fluorescein. Patent Document 3 discloses a method for measuring 25-hydroxyvitamin D using high-performance liquid chromatography (HPLC). Patent Document 4 discloses a method for measuring 25-hydroxyvitamin D using liquid chromatography-mass spectrometry (LC / MS / MS).

Prior Art Documents

Patent Document

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, the above-described method for measuring 25-hydroxyvitamin D has various drawbacks, including a long measurement time, errors in measurement values, high costs, a large sample volume, and reagents that are difficult to handle, and is not optimal for clinical tests. Therefore, there is a need for a method for measuring 25-hydroxyvitamin D that is not very laborious, time-consuming, or costly.

[0011] One object of the present invention is to provide a new quantification method for measuring 25-hydroxyvitamin D, which is a vitamin D derivative, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor.

Means for Solving the Problems

[0012] According to one embodiment of the present invention, there is provided a method for quantifying a vitamin D derivative by adding an oxidoreductase to a sample.

[0013] By adding an oxidoreductase, a mediator may be reduced, the reduced mediator may be reacted with a reagent, and the concentration of the vitamin D derivative may be determined.

[0014] Oxidase reductase is an oxidase, and the concentration of the vitamin D derivative may be determined by quantifying the hydrogen peroxide generated or the oxygen consumed by adding the oxidase.

[0015] Oxidase reductase is an oxidase, and the hydrogen peroxide generated by adding the oxidase may be reacted with a reagent to determine the concentration of the vitamin D derivative.

[0016] According to one embodiment of the present invention, oxidase reductase used in a method for quantifying a vitamin D derivative is provided.

[0017] Oxidase reductase may be an oxidase reductase belonging to EC number 1.1.

[0018] Oxidase reductase may be an oxidase belonging to EC number 1.1.3.

[0019] According to one embodiment of the present invention, a composition for quantifying a vitamin D derivative, which contains oxidase reductase, is provided.

[0020] A mediator reduced by adding oxidase reductase, and a reagent that reacts with the reduced mediator may be further included.

[0021] Oxidase reductase is an oxidase, and may further contain a reagent that reacts with the hydrogen peroxide generated by adding the oxidase.

[0022] According to one embodiment of the present invention, a kit for quantifying a vitamin D derivative, which contains oxidase reductase, a mediator reduced by adding oxidase reductase, and a reagent that reacts with the reduced mediator, is provided.

[0023] According to one embodiment of the present invention, there is provided a kit for quantifying vitamin D derivatives, which includes an oxidoreductase and a reagent that reacts with hydrogen peroxide.

[0024] According to one embodiment of the present invention, there is provided an electrode including an oxidoreductase.

[0025] According to one embodiment of the present invention, there is provided a sensor chip having the electrode as a working electrode.

[0026] According to one embodiment of the present invention, there is provided a sensor including the sensor chip.

Advantages of the Invention

[0027] According to the present invention, there are provided a new quantification method for measuring 25-hydroxyvitamin D, which is a vitamin D derivative, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0029] Hereinafter, a new quantitative method for measuring a vitamin D derivative according to the present invention, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor will be described. However, the new quantitative method for measuring the vitamin D derivative according to the present invention, the enzyme for quantification, the composition for quantification, the kit for quantification, the electrode, the sensor chip, and the sensor are not to be construed as being limited to the descriptions of the following embodiments and examples.

[0030] In one embodiment, the oxidoreductase used in the present invention is an oxidoreductase that acts on a vitamin D derivative as a substrate. An enzyme capable of directly oxidizing or reducing a vitamin D derivative has not been identified as of the filing date of this application. As a result of investigations by the present inventors, it was first found that cholesterol oxidase (ChoF, UniprotKB Entry name Q56DL0_9MICC) derived from Arthrobacter sp. strain F2 acts on 25-hydroxyvitamin D3, which is a vitamin D derivative. Furthermore, it was found that glucose-methanol-choline family oxidoreductase (HeGMCOR, NCBI Reference Sequence: WP_094565544.1) derived from Herbaspirillum sp. strain meg3 acts on 25-hydroxyvitamin D3, which is a vitamin D derivative. In the present specification, cholesterol oxidase derived from Arthrobacter sp. strain F2 is shown and described as an example of the oxidoreductase, but the present invention is not limited thereto and may include those having a certain level or more of reactivity to vitamin D derivatives.

[0031] For example, among the oxidoreductases belonging to EC number 1.1 as oxidoreductases, an enzyme that recognizes a vitamin D derivative as a substrate and has vitamin D derivative oxidoreductase activity can be used. For example, an oxidase belonging to EC number 1.1 that recognizes a vitamin D derivative as a substrate and has vitamin D derivative oxidase activity can be used. For example, an oxidase belonging to EC number 1.1.3 that recognizes a vitamin D derivative as a substrate and has vitamin D derivative oxidase activity can be used. For example, cholesterol oxidase belonging to EC number 1.1.3.6 that recognizes a vitamin D derivative as a substrate and has vitamin D derivative oxidase activity can be used. For example, an oxidoreductase belonging to EC number 1.1 that recognizes a vitamin D derivative as a substrate and has vitamin D derivative dehydrogenase activity can be used. For example, an oxidase belonging to EC number 1.1.3 that recognizes a vitamin D derivative as a substrate and has vitamin D derivative dehydrogenase activity can be used. For example, cholesterol oxidase belonging to EC number 1.1.3.6 that recognizes a vitamin D derivative as a substrate and has vitamin D derivative dehydrogenase activity can be used.

[0032] In one embodiment, the oxidoreductase may be an oxidoreductase produced by a microorganism existing in nature or an oxidoreductase produced by a transformed microorganism. From the viewpoint of efficient mass expression of the enzyme, by using a transformed microorganism, the enzyme can be efficiently mass-expressed.

[0033] In one embodiment, the oxidoreductase may be a multimer or a monomer. For example, among several subunits constituting an oxidoreductase that is a multimer, if only a certain subunit (monomer) catalyzes a dehydrogenation reaction of taking hydrogen from a substrate and transferring it to a hydrogen acceptor, the oxidoreductase used in the present invention may be a multimer or the said subunit (monomer). Also, as long as it has vitamin D derivative oxidoreductase activity, it may be composed of a partial structure of the enzyme.

[0034] As described above, the present inventors have first found that cholesterol oxidase derived from Arthrobacter sp. strain F2 acts on 25-hydroxyvitamin D3, which is a vitamin D derivative. In one embodiment, the oxidoreductase of the present invention includes an oxidoreductase derived from Arthrobacter sp. strain F2, but also includes an oxidoreductase derived from a microorganism classified in the class Actinobacteria, and an oxidoreductase derived from a microorganism classified in the genus Arthrobacter. Also included is an oxidoreductase derived from a microorganism classified in the genus Helicobacter or the genus Pedobacter. In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from a microorganism classified in the genus Corynebacterium, the genus Rhodococcus, the genus Brevibacterium, the genus Nocardia, the genus Vitiosangium, the genus Dietzia, the genus Tomitella, the genus Actinomadura, or the genus Actinoallomurus.In one embodiment, the oxidoreductase of the present invention also includes oxidoreductases derived from microorganisms classified into the genera Amycolatopsis, Actinoplanes, Krasilnikovia, Couchioplanes, Streptosporangium, Nonomuraea, Streptacidiphilus, Nocardioides, Alloactinosynnema, Microbispora, Actinocrispum, Kutzneria, Lentzea, Kibdelosporangium, Catenulispora, Planomonospora, Dyella, Marmoricola, Actinosynnema, Prauserella, Yuhushiella.In addition, oxidoreductases having a high sequence identity (for example, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence of the oxidoreductase described in SEQ ID NO: 1, and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 can be mentioned. Further, the microorganism of the genus Arthrobacter strain F2 is cultured under predetermined conditions (for example, refer to the Journal of the Japanese Society for Bacteriology, 18(1), 1963), and an oxidase reaction reagent or dehydrogenase reaction reagent (described in detail later) containing a vitamin D derivative is mixed with the extract obtained by disrupting the cells, and the presence or absence of reactivity with the reagent is confirmed, whereby the oxidoreductase can also be screened.

[0035] In one embodiment, the present invention provides DNA encoding oxidoreductase. In one embodiment, the present invention provides DNA encoding the amino acid sequence shown in SEQ ID NO: 1, or DNA having the nucleotide sequence shown in SEQ ID NO: 2. In one embodiment, the present invention provides DNA having a nucleotide sequence having 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, and encoding a protein having oxidoreductase activity.

[0036] In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from Arthrobacter sp. strain F2, or an oxidoreductase produced by Escherichia coli transformed with a plasmid containing the oxidoreductase gene derived from Arthrobacter sp. strain F2. However, by using Escherichia coli transformed with a plasmid containing the oxidoreductase gene derived from Arthrobacter sp. strain F2, oxidoreductase can be efficiently expressed in large quantities.

[0037] The present inventors have further first found that a glucose-methanol-choline family oxidoreductase (GMC family oxidoreductase) derived from the Helvibacterium meg3 strain acts on 25-hydroxyvitamin D3, which is a vitamin D derivative. In one embodiment, the oxidoreductase of the present invention includes a GMC family oxidoreductase derived from the Helvibacterium meg3 strain, but may also be a GMC family oxidoreductase derived from a microorganism classified in the genus Pedobacter, or may be a GMC family oxidoreductase derived from a microorganism classified in the genus Pedobacter cryoconitis.In one embodiment, the oxidoreductase of the present invention also includes oxidoreductases derived from microorganisms classified in the genus Rhodococcus, Brevibacterium, Arthrobacter, Dietzia, Actinoallomurus, Nocardia, Chryseobacterium, Streptomyces, Flavobacterium, Kaistella, Ornithobacterium, Psychrobacter, Riemerella, Goodfellowiella, Lentzea, Microscilla, Hymenobacter, Amycolatopsis, Prauserella, Kribbella, Actinobacteria, Soonwooa, Elizabethkingia, Tamaricihabitans, Bizionia, Saccharopolyspora, Weeksella, Harbihabitans, Thalassolituus, Vitiosangium, Streptacidiphilus, Aquabacterium, Kutzneria, Saccharothrix, Actinokineospora.In addition, oxidoreductases having a high sequence identity (for example, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence of the oxidoreductase described in SEQ ID NO: 12, and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 12 can be mentioned. Further, a microorganism of the genus Helicobacter pylori, strain meg3, is cultured under predetermined conditions (see, for example, Journal of the Japanese Society for Bacteriology, 18(1), 1963), and an oxidase reaction reagent or dehydrogenase reaction reagent (details will be described later) containing a vitamin D derivative is mixed with the extract obtained by disrupting the cells, and the presence or absence of reactivity with the reagent is confirmed to screen for oxidoreductase.

[0038] In one embodiment, the present invention provides a DNA encoding an oxidoreductase. In one embodiment, the present invention provides a DNA encoding the amino acid sequence shown in SEQ ID NO: 12, or a DNA having the nucleotide sequence shown in SEQ ID NO: 9. In one embodiment, the present invention provides a DNA having a nucleotide sequence having 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 9, and encoding a protein having oxidoreductase activity.

[0039] In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from the meg3 strain of the genus Helicobacter pylori, or an oxidoreductase produced by Escherichia coli transformed with a plasmid containing the oxidoreductase gene derived from the meg3 strain of the genus Helicobacter pylori. However, by using Escherichia coli transformed with a plasmid containing the oxidoreductase gene derived from the meg3 strain of the genus Helicobacter pylori, the oxidoreductase can be efficiently expressed in large quantities.

[0040] In one embodiment, the reaction conditions for the oxidoreductase may be any conditions as long as they act on the vitamin D derivative and efficiently catalyze the oxidation reaction or the reduction reaction. Generally, an enzyme has an optimum temperature and an optimum pH at which it exhibits the highest activity. Therefore, the reaction conditions are preferably near the optimum temperature and the optimum pH. In one embodiment, as the reaction conditions for the oxidoreductase, the conditions are comprehensively considered based on compositions other than the enzyme, such as components of a chromogenic reagent, a mediator, a stabilizer for the enzyme, a stabilizer for the measurement sample, etc., and the compatibility with the measuring device. A method for quantifying the vitamin D derivative under conditions other than the optimum conditions for the enzyme alone is also included in the measuring method of the present invention.

[0041] [Vector] As the vector that can be used in the present invention, for example, any vector known to those skilled in the art, such as bacteriophage, cosmid, etc., can be used. Specifically, for example, pUC18 (manufactured by Takara Bio Inc.), pBluescriptII SK+ (manufactured by STRATAGENE), pET-22b(+) (manufactured by Merck), pKK223-3 (manufactured by addgene), etc. are preferable.

[0042] [Construction of expression plasmid] The plasmid for oxidoreductase expression according to the present invention is obtained by a commonly used method. For example, DNA is extracted from a microorganism that produces the oxidoreductase according to the present invention, and a DNA library is prepared. From the prepared DNA library, a DNA fragment encoding the oxidoreductase according to the present invention is identified and isolated. Using complementary primers with the isolated DNA fragment as a template, the DNA fragment is amplified by polymerase chain reaction (PCR), and the gene encoding the oxidoreductase according to the present invention is cloned. The amplified DNA fragment is ligated to a vector to obtain a plasmid having a DNA fragment encoding the oxidoreductase according to the present invention.

[0043] Alternatively, a DNA fragment encoding the oxidoreductase according to the present invention is chemically synthesized, and the DNA fragment is ligated to a vector to obtain a plasmid having the DNA encoding the oxidoreductase according to the present invention.

[0044] [Mutation treatment of oxidoreductase gene] The mutation treatment of the oxidoreductase gene can be carried out by any known method according to the intended mutation form. That is, methods that make full use of genetic engineering techniques or protein engineering techniques can be widely used.

[0045] As a method of making full use of protein engineering techniques, generally, a technique known as Site-Specific Mutagenesis can be used. For example, the Kramer method (Nucleic Acids Res., 12, 9441 (1984); Methods Enzymol., 154, 350 (1987); Gene, 37, 73 (1985)), the Eckstein method (Nucleic Acids Res., 13, 8749 (1985); Nucleic Acids Res., 13, 8765 (1985); Nucleic Acids Res., 14, 9679 (1986)), the Kunkel method (Proc. Natl. Acid. Sci. U.S.A., 82, 488 (1985); Methods Enzymol., 154, 367 (1987)), etc. can be mentioned. As a specific method for converting the base sequence in DNA, for example, the use of commercially available kits (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene, Quick Change Site Directed Mutagenesis Kit; manufactured by Stratagene, etc.) can be mentioned.

[0046] In addition, a technique known as the general PCR method (Polymerase Chain Reaction) can also be used (Technique, 1, 11(1989)). In addition to the above gene modification method, a desired modified oxidoreductase gene can also be directly synthesized by an organic synthesis method or an enzyme synthesis method.

[0047] When determining or confirming the DNA base sequence of the oxidoreductase gene obtained by the above method, for example, it can be carried out by using an Applied Biosystems 3730xl DNA analyzer (manufactured by Thermo Fisher Scientific) or the like.

[0048] [Transformation·Transduction] The oxidoreductase gene obtained as described above is incorporated into a vector such as a bacteriophage, a cosmid, or a plasmid used for transformation of prokaryotic or eukaryotic cells by a conventional method, and the host corresponding to each vector can be transformed or transduced by a conventional method. For example, using the obtained recombinant DNA, any host, for example, a microorganism belonging to the genus Escherichia, specific examples include Escherichia coli K-12 strain, preferably Escherichia coli JM109 strain, Escherichia coli DH5α strain (both manufactured by Takara Bio Inc.) and Escherichia coli B strain, preferably Escherichia coli BL21 strain (manufactured by Nippon Gene Co., Ltd.) etc. can be transformed or transduced to obtain each strain.

[0049] Also, for example, as an example of a eukaryotic host cell, yeast can be mentioned. Examples of microorganisms classified as yeast include yeast belonging to the genus Zygosaccharomyces, Saccharomyces, Pichia, Candida, etc. The inserted gene may contain a marker gene for enabling selection of the transformed cells. Examples of the marker gene include genes that complement the auxotrophy of the host, such as URA3 and TRP1. Further, it is desirable that the inserted gene contains a promoter or other control sequences (for example, a secretion signal sequence, an enhancer sequence, a terminator sequence, a polyadenylation sequence, etc.) capable of expressing the gene of the present invention in the host cell. Specific examples of the promoter include, for example, the GAL1 promoter, the ADH1 promoter, etc. As a method for transforming yeast, known methods, for example, a method using lithium acetate (Methods Mol. Cell. Biol., 5, 255-269 (1995)) or electroporation (J Microbiol Methods 55 (2003) 481-484) etc. can be preferably used, but it is not limited thereto, and transformation may be performed using various optional techniques including the spheroplast method and the glass bead method.

[0050] In addition, for example, other examples of eukaryotic host cells include filamentous fungi such as the genus Aspergillus and the genus Tricoderma. The method for producing a transformant of filamentous fungi is not particularly limited. For example, according to a conventional method, there is a method of inserting into a host filamentous fungus in a manner in which a gene encoding an oxidoreductase is expressed. Specifically, a DNA construct in which a gene encoding an oxidoreductase is inserted between an expression-inducing promoter and a terminator is prepared, and then a host filamentous fungus is transformed with the DNA construct containing the gene encoding the oxidoreductase, whereby a transformant overexpressing the gene encoding the oxidoreductase can be obtained. In the present specification, a DNA fragment consisting of an expression-inducing promoter - a gene encoding an oxidoreductase - a terminator prepared for transforming a host filamentous fungus and a recombinant vector containing the DNA fragment may be collectively referred to as a DNA construct.

[0051] The method of inserting into a host filamentous fungus in a manner in which a gene encoding an oxidoreductase is expressed is not particularly limited. For example, there are a method of directly inserting onto the chromosome of a host organism by utilizing homologous recombination, a method of introducing into a host filamentous fungus by ligating onto a plasmid vector, and the like.

[0052] In the method utilizing homologous recombination, a DNA construct can be ligated between sequences homologous to the upstream region and the downstream region of a recombination site on the chromosome and inserted into the genome of the host filamentous fungus. By overexpressing in the host filamentous fungus under the control of a high-expression promoter of the host filamentous fungus itself, a transformant by self-cloning can be obtained. The high-expression promoter is not particularly limited. For example, there are a promoter region of the TEF1 gene (tef1) which is a translation elongation factor, a promoter region of the α-amylase gene (amy), a promoter region of the alkaline protease gene (alp), and the like.

[0053] In the method using a vector, a DNA construct can be incorporated into a plasmid vector used for transformation of filamentous fungi by a conventional method, and the corresponding host filamentous fungi can be transformed by a conventional method.

[0054] Such a suitable vector-host system is not particularly limited as long as it can produce oxidoreductase in the host filamentous fungi. Examples include the pUC19 and filamentous fungi system, the pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and filamentous fungi system, and the like.

[0055] The DNA construct is preferably introduced into the chromosome of the host filamentous fungi. As another method, the DNA construct can be incorporated into a self-replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)) and used without being introduced into the chromosome.

[0056] The DNA construct may contain a marker gene for enabling selection of the transformed cells. The marker gene is not particularly limited, and examples include genes that complement the auxotrophy of the host, such as pyrG, niaD, adeA, and drug resistance genes against drugs such as pyrithiamine, hygromycin B, and oligomycin. In addition, the DNA construct preferably contains a promoter, a terminator, and other regulatory sequences (such as enhancers, polyadenylation sequences, etc.) that enable overexpression of the gene encoding oxidoreductase in the host cell. The promoter is not particularly limited, and examples include appropriate expression-inducing promoters and constitutive promoters, such as the tef1 promoter, alp promoter, amy promoter, and the like. The terminator is also not particularly limited, and examples include the alp terminator, amy terminator, tef1 terminator, and the like.

[0057] In a DNA construct, the expression control sequence of the gene encoding the oxidoreductase is not necessarily required when the DNA fragment containing the gene encoding the oxidoreductase to be inserted contains a sequence having an expression control function. Also, when performing transformation by the co-transformation method, the DNA construct may not have a marker gene in some cases.

[0058] One embodiment of the DNA construct is, for example, a DNA construct in which a tef1 promoter, a gene encoding an oxidoreductase, an alp terminator, and a pyrG marker gene are ligated to the In-Fusion Cloning Site in the multi-cloning site of pUC19.

[0059] As a method for transforming filamentous fungi, a method known to those skilled in the art can be appropriately selected. For example, after preparing protoplasts of the host filamentous fungus, the protoplast PEG method using polyethylene glycol and calcium chloride (see, for example, Mol. Gen. Genet. 218, 99-104, 1989, JP-A-2007-222055, etc.) can be used. The medium for regenerating the transformed filamentous fungus is appropriately selected according to the host filamentous fungus used and the transformation marker gene. For example, when Aspergillus sojae is used as the host filamentous fungus and the pyrG gene is used as the transformation marker gene, the regeneration of the transformed filamentous fungus can be carried out, for example, in a Czapek-Dox minimal medium (manufactured by Difco) containing 0.5% agar and 1.2 M sorbitol.

[0060] [Identity or similarity of amino acid sequences] The identity or similarity of amino acid sequences can be calculated by programs such as maximum matching and search homology in GENETYX Ver.11 or Ver.14 (manufactured by Genetics) or programs such as maximum matching and multiple alignment in DNASIS Pro (manufactured by Hitachi Solutions). To calculate the amino acid sequence identity, when aligning two or more oxidoreductases, the positions of amino acids that are identical in the two or more oxidoreductases can be examined. Based on such information, the identical regions in the amino acid sequence can be determined.

[0061] Also, the positions of amino acids that are similar in two or more oxidoreductases can be examined. For example, multiple amino acid sequences can be aligned using CLUSTALW. In this case, Blosum62 is used as the algorithm, and amino acids that are determined to be similar when multiple amino acid sequences are aligned may be called similar amino acids. In the variant of the present invention, the amino acid substitution may be due to substitution between such similar amino acids. Through such alignment, for multiple amino acid sequences, the regions where the amino acid sequences are identical and the positions occupied by similar amino acids can be examined. Based on such information, the homologous regions (conserved regions) in the amino acid sequence can be determined.

[0062] [Method for Preparing Enzyme] Hereinafter, the method for preparing the oxidoreductase according to the present invention will be described.

[0063] A strain such as Escherichia coli is transformed with a plasmid having DNA encoding the oxidoreductase according to the present invention to obtain a strain such as Escherichia coli having DNA encoding the oxidoreductase according to the present invention.

[0064] [Recombinant Expression of Enzyme] A strain such as Escherichia coli having a DNA encoding the oxidoreductase of the present invention is cultured in a medium. The microbial host cells may be cultured at a culture temperature of 10 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. Any of the usual culture media for culturing filamentous fungi, that is, synthetic and natural media, can be used as long as they contain carbon sources, nitrogen sources, inorganic substances, and other nutrients in appropriate ratios. In addition, as the culture medium for culturing the microbial host cells, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran leachate are added with one or more inorganic salts such as sodium chloride, potassium monophosphate, potassium diphosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate, and further, if necessary, carbohydrate raw materials, vitamins, etc. are appropriately added.

[0065] The culture conditions may be those for filamentous fungi generally known to those skilled in the art, and may be appropriately set, for example, by adjusting the initial pH of the medium to 5 to 10, the culture temperature to 20 to 40°C, and the culture time to several hours to several days, preferably 1 to 7 days, more preferably 2 to 5 days. The culture means is not particularly limited, and may be submerged culture with aeration and stirring, shaking culture, static culture, etc., but it is preferable to culture under conditions that provide sufficient dissolved oxygen. For example, an example of a medium and culture conditions for culturing an Aspergillus microorganism is shaking culture at 30°C and 160 rpm for 3 to 5 days using DPY medium.

[0066] After completion of the culture, the oxidoreductase of the present invention is collected from the culture. For this, ordinary known enzyme collection means may be used. For example, the culture supernatant fraction may be recovered, or the cells may be disrupted by ultrasonic treatment, grinding treatment, etc. according to a conventional method, or this enzyme may be extracted using a lytic enzyme such as lysozyme or mutanolysin, or lysis may be carried out by shaking or leaving in the presence of toluene or the like to discharge this enzyme outside the cells. Then, this solution is filtered, centrifuged, etc. to remove the solid part, and if necessary, after removing nucleic acids with streptomycin sulfate, protamine sulfate, or manganese sulfate, etc., ammonium sulfate, alcohol, acetone, etc. are added thereto for fractionation, and the precipitate is collected to obtain a crude enzyme of the oxidoreductase of the present invention.

[0067] [Purification of enzyme] The method for purifying the enzyme may be any method as long as it can purify the enzyme from the crude enzyme solution. For example, gel filtration using Sephadex, Ultrogel, or Bio-Gel, etc., adsorption elution using an ion exchanger, electrophoresis using polyacrylamide gel, etc., adsorption elution using hydroxyapatite, sedimentation methods such as sucrose density gradient centrifugation, affinity chromatography, fractionation using a molecular sieve membrane or a hollow fiber membrane, etc. are appropriately selected, or these are combined and carried out to obtain a purified enzyme preparation of the oxidoreductase of the present invention.

[0068] [Measurement of enzyme activity] The method for measuring the activity of an enzyme may be any method as long as it directly or indirectly measures the product of the oxidation-reduction reaction catalyzed by the enzyme. For example, when the enzyme catalyzes an oxidation-reduction reaction to generate a reduced product, the enzyme activity can be measured by measuring the current value generated when the reduced product transfers electrons to an electrode. Also, when the enzyme catalyzes an oxidation-reduction reaction and oxygen is consumed, the enzyme activity can be measured from the amount of oxygen consumed by, for example, an electrochemical method using an oxygen electrode. Preferably, the enzyme activity can be measured by reacting a reduced product of the oxidation-reduction reaction catalyzed by the enzyme with a reagent containing an absorbent substance that reacts with the reduced product (hereinafter, "absorbing reagent") and measuring the absorbance.

[0069] [Composition Containing Oxidoreductase and Kit for Quantifying Vitamin D Derivative] The method for quantifying a vitamin D derivative using an oxidoreductase according to the present invention may be carried out by providing a composition containing an oxidoreductase and a product reaction reagent, or by combining an oxidoreductase with a commercially available product reaction reagent.

[0070] The method for quantifying a vitamin D derivative, the oxidoreductase for quantification, the composition for quantification, and the kit for quantification according to the present invention can provide a new method for quantifying a vitamin D derivative that is an indicator of a disease associated with vitamin D deficiency, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor by containing an oxidoreductase.

[0071] [Sensor Chip and Electrode] FIG. 1(a) is a schematic diagram of a sensor chip 10 according to an embodiment of the present invention, and FIGS. 1(b) to 1(d) are schematic diagrams showing members constituting the sensor chip 10. The sensor chip 10 includes two or more electrodes disposed on a base material 11. The base material 11 is made of an insulating material. In FIGS. 1(a) and 1(b), as an example, a working electrode 1, a counter electrode 3, and a reference electrode 5 are disposed on the base material 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 side opposite to the wiring direction of each electrode. The working electrode 1, the counter electrode 3, and the reference electrode 5 are spaced apart from each other. Further, the working electrode 1, the counter electrode 3, and the reference electrode 5 are preferably formed integrally with the wiring portion 7 and the terminal 9. Also, the counter electrode 3 and the reference electrode 5 may be integrated.

[0072] As shown in FIGS. 1(a) and 1(c), a spacer 13 is disposed at an end of the base material 11 parallel to the wiring portion 7, and a cover 15 that covers the working electrode 1, the counter electrode 3, the reference electrode 5, and the spacer 13 is disposed. The spacer 13 and the cover 15 are made of an insulating material. The spacer 13 has a thickness substantially equal to that of the working electrode 1, the counter electrode 3, and the reference electrode 5, and preferably adheres to the working electrode 1, the counter electrode 3, and the reference electrode 5. Also, the spacer 13 and the cover 15 may be formed integrally. The cover 15 is a protective layer that prevents the wiring portion 7 from being exposed to the outside air and deteriorating, or short-circuiting due to infiltration of the measurement sample.

[0073] In one embodiment, the oxidoreductase of the present invention may be applied, adsorbed, or immobilized on an electrode. Preferably, the oxidoreductase of the present invention is applied, adsorbed, or immobilized on the working electrode. In another embodiment, a mediator may also be applied, adsorbed, or immobilized on the electrode together with the oxidoreductase. The oxidoreductase, or the oxidoreductase and the mediator, may be included in a reaction layer disposed on the working electrode, counter electrode, and reference electrode. As the electrode, a carbon electrode, a metal electrode such as platinum, gold, silver, nickel, and palladium, etc. can be used. 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. For example, an enzyme can be immobilized on the working electrode. Examples of the reference electrode 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 viewpoints of stability and reproducibility, it is preferable to use Ag / AgCl.

[0074] The enzyme may be immobilized on the electrode 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, etc. The enzyme may also be immobilized in a polymer together with a mediator or adsorbed and immobilized on the electrode, and these methods may be combined.

[0075] The mediator (also referred to as an artificial electron mediator, artificial electron acceptor, or electron mediator) used in the composition, kit, electrode, or sensor chip of the present invention is not particularly limited as long as it can receive electrons from oxidoreductase. Examples of mediators include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides such as potassium ferricyanide, ferredoxins, ferrocene, osmium complexes and their derivatives, etc. Examples of phenazine compounds include, but are not limited to, 5-Methylphenazinium methosulfate (PMS) and methoxy PMS.

[0076] The oxidoreductase of the present invention 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, when the oxidoreductase reacts with a vitamin D derivative, the current value generated by applying +600 mV to +1000 mV (vs. Ag / AgCl) to hydrogen peroxide generated by a hydrogen peroxide electrode can be measured, and thus the concentration of the vitamin D derivative in the sample can be calculated. For example, calibration curves can be created by measuring the current values for known vitamin D derivative concentrations (0, 5, 10, 50 μM) and plotting them against the vitamin D derivative concentration. The concentration of the vitamin D derivative can be obtained from the calibration curve by measuring the current value of the unknown vitamin D derivative. As the hydrogen peroxide electrode, for example, a carbon electrode or a platinum electrode can be used. Also, instead of the hydrogen peroxide electrode, an electrode immobilized with a reductase such as peroxidase or catalase can be used to measure the reduction current value generated by applying -400 mV to +100 mV (vs. Ag / AgCl) to quantify the amount of hydrogen peroxide and measure the value of the vitamin D derivative.

[0077] Further, a mediator is mixed into the reaction solution, and for example, by the amperometry method, when the oxidoreductase reacts with the vitamin D derivative, the electrons generated are transferred to the oxidized mediator to generate a reduced mediator, and the current value generated by applying a voltage of -1000 mV to +500 mV (vs. Ag / AgCl) is measured to calculate the concentration of the vitamin D derivative in the sample. As the counter electrode, a carbon electrode or a platinum electrode is preferred. For example, calibration curves can be created by measuring the current values for known vitamin D derivative concentrations (0, 100, 200, 500 μM) and plotting them against the vitamin D derivative concentration. The concentration of the vitamin D derivative can be obtained from the calibration curve by measuring the current value of the unknown vitamin D derivative.

[0078] 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 electrode is preferably formed on a substrate composed of an insulating substrate. Specifically, it is desirable that the electrode is formed on the substrate by photolithography technology or printing technologies such as screen printing, gravure printing, and flexographic printing. Examples of the material of the insulating substrate include silicon, glass, ceramics, polyvinyl chloride, polyethylene, polypropylene, and polyester, but it is more preferable to use a material with strong resistance to various solvents and chemicals.

[0079] [Vitamin D Derivative Measurement Sensor] In one embodiment, a vitamin D derivative measurement sensor using the oxidoreductase of the present invention is provided. FIG. 2(a) is a schematic diagram of a sensor 100 according to one embodiment of the present invention. The sensor is a vitamin D derivative measurement device using the oxidoreductase of the present invention, and includes a sensor chip containing the oxidoreductase and a measurement unit. The measurement unit 30 may include, for example, a switch 31 which is an input unit and a display 33 which is 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, or to control the start or interruption of the measurement of vitamin D derivatives by the sensor 100. The display 33 may display, for example, the measured value of the vitamin D derivative, or may be provided with a touch panel as an input unit for controlling the measurement unit 30.

[0080] FIG. 2(b) is a block configuration diagram of the sensor 100 according to one 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, and these may be electrically connected to each other by wiring 190. Also, the terminals of the sensor chip 10 described later and the terminals of the measurement unit 30 are electrically connected, and the current generated in the sensor chip 10 is detected by the control unit 110. The control unit 110 is a control device for controlling the sensor 100, and is composed of, for example, a known central processing unit (CPU) and an operation program for controlling 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 measuring vitamin D derivatives.

[0081] The display unit 120 includes, for example, a known display 33, and may display the measured value of the vitamin D derivative, the state of the measurement unit 30, or an operation request to the measurer. The input unit 130 is an input device for the measurer to operate the sensor 100, and may be, for example, a switch 31 or a touch panel arranged on the display 33. A plurality of switches 31 may be arranged in the measurement unit 30.

[0082] The storage unit 140 is composed of a main storage device (memory), and an auxiliary storage device (hard disk) may be externally arranged. The main storage device (memory) may be composed of a read-only memory (ROM) and / or a random access memory (RAM). An operation program, an operating system, an application program, or a module is stored in the storage unit 140 and executed by the central processing unit to constitute the control unit 110. Also, measured values and current values can be stored in the storage unit 140.

[0083] The communication unit 150 is a known communication device that connects the sensor 100 or the measurement unit 30 and an external device (computer, printer, or network). The communication unit 150 and the external device are connected by wired or wireless communication. Also, the power supply 160 is a known power supply device that supplies power to the sensor 100 or the measurement unit 30.

[0084] As described above, the method for quantifying a vitamin D derivative, the oxidoreductase for quantification, the composition for quantification, and the kit for quantification according to the present invention can provide a new method for quantifying the vitamin D derivative concentration, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor by containing an oxidoreductase.

[0085] [Method for Quantifying Vitamin D Derivative Using Dehydrogenase Activity] The dehydrogenase used in the present invention is an enzyme that acts on a vitamin D derivative as a substrate, oxidizes the vitamin D derivative, and transfers the extracted electrons to various mediators. However, until the filing of the present application, a dehydrogenase that acts on a vitamin D derivative has not been identified. Here, in this specification, an oxidoreductase having dehydrogenase activity may be referred to as dehydrogenase.

[0086] In one embodiment, the dehydrogenase is selected from the oxidoreductases described above, or may be an oxidoreductase having high vitamin D derivative dehydrogenase activity among the oxidoreductases described above. In one embodiment, the dehydrogenase is the cholesterol oxidase derived from Arthrobacter sp. F2 strain described above. In one embodiment, examples of the dehydrogenase of the present invention include oxidoreductases derived from Arthrobacter sp. F2 strain, but also include oxidoreductases derived from microorganisms classified in Actinobacteria, and also include oxidoreductases derived from microorganisms classified in the genus Arthrobacter. In one embodiment, the dehydrogenase of the present invention may be an oxidoreductase derived from a microorganism classified in the genus Corynebacterium, Rhodococcus, Brevibacterium, Nocardia, Vitiosangium, Dietzia, Tomitella, Actinomadura, Actinoallomurus.In one embodiment, the oxidoreductase of the present invention includes oxidoreductases derived from microorganisms classified into the genera Amycolatopsis, Actinoplanes, Krasilnikovia, Couchioplanes, Streptosporangium, Nonomuraea, Streptacidiphilus, Nocardioides, Alloactinosynnema, Microbispora, Actinocrispum, Kutzneria, Lentzea, Kibdelosporangium, Catenulispora, Planomonospora, Dyella, Marmoricola, Actinosynnema, Prauserella, and Yuhushiella. Also, oxidoreductases having a high sequence identity (e.g., 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence set forth in SEQ ID NO: 1, and oxidoreductases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 1 can be mentioned.

[0087] In one embodiment, the dehydrogenase may be an oxidase derived from Arthrobacter sp. strain F2, or an oxidase produced by Escherichia coli transformed with a plasmid containing the oxidase gene derived from Arthrobacter sp. strain F2. However, by using Escherichia coli transformed with a plasmid containing the oxidase gene derived from Arthrobacter sp. strain F2, the oxidase can be efficiently overexpressed.

[0088] In one embodiment, as the reaction conditions of the dehydrogenase, any conditions may be used as long as they act on the vitamin D derivative and efficiently catalyze the dehydrogenation reaction. Generally, an enzyme has an optimum temperature and an optimum pH at which it exhibits the highest activity. Therefore, the reaction conditions may preferably be near the optimum temperature and the optimum pH.

[0089] In one embodiment, as the reaction process of the dehydrogenase, various chemical substances may be involved when the dehydrogenase of the present invention acts on the vitamin D derivative. For example, when the dehydrogenase acts on the vitamin D derivative, the transfer of electrons may be involved in the oxidation-reduction reaction.

[0090] In one embodiment, a method for measuring a vitamin D derivative is provided, in which a dehydrogenase acting on the vitamin D derivative is allowed to act to oxidize the vitamin D derivative, the extracted electrons reduce a mediator, and the reduced mediator is further reacted with a chromogenic or decolorizing reagent. Examples of the chromogenic or decolorizing substrate used in the present invention include, in addition to DCIP (2,6-Dichlorophenolindophenol), for example, tetrazolium compounds (Tetrazolium blue, Nitro-tetrazolium blue, Water soluble tetrazolium (WST)-1, WST-3, WST-4, WST-5, WST-8, WST-9), and the like.

[0091] In one embodiment, when quantifying a vitamin D derivative using blood as a sample, depending on the vitamin D derivative to be measured, the sample can be arbitrarily selected from whole blood, plasma, or serum. Also, a composition for quantifying a vitamin D derivative containing dehydrogenase or dehydrogenase may be directly mixed with the sample, or the sample may be pretreated before mixing with the composition for quantifying a vitamin D derivative containing dehydrogenase or dehydrogenase. For example, after decomposing vitamin D binding protein with protease to release the vitamin D derivative, it may be mixed with the composition for quantifying a vitamin D derivative containing dehydrogenase or dehydrogenase.

[0092] [Method for preparing enzyme] Since the dehydrogenase according to the present invention can be prepared by the same preparation method as the preparation method of the oxidoreductase described above, detailed description is omitted.

[0093] [Measurement of enzyme activity] The method for measuring the activity of an enzyme may be any method as long as it directly or indirectly measures the product of the reaction catalyzed by the enzyme. For example, if a reagent that reacts with the product of the reaction catalyzed by the enzyme (hereinafter, "product reaction reagent") is reacted with the product of the reaction catalyzed by the enzyme and the absorbent substance generated by the reaction is measured, the enzyme activity can be measured by measuring the absorbance.

[0094] [Composition containing dehydrogenase and kit for quantifying vitamin D derivative] In one embodiment, the vitamin D derivative may be quantified using the dehydrogenase according to the present invention. The method for quantifying a vitamin D derivative using the dehydrogenase according to the present invention may be performed by providing a composition containing the dehydrogenase and a product reaction reagent, or by combining the dehydrogenase with a commercially available product reaction reagent. For example, it may be provided as a composition for quantifying a vitamin D derivative containing a composition for quantifying a vitamin D derivative containing dehydrogenase, a mediator reduced by adding dehydrogenase, and a reagent that reacts with the reduced mediator. Further, it may be provided as a kit for quantifying a vitamin D derivative containing dehydrogenase, a mediator reduced by adding dehydrogenase, and a reagent that reacts with the reduced mediator.

[0095] The mediator (also referred to as an artificial electron mediator, artificial electron acceptor, or electron mediator) used in the measurement method or quantification kit of the present invention is not particularly limited as long as it can receive electrons from dehydrogenase. Examples of mediators include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides such as potassium ferricyanide, ferredoxins, ferrocene, osmium complexes and their derivatives, etc. Examples of phenazine compounds include, but are not limited to, PMS, methoxy PMS, 5-Methylphenazinium ethylsulfate (PES), and methoxy PES.

[0096] [Sensor Chip and Electrode] In one embodiment, the dehydrogenase of the present invention may be applied, adsorbed, or immobilized on an electrode. Preferably, the dehydrogenase of the present invention is applied, adsorbed, or immobilized on a working electrode. Since the configuration of the electrode can be the same as the configuration described for the electrode using oxidoreductase, a detailed description thereof will be omitted. Further, the dehydrogenase can be immobilized on the electrode 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.

[0097] The dehydrogenase of the present invention can be applied to various electrochemical measurement methods by using a potentiostat, a galvanostat, or the like. Examples of the electrochemical measurement method include various methods such as amperometry, potentiometry, and coulometry. Further, a mediator is mixed in the reaction solution, and for example, by the amperometry method, the electrons generated when the dehydrogenase reacts with the vitamin D derivative are transferred to the oxidized mediator to generate a reduced mediator, and the current value generated by applying -1000 mV to +500 mV (vs. Ag / AgCl) is measured, whereby the concentration of the vitamin D derivative in the sample can be calculated. As the counter electrode, a carbon electrode or a platinum electrode is preferable. For example, a calibration curve can be created by measuring the current values for known vitamin D derivative concentrations (0, 100, 200, 500 μM) and plotting them against the vitamin D derivative concentration. The concentration of the vitamin D derivative can be obtained from the calibration curve by measuring the current value of the unknown vitamin D derivative.

[0098] Furthermore, in order to reduce the amount of solution required for the measurement, a printed electrode (sensor chip) can also be used. In this case, the electrode is preferably formed on a substrate composed of an insulating substrate. The configuration of the sensor chip using dehydrogenase may be the same as the configuration of the sensor chip using oxidoreductase, and a detailed description thereof will be omitted.

[0099] [Vitamin D Derivative Measurement Sensor] In one embodiment, a vitamin D derivative measurement sensor using the dehydrogenase of the present invention is provided. The sensor is a vitamin D derivative measurement device using the dehydrogenase of the present invention, and includes a sensor chip containing dehydrogenase and a measurement unit. The configuration of the vitamin D derivative measurement sensor using dehydrogenase may be the same as the configuration of the vitamin D derivative measurement sensor using oxidoreductase, and detailed description thereof will be omitted.

[0100] As described above, the method for quantifying vitamin D derivatives, the dehydrogenase for quantification, the composition for quantification, and the kit for quantification according to the present invention can provide a new method for quantifying vitamin D derivative concentration, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor by containing dehydrogenase.

[0101] [Method for Quantifying Vitamin D Derivatives Using Oxidase Activity] The oxidase used in the present invention is an oxidase that acts on vitamin D derivatives as a substrate. However, until the filing of the present application, no oxidase acting on vitamin D derivatives has been identified.

[0102] In one embodiment, the oxidase is selected from the oxidoreductases described above, or may be an oxidase having high vitamin D derivative oxidase activity among the oxidoreductases described above. In one embodiment, the oxidase is cholesterol oxidase derived from Arthrobacter sp. F2 strain. In one embodiment, the oxidase of the present invention includes oxidases derived from Arthrobacter sp. F2 strain, but also includes oxidases derived from microorganisms classified in the class Actinobacteria, and oxidases derived from microorganisms classified in the genus Arthrobacter. Oxidoreductases derived from microorganisms classified in the genus Herbaspirillum or Pedobacter are also included. In one embodiment, the oxidase of the present invention may be an oxidase derived from microorganisms classified in the genus Corynebacterium, Rhodococcus, Brevibacterium, Nocardia, Vitiosangium, Dietzia, Tomitella, Actinomadura, Actinoallomurus.In one embodiment, the oxidase of the present invention also includes oxidoreductases derived from microorganisms classified into the genera Amycolatopsis, Actinoplanes, Krasilnikovia, Couchioplanes, Streptosporangium, Nonomuraea, Streptacidiphilus, Nocardioides, Alloactinosynnema, Microbispora, Actinocrispum, Kutzneria, Lentzea, Kibdelosporangium, Catenulispora, Planomonospora, Dyella, Marmoricola, Actinosynnema, Prauserella, and Yuhushiella. In addition, oxidases having a high sequence identity (e.g., 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more) with respect to the amino acid sequence set forth in SEQ ID NO: 1, and oxidases having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 1 can also be mentioned.

[0103] In one embodiment, the oxidase may be an oxidase derived from Arthrobacter sp. strain F2, or an oxidase produced by Escherichia coli transformed with a plasmid containing the oxidase gene derived from Arthrobacter sp. strain F2. However, by using Escherichia coli transformed with a plasmid containing the oxidase gene derived from Arthrobacter sp. strain F2, the oxidase can be efficiently expressed in large quantities.

[0104] In one embodiment, as the reaction conditions for the oxidase, any conditions may be used as long as they act on the vitamin D derivative and efficiently catalyze the oxidation reaction. Generally, enzymes have an optimum temperature and an optimum pH at which they exhibit the highest activity. Therefore, the reaction conditions may preferably be near the optimum temperature and the optimum pH.

[0105] In one embodiment, in the reaction process of the oxidase, various chemical substances may be involved when the oxidase of the present invention acts on the vitamin D derivative. For example, when the oxidase acts on the vitamin D derivative, oxygen may be involved as an electron acceptor in the oxidation-reduction reaction.

[0106] In one embodiment, examples of the compound produced by allowing the oxidase to act on the vitamin D derivative include hydrogen peroxide and the like. The amount of hydrogen peroxide produced by allowing the oxidase to act on the vitamin D derivative can be measured, for example, by a colorimetric method using the catalytic reaction of peroxidase. Examples of the compound consumed by allowing the oxidase to act on the vitamin D derivative include oxygen and the like. The amount of oxygen consumed by allowing the oxidase to act on the vitamin D derivative can be measured, for example, by an electrochemical method using an oxygen electrode.

[0107] In one embodiment, when quantifying a vitamin D derivative using blood as a sample, the sample can be arbitrarily selected from whole blood, plasma, or serum according to the vitamin D derivative to be measured. Further, a composition for quantifying a vitamin D derivative containing an oxidase or an oxidase may be directly mixed with the sample, or the sample may be pretreated before mixing with the composition for quantifying a vitamin D derivative containing an oxidase or an oxidase. For example, after decomposing vitamin D-binding protein with a protease to release the vitamin D derivative, it may be mixed with the composition for quantifying a vitamin D derivative containing an oxidase or an oxidase.

[0108] [Method for preparing enzyme] Since the oxidase according to the present invention can be prepared by the same preparation method as the above-described method for preparing an oxidoreductase, detailed description thereof is omitted.

[0109] [Measurement of enzyme activity] The method for measuring the activity of an enzyme may be any method as long as it directly or indirectly measures the product of the reaction catalyzed by the enzyme. For example, if a reagent that reacts with the product of the reaction catalyzed by the enzyme (hereinafter, "product reaction reagent") is reacted with the product of the reaction catalyzed by the enzyme and the absorbent substance generated by the reaction is measured, the enzyme activity can be measured by measuring the absorbance.

[0110] [Composition containing oxidase and kit for quantifying vitamin D derivative] In one embodiment, the vitamin D derivative may be quantified using the oxidase according to the present invention. The method for quantifying the vitamin D derivative using the oxidase according to the present invention may be performed by providing a composition containing the oxidase and a product reaction reagent, or by combining the oxidase with a commercially available product reaction reagent. For example, it may be provided as a composition for quantifying a vitamin D derivative containing a composition for quantifying a vitamin D derivative containing an oxidase, or a reagent that reacts with hydrogen peroxide generated by adding an oxidase. Further, it may be provided as a kit for quantifying a vitamin D derivative containing an oxidase and a reagent that reacts with hydrogen peroxide generated by adding an oxidase.

[0111] [Sensor chip and electrode] In one embodiment, the oxidase of the present invention may be applied, adsorbed, or immobilized on the electrode. Preferably, the oxidase of the present invention is applied, adsorbed, or immobilized on the working electrode. Since the configuration of the electrode can be the same as the configuration described for the electrode using the oxidoreductase, a detailed description thereof is omitted. Further, the oxidase can be immobilized on the electrode 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.

[0112] The oxidase of the present invention can be applied to various electrochemical measurement methods by using a potentiostat, a galvanostat, or the like. Examples of the electrochemical measurement methods include various methods such as amperometry, potentiometry, and coulometry. For example, by the amperometry method, when the oxidase reacts with a vitamin D derivative, the current value generated by applying +600 mV to +1000 mV (vs. Ag / AgCl) to hydrogen peroxide generated by a hydrogen peroxide electrode is measured, and thus the concentration of the vitamin D derivative in the sample can be calculated. For example, a calibration curve can be created by measuring the current values for known vitamin D derivative concentrations (0, 5, 10, 50 μM) and plotting them against the vitamin D derivative concentration. The concentration of the vitamin D derivative can be obtained from the calibration curve by measuring the current value of the unknown vitamin D derivative. As the hydrogen peroxide electrode, for example, a carbon electrode or a platinum electrode can be used. Further, instead of the hydrogen peroxide electrode, an electrode immobilized with a 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 -400 mV to +100 mV (vs. Ag / AgCl), and the value of the vitamin D derivative can also be measured.

[0113] Furthermore, in order to reduce the amount of solution required for the measurement, a printed electrode (sensor chip) can also be used. In this case, the electrode is preferably formed on a substrate composed of an insulating substrate. The configuration of the sensor chip using oxidase may be the same as the configuration of the sensor chip using oxidoreductase, and a detailed description thereof will be omitted.

[0114] [Vitamin D Derivative Measurement Sensor] In one embodiment, a vitamin D derivative measurement sensor using the oxidase of the present invention is provided. The sensor is a vitamin D derivative measurement device using the oxidase of the present invention, and includes a sensor chip containing the oxidase and a measurement unit. The configuration of the vitamin D derivative measurement sensor using the oxidase may be the same as the configuration of the vitamin D derivative measurement sensor using the oxidoreductase, and a detailed description thereof will be omitted.

[0115] As described above, the method for quantifying a vitamin D derivative, the oxidase for quantification, the composition for quantification, and the kit for quantification according to the present invention can provide a new method for quantifying the vitamin D derivative concentration, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor by containing the oxidase.

Examples

[0116] Specific examples and test results of the quantification method, the oxidoreductase for quantification, the composition for quantification, and the kit for quantification according to the present invention described above will be shown and described in more detail.

[0117] [Preparation of Recombinant Plasmid pUC18-ChoF] The DNA fragment of vector pUC18 was amplified by PCR using primers of SEQ ID NOs: 3 and 4 with pUC18 as a template. 1.0 μl of DpnI (manufactured by New England BioLabs) was added to the PCR reaction solution and treated at 37°C for 1 hour, and then subjected to agarose gel electrophoresis. The gel containing the target DNA fragment (about 2.7 kbp) was cut out. The target DNA fragment was extracted from the gel using illustra (registered trademark) GFX PCR DNA and Gel Band Purification Kit (manufactured by GE Healthcare).

[0118] The ChoF gene having the base sequence of SEQ ID NO:2 was divided into the first half (choF-f1) described in SEQ ID NO:5 and the second half (choF-f2) described in SEQ ID NO:6, and synthesis was entrusted to Integrated DNA Technologies. The 15 bases on the 3'-end side of choF-f1 and the 15 bases on the 5'-end side of choF-f2 (ACAACCTTGCATCGC) show a sequence overlapping between the first half and the second half of the ChoF gene. Here, the ChoF gene according to this embodiment is one in which the N-terminal 45 a.a. predicted to be the signal peptide of cholesterol oxidase (ChoF, UniprotKB Entry name Q56DL0_9MICC) derived from Arthrobacter sp. F2 strain is deleted.

[0119] An in-fusion reaction (50°C, 15 min) was carried out using the DNA fragment of vector pUC18 and two ChoF gene fragments according to the manual of the In-Fusion (registered trademark) HD ​​Cloning Kit to obtain an expression plasmid for ChoF (pUC18-ChoF). The resulting plasmid was used to transform E. coli JM109 strain.

[0120] [Expression of ChoF] The E. coli JM109 (pUC18-ChoF) strain carrying the recombinant plasmid was inoculated into 2.5 ml of LB-amp medium [1% (w / v) bactotryptone, 0.5% (w / v) yeast extract, 0.5% (w / v) NaCl, 50 μg / ml ampicillin] and cultured with shaking at 37°C for 24 hours to obtain a culture.

[0121] One ml of the seed culture was inoculated into 50 ml of LB-amp medium [1% (w / v) bactotryptone, 0.5% (w / v) yeast extract, 0.5% (w / v) NaCl, 50 μg / ml ampicillin] containing 0.1 mM IPTG placed in a Sakaguchi flask and cultured at 25°C for 16 hours.

[0122] The culture solution was collected by centrifugation at 6,500×g for 10 minutes. The obtained cells were washed with 10 mM potassium phosphate buffer (pH 7.0) and resuspended. After sonicating the cell suspension, the supernatant obtained by centrifugation at 20,400×g for 15 minutes was dialyzed against 10 mM potassium phosphate buffer (pH 6.0) using Amicon (registered trademark) Ultra Ultracel-30K (manufactured by Millipore) to obtain a crude enzyme solution of ChoF.

[0123] [Purification of ChoF] The crude enzyme solution of ChoF was injected into HiScreen Capto Q (manufactured by GE Healthcare, carrier volume 4.7 ml) equilibrated with 10 mM potassium phosphate buffer pH 6.0, and the fraction that did not bind to the anion exchange column was collected.

[0124] The collected fraction was dialyzed against 10 mM CHES-NaOH buffer (p9.5) using Amicon Ultra Ultracel-30K, and then injected into HiScreen Capto Q (manufactured by GE Healthcare, carrier volume 4.7 ml) equilibrated with 10 mM CHES-NaOH buffer (pH 9.5) to bind to the anion exchange column. Then, the column was washed with 10 mM CHES-NaOH buffer (pH 9.5), and ChoF bound to the column was eluted with 10 mM CHES-NaOH buffer (pH 9.5) containing NaCl with a concentration gradient from 0 mM to 250 mM.

[0125] The eluted fraction was concentrated using Amicon Ultra Ultracel-30K. It was fractionated using HiLoad 26 / 60 Superdex 200 (manufactured by GE Healthcare) equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 150 mM NaCl. The purity of each eluted fraction was evaluated by polyacrylamide gel electrophoresis (SDS-PAGE), and the fraction containing no contaminating proteins was collected as a purified standard of ChoF.

[0126] The protein concentration of the purified ChoF was the absorbance (A) at 280 nm 280) was measured by the ultraviolet absorption method (see Protein Sci. 4, 2411-23, 1995). The molecular weight of ChoF calculated from the amino acid sequence is 55.6 kDa, and since ChoF contains 19 residues of tyrosine and 9 residues of tryptophan, the A of a 1.0 mg / ml ChoF solution 280 shows 1.4.

[0127] [Quantification of vitamin D derivatives by oxidase activity] Using ChoF obtained by the method described above, the oxidase activity using 25-hydroxyvitamin D3 (calcidiol), a vitamin D derivative, as a substrate was measured.

[0128] [Substrate reagent] 4.19 mg of 25-hydroxyvitamin D3 was dissolved in 1 ml of DMSO (final concentration 10 mM). Since 25-hydroxyvitamin D3 is lipophilic, it was diluted to 5, 10, 20, 100 μM (measurement concentration 2.5, 5, 10, 50 μM) using a 3.9% (v / v) Triton X-100 solution to maintain solubility.

[0129]

Table 1

[0130] After incubating 450 μl of the measurement reagent having the composition shown in Table 1 at 37°C for 5 minutes, 450 μl of the substrate reagent was added, and the absorbance (Abs) at a wavelength of 555 nm was measured using a spectrophotometer (U-3900, manufactured by Hitachi High-Technologies Corporation). The relationship between the vitamin D derivative concentration and the absorbance (A555) after reacting for 5 minutes is shown in Figure 3.

[0131] In the range of 2.5 μM to 50 μM, ChoF has a coefficient of determination (R 2 ) of 0.9993, which is an index of the correlation between the vitamin D derivative concentration and the absorbance. Thus, it was shown that the vitamin D derivative can be quantified using the oxidase activity of ChoF. EXAMPLES

[0132] [Preparation of recombinant plasmid pKK223-3 HeGMCOR] The DNA fragment of the vector pKK223-3 was amplified by PCR using primers of SEQ ID NO: 7 and SEQ ID NO: 8 with pKK223-3 as a template. 1.0 μl of DpnI (New England BioLabs) was added to the PCR reaction solution, which was then treated at 37° C. for 1 hour, and subjected to agarose gel electrophoresis, and the gel containing the target DNA fragment (about 4.6 kbp) was excised. The target DNA fragment was extracted from the gel using illustra (registered trademark) GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).

[0133] The HeGMCOR gene having the base sequence of SEQ ID NO: 9 was divided into the first half (HeGMCOR-f1) described in SEQ ID NO: 10 and the second half (HeGMCOR-f2) described in SEQ ID NO: 11, and synthesis was entrusted to Integrated DNA Technologies. The 15 bases on the 3'-end side of HeGMCOR-f1 and the 15 bases on the 5'-end side of HeGMCOR-f2 (GAAGGAAATGGCTAT) show a sequence overlapping between the first half and the second half of the HeGMCOR gene. Here, the HeGMCOR gene according to this embodiment is glucose-methanol-choline family oxidoreductase (HeGMCOR, NCBI Reference Sequence: WP_094565544.1) derived from Herbaspirillum sp. meg3 strain, and has an amino acid sequence identity of 50% with ChoF of Example 1.

[0134] An in-fusion reaction (50°C, 15 minutes) was carried out using the DNA fragment of vector pKK223-3 and two HeGMCOR gene fragments according to the manual of the In-Fusion (registered trademark) HD ​​Cloning Kit to obtain an expression plasmid for HeGMCOR (pKK223-3_HeGMCOR). The resulting plasmid was transformed into E. coli BL21 (DE3).

[0135] [Expression of HeGMCOR] E. coli BL21(DE3)(pKK223-3_HeGMCOR) strain with the recombinant plasmid was inoculated into 2.5 ml of LB-amp medium [1% (W / V) Bactotryptone, 0.5% (W / V) yeast extract, 0.5% (W / V) NaCl, 50 μg / ml Ampicillin] and cultured with shaking at 37 °C for 24 hours to obtain a culture.

[0136] 1.5 ml of the seed culture was inoculated into 150 ml of LB-amp medium [1% (W / V) Bactotryptone, 0.5% (W / V) yeast extract, 0.5% (W / V) NaCl, 50 μg / ml Ampicillin] containing 0.1 mM IPTG in a Sakaguchi flask and cultured at 25 °C for 20 hours.

[0137] The culture was centrifuged at 6,500×g for 10 minutes to collect the bacteria. The obtained bacterial cells were washed with 12 ml of 10 mM potassium phosphate buffer (pH 7.0) and resuspended. After ultrasonically disrupting the cell suspension, the supernatant obtained by centrifugation at 20,400×g for 15 minutes was collected as the cell extract. Ammonium sulfate was added to the obtained cell extract to a saturation concentration of 35%, and it was thoroughly vortexed to dissolve the ammonium sulfate. The supernatant obtained by centrifugation at 20360×g for 5 minutes was used as the crude enzyme solution.

[0138] [Purification of HeGMCOR] Subsequently, the crude enzyme solution was injected into HiScreen Butyl HP (manufactured by GE Healthcare, column volume 4.7 ml) equilibrated with 10 mM potassium phosphate buffer (pH 7.5) containing 500 mM ammonium sulfate and bound to the column. Then, the column was washed with 10 mM potassium phosphate buffer (pH 7.5) containing 250 mM ammonium sulfate, and HeGMCOR bound to the column was eluted with 10 mM potassium phosphate buffer (pH 7.5) containing ammonium sulfate with a concentration gradient from 250 mM to 0 mM. The purity of each eluted fraction was evaluated by polyacrylamide gel electrophoresis (SDS-PAGE), and the fraction with fewer contaminating proteins was collected.

[0139] The obtained elution fraction was concentrated using Amicon Ultra Ultracel-30K and fractionated with HiLoad 26 / 60 Superdex 200 (manufactured by GE Healthcare) equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 150 mM NaCl. The purity of each eluted fraction was evaluated by polyacrylamide gel electrophoresis (SDS-PAGE), and the fractions containing no contaminating proteins were collected and used as a purified sample of HeGMCOR.

[0140] The protein concentration of the purified HeGMCOR was measured by the ultraviolet absorption method using the absorbance (A 280 ) at 280 nm (see Protein Sci. 4, 2411-23, 1995). The molecular weight of HeGMCOR calculated from the amino acid sequence is 54.3 kDa, and since HeGMCOR contains 22 residues of tyrosine and 6 residues of tryptophan, the A 280 of a 1.0 mg / ml HeGMCOR solution is 1.2.

[0141] [Quantification of Vitamin D Derivatives by Oxidase Activity] Using HeGMCOR obtained by the method described above, the oxidase activity was measured with 25-hydroxyvitamin D3 (calcidiol), a vitamin D derivative, as a substrate.

[0142] [Substrate Reagent] 4.19 mg of 25-hydroxyvitamin D3 was dissolved in 1 ml of DMSO (final concentration 10 mM). Since 25-hydroxyvitamin D3 is lipophilic, it was diluted to 2, 5, 20 μM (measurement concentration 1, 2.5, 10 μM) using a 3.9% (v / v) TritonX-100 solution to maintain solubility.

[0143]

Table 2

[0144] After incubating 375 μl of the measurement reagent composed of the composition shown in Table 2 at 37°C for 5 minutes, 375 μl of the substrate reagent was added, and the absorbance (Abs) at a wavelength of 555 nm was measured using a spectrophotometer (U-3900, manufactured by Hitachi High-Tech Sciences). The relationship between the vitamin D derivative concentration and the absorbance (A555) after reacting for 5 minutes is shown in Figure 4.

[0145] HeGMCOR has a coefficient of determination (R 2 ) of 0.9927 in the range of 1 μM to 10 μM, indicating a correlation between the vitamin D derivative concentration and the absorbance. Therefore, it was shown that the vitamin D derivative can be quantified using the oxidase activity of HeGMCOR.

Example

[0146] [Quantification of Vitamin D Derivative by Dehydrogenase Activity] Using ChoF obtained in Example 1, the dehydrogenase activity using 25-hydroxyvitamin D3 (calcidiol), a vitamin D derivative, as a substrate was measured.

[0147] [Substrate Reagent] 4.19 mg of 25-hydroxyvitamin D3 was dissolved in 1 ml of DMSO (final concentration 10 mM). Since 25-hydroxyvitamin D3 is lipophilic, it was diluted to 200, 400, 500, 1000 μM (measurement concentration 100, 200, 250, 500 μM) using a 3.9% (v / v) Triton X-100 solution to maintain solubility.

[0148]

Table 3

[0149] After incubating 375 μl of the measurement reagent composed of the composition in Table 3 at 37°C for 5 minutes, 375 μl of the substrate reagent was added, and the absorbance (Abs) at a wavelength of 600 nm was measured using a spectrophotometer (U-3900, manufactured by Hitachi High-Tech Science). Figure 5 shows the relationship between the vitamin D derivative concentration after reacting for 3 minutes and the change in absorbance (A600) per minute.

[0150] ChoF is a coefficient of determination (R 2 ) that is an index of the correlation between the vitamin D derivative concentration and absorbance in the range of 100 μM to 500 μM. As a result, it was found that there is a correlation between the vitamin D derivative concentration and absorbance. Therefore, it was shown that the vitamin D derivative can be quantified using the dehydrogenase activity of ChoF.

[0151] As described above, a method for quantifying a vitamin D derivative by adding an oxidase to a sample containing a vitamin D derivative according to the present invention, an oxidase for quantifying a vitamin D derivative added to a sample containing a vitamin D derivative, a composition for quantifying a vitamin D derivative containing an oxidase added to a sample containing a vitamin D derivative, and a kit for quantifying a vitamin D derivative containing an oxidase added to a sample containing a vitamin D derivative can provide a new method for quantifying the vitamin D derivative concentration, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor. Further, a method for quantifying a vitamin D derivative by adding an oxidoreductase to a sample containing a vitamin D derivative according to the present invention, an oxidoreductase for quantifying a vitamin D derivative added to a sample containing a vitamin D derivative, a composition for quantifying a vitamin D derivative containing an oxidoreductase added to a sample containing a vitamin D derivative, and a kit for quantifying a vitamin D derivative containing an oxidoreductase added to a sample containing a vitamin D derivative can provide a new method for quantifying the vitamin D derivative concentration, an enzyme for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor.

Explanation of Symbols

[0152] 1 working electrode, 3 counter electrodes, 5 reference electrodes, 7 wiring parts, 9 terminals, 10 sensor chips, 11 base materials, 13 spacers, 15 covers, 19 reaction layers, 30 measurement parts, 31 switches, 33 displays, 100 sensors, 110 control parts, 120 display parts, 130 input parts, 140 memory parts, 150 communication parts, 160 power supplies, 190 wirings

Claims

**Claim 1** A method for quantifying 25-hydroxyvitamin D3, comprising adding an oxidoreductase having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 12 and acting on 25-hydroxyvitamin D3 to a sample, transferring electrons generated when the oxidoreductase reacts with 25-hydroxyvitamin D3 to an oxidized mediator to generate a reduced mediator, and reacting the reduced mediator with a reagent to determine the concentration of 25-hydroxyvitamin D3. **Claim 2** A method for quantifying 25-hydroxyvitamin D3, comprising adding an oxidoreductase having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 12 and acting on 25-hydroxyvitamin D3 to a sample, wherein the oxidoreductase is an oxidase, and determining the concentration of 25-hydroxyvitamin D3 by quantifying hydrogen peroxide generated or oxygen consumed by adding the oxidase. **Claim 3** The method for quantifying 25-hydroxyvitamin D3 according to claim 2, wherein hydrogen peroxide generated by adding the oxidase is reacted with a reagent to determine the concentration of 25-hydroxyvitamin D3. **Claim 4** An oxidoreductase used in the method for quantifying 25-hydroxyvitamin D3 according to any one of claims 1 to 3, a mediator reduced by receiving electrons generated when the oxidoreductase reacts with 25-hydroxyvitamin D3, and a reagent that reacts with the reduced mediator, and a composition for quantifying 25-hydroxyvitamin D3 comprising the same. **Claim 5** The oxidoreductase which is an oxidase used in the method for quantifying 25-hydroxyvitamin D3 according to any one of claims 1 to 3, A composition for the quantification of 25-hydroxyvitamin D3, comprising a reagent that reacts with hydrogen peroxide generated by adding the oxidase.

6. In the method for quantifying 25-hydroxyvitamin D3 according to any one of Claims 1 to 3, the oxidoreductase used, A mediator that is reduced by receiving electrons generated when the oxidoreductase reacts with 25-hydroxyvitamin D3, A reagent that reacts with the reduced mediator, and a kit for the quantification of 25-hydroxyvitamin D3.

7. In the method for quantifying 25-hydroxyvitamin D3 according to any one of Claims 1 to 3, the oxidoreductase used, A reagent that reacts with hydrogen peroxide, And a kit for the quantification of 25-hydroxyvitamin D3.

8. An electrode comprising the oxidoreductase used in the method for quantifying 25-hydroxyvitamin D3 according to any one of Claims 1 to 3.

9. A sensor chip provided with the electrode according to Claim 8 as a working electrode.

10. A sensor provided with the sensor chip according to Claim 9.

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