Method for quantifying hormone or hormone precursor, enzyme for quantification, composition for quantification, and kit for quantification

Abstract

A method for quantifying a hormone or a hormone precursor according to one embodiment of the present invention comprises: adding an oxidoreductase and an oxidized first coenzyme to a sample, whereby the oxidoreductase oxidizes the hormone or the hormone precursor contained in the sample to produce an oxidized hormone or an oxidized hormone precursor, and transfers electrons withdrawn from the hormone or the hormone precursor to the oxidized first coenzyme to produce a reduced first coenzyme; and determining the concentration of the hormone or the hormone precursor from the concentration of the reduced first coenzyme.

Description

Method for quantifying hormones or hormone precursors, enzymes for quantification, compositions for quantification, and kits for quantification.

[0001] The present invention relates to a method for quantifying hormones or hormone precursors, an enzyme for quantification, a composition for quantification, and a kit for quantification.

[0002] Hormones are physiologically active substances that act on diverse biological processes within the body. In response to information received by the body, hormones are synthesized and secreted in specific organs, circulate throughout the body via the bloodstream, and exert their effects in specific cells. Therefore, there is a wide variety of hormones, and their types also change depending on the stage of the body's growth. Hormones are substances that transmit information to regulate the function of specific organs within the body, and their concentration in body fluids is extremely low. Examples of human hormones include polypeptides, amines, catecholamines, and steroids.

[0003] Furthermore, 25-hydroxyvitamin D (calcifediol), a hormone precursor, is known to be involved in bone and mineral metabolism. As will be discussed later, it is known as an indicator of calcium and phosphate concentrations in the body, as well as bone formation. Compounds that are thought to be closely related to various diseases such as breast cancer, uterine cancer, gastrointestinal cancer, cardiovascular disorders, congenital disorders of sex hormone metabolism, precocious puberty, and osteoporosis include 17β-hydroxysteroids, such as estrogen. Among estrogens, estradiol has the strongest effect and is therefore known as an indicator for understanding metabolism and disease status.

[0004] Estrogen, a 17β-hydroxysteroid, is known to play a crucial role in maintaining bone homeostasis throughout life, including longitudinal bone growth, bone healing, and adaptation to mechanical forces. Furthermore, estrogen has been observed to crosstalk with other signaling pathways, suggesting that it has broader effects beyond just bone homeostasis. The rapid decline in estrogen production experienced by postmenopausal women is associated with a rapid decrease in bone mass, ultimately leading to osteoporosis; therefore, estrogen is an important biomarker involved in bone formation.

[0005] Vitamin D is divided into vitamin D ２ (ergocalciferol) and vitamin D ３ (cholecalciferol). Sources of vitamin D include biosynthesis of vitamin D ３ in the skin and intake of vitamin D ２ and vitamin D ３ from foods, supplements, and the like. Here, vitamin D ２ and vitamin D ３ undergo the same metabolism and exert the same action, therefore, when not distinguishing between vitamin D ２ and vitamin D ３ , they are referred to as vitamin D.

[0006] Vitamin D taken into a living body is hydroxylated in the liver, converted into 25-hydroxyvitamin D, and stored in hepatocytes. 25-hydroxyvitamin D binds to vitamin D-binding protein and is released into the blood. Since the half-life of 25-hydroxyvitamin D in blood is as long as about 2 to 3 weeks, the blood 25-hydroxyvitamin D concentration reflects the vitamin D concentration in the living body, and thus is an important biomarker associated with bone formation.

[0007] 25-hydroxyvitamin D in blood is taken up into cells via endocytosis mediated by megalin receptors. 25-hydroxyvitamin D transported to the renal tubules is hydroxylated and converted into active 1,25-dihydroxyvitamin D. 1,25-dihydroxyvitamin D released back into blood binds to the vitamin D receptor in target cells and acts as a transcription factor that regulates the expression of various genes involved in calcium transport and utilization. The half-life of 1,25-dihydroxyvitamin D in blood is as short as about 15 hours, and the blood 1,25-dihydroxyvitamin D concentration is strictly regulated by parathyroid hormone, calcium, and phosphate. For this reason, it is considered that the blood 1,25-dihydroxyvitamin D concentration does not change unless extreme vitamin D deficiency or excess occurs.

[0008] Vitamin D plays an essential role in regulating calcium and phosphate concentrations in the body. It primarily affects intestinal and osteocytes, assisting in calcium uptake regulation in the former and regulating skeletal formation and maintenance in the latter. Vitamin D is also known to be involved in cell proliferation and differentiation, as well as the immune system. Vitamin D deficiency or excess can have various consequences. In particular, vitamin D deficiency has been linked to serious diseases such as rickets, osteomalacia, osteoporosis, chronic renal failure, hyperparathyroidism, and psoriasis.

[0009] The criteria for determining vitamin D deficiency or lack thereof are as follows: a serum 25-hydroxyvitamin D concentration of 30 ng / ml or higher is considered vitamin D sufficiency, 20 ng / ml to less than 30 ng / ml is considered vitamin D deficiency, and less than 20 ng / ml is considered vitamin D deficiency (according to the guidelines for determining vitamin D deficiency and lack thereof by the Japanese Society for Bone and Mineral Research and the Japanese Endocrine Society). It is currently estimated that there are 1 billion people worldwide suffering from vitamin D deficiency. Early detection of vitamin D deficiency is particularly useful in modern society where people tend to avoid direct sunlight, and in Japan, electrochemiluminescence immunoassay (ECLIA), chemiluminescence enzyme immunoassay (CLEIA), and chemiluminescence immunoassay (CLIA) for serum 25-hydroxyvitamin D are covered by insurance. All of these are immunological assays using anti-25-hydroxyvitamin D antibodies.

[0010] 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 this anti-25-hydroxyvitamin D antibody, the detection limit for 25-hydroxyvitamin D is less than 3.0 ng / ml, indicating high sensitivity, and therefore it is considered useful for the early diagnosis of vitamin D deficiency. However, because it is a test method that uses an immunological assay, it has the drawbacks of being time-consuming and costly.

[0011] 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 a vitamin D-binding protein using 25-hydroxyvitamin D labeled with biotin or fluorocein. 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 a liquid chromatography-mass spectrometer (LC / MS / MS).

[0012] Patent documents 5 and 6 disclose methods for measuring 25-hydroxyvitamin D using enzymes.

[0013] Japanese Patent Publication No. 2017-40659, U.S. Patent No. 5981779, Japanese Patent Publication No. 2009-540275, Japanese Patent Publication No. 2018-81023, International Publication No. 2021 / 117790, International Publication No. 2022 / 264946

[0014] M. Harkonen et al. , J. Steroid. Biochem. , 11, 1205-1208 (1979).

[0015] However, the aforementioned methods for measuring 25-hydroxyvitamin D have various drawbacks, including long measurement times, measurement errors, high costs, large sample volumes, and the use of difficult-to-handle reagents, making them unsuitable for clinical testing. Therefore, there is a need for a method of measuring 25-hydroxyvitamin D that is less time-consuming, less laborious, and less expensive.

[0016] On the other hand, Non-Patent Document 1 discloses that 3α,20β-hydroxysteroid dehydrogenase from Streptomyces exfoliatus reacts with 25-hydroxyvitamin D.

[0017] One of the objectives of the present invention is to provide a novel quantitative method, quantitative enzyme, quantitative composition, and quantitative kit for measuring 25-hydroxyvitamin D or 17β-hydroxysteroid, which are hormones or hormone precursors, as biomarkers.

[0018] According to one embodiment of the present invention, a method for quantifying a hormone or hormone precursor is provided, in which oxidoreductase and oxidized coenzyme I are added to a sample, the oxidoreductase oxidizes the hormone or hormone precursor contained in the sample to produce an oxidized hormone or oxidized hormone precursor, the electrons extracted from the hormone or hormone precursor are transferred to oxidized coenzyme I to produce reduced coenzyme I, and the concentration of the hormone or hormone precursor is determined from the concentration of reduced coenzyme I.

[0019] By further adding a mediator and a reagent to the sample, the mediator may transfer electrons from the reduced coenzyme I to the reagent, and the concentration of the hormone or hormone precursor may be determined from the change in the reagent.

[0020] The mediator may oxidize the reduced coenzyme I to produce the oxidized coenzyme I.

[0021] Oxidized coenzyme I is NAD ＋ It may include.

[0022] The mediator may be selected from the group consisting of quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides, such as potassium ferricyanide, ferredoxins, ferrocene, osmium complexes, and their derivatives.

[0023] The reagent may be selected from the group consisting of DCIP, Tetrazolium blue, Nitro-tetrazolium blue, Water-soluble tetrazolium (WST)-1, WST-3, WST-4, WST-5, WST-8, and WST-9.

[0024] By adding a larger amount of reduced coenzyme II than reduced coenzyme I to the sample, the oxidoreductase may oxidize reduced coenzyme II to produce oxidized coenzyme II, and then transfer the electrons extracted from reduced coenzyme II to an oxidized hormone or oxidized hormone precursor to produce a hormone or hormone precursor.

[0025] The amount of oxidized coenzyme I may be greater than the amount of oxidized coenzyme II.

[0026] The oxidized form of coenzyme I is Thio-NAD ＋ It contains, and the reduced coenzyme 2 may contain NADH.

[0027] The hormone or hormone precursor may contain 25-hydroxyvitamin D or 17β-hydroxysteroid.

[0028] The oxidoreductase may have (1) sequence identity of 70% or more to the full-length amino acid sequence and 90% or more to the amino acid sequence of the homologous region, or (2) sequence identity of 90% or more to the full-length amino acid sequence, and may also have hormone or hormone precursor oxidoreductase activity.

[0029] The oxidoreductase may be one in which the amino acid residue at the position corresponding to position 187 of the amino acid sequence of the oxidoreductase of SEQ ID NO: 3 is substituted with phenylalanine, compared to the amino acid sequence of the oxidoreductase of SEQ ID NO: 1 to 12.

[0030] According to one embodiment of the present invention, a quantitative enzyme is provided which has (1) sequence identity of 70% or more with respect to the full-length amino acid sequence and 90% or more with respect to the amino acid sequence of the homologous region, or (2) sequence identity of 90% or more with respect to the full-length amino acid sequence, and which also has hormone or hormone precursor oxidoreductase activity.

[0031] The oxidoreductase may be one in which the amino acid residue at the position corresponding to position 187 of the amino acid sequence of the oxidoreductase of SEQ ID NO: 3 is substituted with phenylalanine, compared to the amino acid sequence of the oxidoreductase of SEQ ID NO: 1 to 12.

[0032] According to one embodiment of the present invention, a composition for quantifying hormones or hormone precursors is provided, comprising a quantitative enzyme and an oxidized coenzyme I, wherein the oxidoreductase oxidizes the hormone or hormone precursor contained in the sample to produce an oxidized hormone or oxidized hormone precursor, and transfers electrons extracted from the hormone or hormone precursor to the oxidized coenzyme I to produce a reduced coenzyme I.

[0033] The solution further comprises a mediator and a reagent, wherein the mediator may transfer electrons from the reduced first coenzyme to the reagent.

[0034] The oxidoreductase may further contain a larger amount of reduced coenzyme II than reduced coenzyme I, and the oxidoreductase may oxidize reduced coenzyme II to produce oxidized coenzyme II, and then transfer the electrons extracted from reduced coenzyme II to an oxidized hormone or oxidized hormone precursor to produce a hormone or hormone precursor.

[0035] The amount of oxidized coenzyme I may be greater than the amount of oxidized coenzyme II.

[0036] According to one embodiment of the present invention, a kit for quantifying hormones or hormone precursors is provided, comprising a quantitative enzyme and an oxidized coenzyme I, wherein the oxidoreductase oxidizes the hormone or hormone precursor contained in the sample to produce an oxidized hormone or oxidized hormone precursor, and transfers electrons extracted from the hormone or hormone precursor to the oxidized coenzyme I to produce a reduced coenzyme I.

[0037] According to the present invention, a novel quantitative method, a quantitative enzyme, a quantitative composition, and a quantitative kit are provided for measuring 25-hydroxyvitamin D or 17β-hydroxysteroid, which are hormones or hormone precursors, as biomarkers.

[0038] This figure shows the alignment of the amino acid sequences of ChnA (SEQ ID NO: 2) and SeHSD (SEQ ID NO: 1). This figure also shows the alignment of the amino acid sequences of PtHSD (SEQ ID NO: 3), CfHSD (SEQ ID NO: 4), CgHSD (SEQ ID NO: 5), CrHSD (SEQ ID NO: 6), CtHSD (SEQ ID NO: 7), DcHSD (SEQ ID NO: 8), SmHSD (SEQ ID NO: 9), PfHSD (SEQ ID NO: 10), and SbHSD (SEQ ID NO: 11). This figure shows the sequence identity of the amino acid sequences of PtHSD (SEQ ID NO: 3), CfHSD (SEQ ID NO: 4), CgHSD (SEQ ID NO: 5), CrHSD (SEQ ID NO: 6), CtHSD (SEQ ID NO: 7), DcHSD (SEQ ID NO: 8), SmHSD (SEQ ID NO: 9), PfHSD (SEQ ID NO: 10), SbHSD (SEQ ID NO: 11), PpHSD (SEQ ID NO: 12), ChnA (SEQ ID NO: 2), and SeHSD (SEQ ID NO: 1). This figure shows the alignment of the amino acid sequences of PtHSD (SEQ ID NO: 3) and ChnA (SEQ ID NO: 2). (a) is a schematic diagram of a sensor chip 10 according to one embodiment of the present invention, and (b) to (d) are schematic diagrams showing the components constituting the sensor chip 10. (a) is a schematic diagram of sensor 100 according to one embodiment of the present invention, and (b) is a block diagram of sensor 100 according to one embodiment of the present invention. This figure shows the results of analyzing the purity of purified PtHSD-1M mutant (PtHSD A187F mutant) using SDS-PAGE. This figure explains the principle of the activity measurement method. This figure shows the dehydrogenase activity of wild-type (wt), M203V mutant, and Y189L mutant of ChnA. This figure shows the dehydrogenase activity of wild-type (wt), Q75F mutant, and A187F mutant of PtHSD. This figure shows the dehydrogenase activity of wild-type and mutant versions of each oxidoreductase.This figure shows the quantitative analysis of calcifediol by dehydrogenase activity of the PtHSD-5M mutant (A187F / V115F / Y202L / G196F / S205A mutant (PtHSD-5M)). This figure shows the dehydrogenase activity of mutants into which amino acid substitutions have been introduced to the PtHSD-5M mutant. This figure shows the dehydrogenase activity of mutants into which amino acid substitutions have been introduced to the PtHSD-7M mutant. This figure shows the dehydrogenase activity of mutants into which amino acid substitutions have been introduced to the PtHSD-A187F mutant. This figure shows the dehydrogenase activity of the PtHSD-7M / H119D mutant. This figure shows the dehydrogenase activity of the CfHSD-7M mutant into which the amino acid substitution N152H / Q201N has been introduced to the CfHSD-5M mutant. This figure shows the dehydrogenase activity of the SD-7M mutant. This figure shows the dehydrogenase activity of the CtHSD-7M mutant, which was created by introducing the amino acid substitution N152H / R201N into CtHSD-5M. This figure shows the dehydrogenase activity of the DcHSD-7M mutant, which was created by introducing the amino acid substitution N152H / R201N into DcHSD-5M. This figure shows the dehydrogenase activity of the introduced SmHSD-6M mutant. This figure shows the dehydrogenase activity of the PfHSD-6M mutant, which was created by introducing the R199N amino acid substitution to PfHSD-5M. This figure shows the quantitative analysis of calcifediol by the dehydrogenase activity of the PtHSD-5M mutant, PtHSD-7M mutant, PtHSD-5M / N152H / Y202H mutant, or CfHSD-7M mutant.

[0039] The following describes a new quantitative method, quantitative enzyme, quantitative composition, and quantitative kit for measuring hormones or hormone precursors according to the present invention. However, the new quantitative method, quantitative enzyme, quantitative composition, and quantitative kit for measuring hormones or hormone precursors according to the present invention shall not be construed as being limited to the contents of the embodiments and examples shown below.

[0040] In one embodiment, the quantitative enzyme used in the present invention is an oxidoreductase, which is an oxidoreductase that acts on a hormone or hormone precursor substrate. For example, an enzyme capable of directly acting on 25-hydroxyvitamin D, a hormone precursor, is disclosed in Non-Patent Document 1. However, as a result of our investigation, we found that the 3-α-(or 20-β)-hydroxysteroid dehydrogenase (SeHSD: UniProt Access No. P19992, SEQ ID NO: 1) of Streptomyces exfoliatus, the SeHSD homolog of Acinetobacter sp. (ChnA: GenBank Access No. MBD1220617.1, SEQ ID NO: 2), and Pseudomonas testosterone When the 3-α-hydroxysteroid dehydrogenase (PtHSD: UniProt Access No. P80702, SEQ ID NO. 3) of testosteroni was used in a redox reaction, its activity could not be detected. Therefore, mutations were introduced into ChnA and PtHSD respectively to improve the reactivity of hormones or hormone precursors. As a result, a new quantitative method, quantitative enzyme, quantitative composition, and quantitative kit for measuring 25-hydroxyvitamin D or 17β-hydroxysteroid, which are hormones or hormone precursors, as biomarkers, were established.

[0041] Examples of substrates used in the present invention include hormones or hormone precursors used as biomarkers. For example, 25-hydroxyvitamin D (calcifediol) and 17β-hydroxysteroids can be used. Examples of hormones corresponding to 17β-hydroxysteroids include estradiol, estriol, estetrol, testosterone, dihydrotestosterone, and androstenediol. 25-hydroxyvitamin D or estradiol are preferred substrates used in the present invention.

[0042] In the present invention, the hormone or hormone precursor oxidoreductase activity is preferably, for example, oxidoreductase activity with 25-hydroxyvitamin D or estradiol as a substrate. When detecting 25-hydroxyvitamin D or estradiol in serum as a substrate, cross-reactivity with other hormones or hormone precursors becomes a problem. For this reason, the hormone or hormone precursor oxidoreductase activity is preferably greater than, for example, the oxidoreductase activity for androsterone or cholecalciferol.

[0043] In the present invention, the hormone or hormone precursor dehydrogenase activity is preferably, for example, dehydrogenase activity using 25-hydroxyvitamin D or estradiol as a substrate. When detecting 25-hydroxyvitamin D or estradiol in serum as a substrate, cross-reactivity with other hormones or hormone precursors becomes a problem. For this reason, the hormone or hormone precursor dehydrogenase activity is preferably greater than, for example, the oxidoreductase activity for androsterone or cholecalciferol.

[0044] For example, the oxidoreductase used in the present invention can be an enzyme belonging to EC number 1.1 that recognizes a hormone or hormone precursor as a substrate and has hormone or hormone precursor oxidoreductase activity. For example, an oxidoreductase belonging to EC number 1.1 that recognizes a hormone or hormone precursor as a substrate and has hormone or hormone precursor dehydrogenase activity can be used. For example, a dehydrogenase belonging to EC number 1.1.1 that recognizes a hormone or hormone precursor as a substrate and has hormone or hormone precursor dehydrogenase activity can be used. For example, a 3-α-(or 20-β)-hydroxysteroid dehydrogenase belonging to EC number 1.1.1.53 that recognizes a hormone or hormone precursor as a substrate and has hormone or hormone precursor dehydrogenase activity can be used. For example, an oxidoreductase belonging to EC number 1.1 that recognizes a hormone or hormone precursor as a substrate and has hormone or hormone precursor oxidase activity can be used. For example, a dehydrogenase belonging to EC number 1.1.3, which recognizes hormones or hormone precursors as substrates and has hormone or hormone precursor oxidase activity, can be used.

[0045] In one embodiment, the oxidoreductase may be an oxidoreductase produced by a naturally occurring microorganism, or an oxidoreductase produced by a transformed microorganism. From the viewpoint of efficient large-scale expression of the enzyme, using a transformed microorganism allows for efficient large-scale expression of the enzyme.

[0046] In one embodiment, the oxidoreductase may be a polymer or a monomer. For example, if even just one subunit (monomer) among several subunits constituting a polymeric oxidoreductase catalyzes a dehydrogenation reaction that removes hydrogen from a substrate and passes it to a hydrogen acceptor, the oxidoreductase used in the present invention may be a polymer or the subunit (monomer) in question. Furthermore, it may be composed of a partial structure of an enzyme, as long as it has hormone or hormone precursor oxidoreductase activity.

[0047] As described above, the inventors introduced mutations into ChnA to improve the reactivity of hormones or hormone precursors, and found that several mutants act on 25-hydroxyvitamin D, which is a hormone or hormone precursor. In one embodiment, the oxidoreductase of the present invention is an oxidoreductase derived from the genus Acinetobacter (ChnA: GenBank Accession No. MBD1220617.1, SEQ ID NO: 2), but oxidoreductases derived from microorganisms classified in the family Moraxellaceae are also examples, as are oxidoreductases derived from microorganisms classified in the order Pseudomonadales. In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from a microorganism classified in the genus Streptomyces, and may be an oxidoreductase derived from Streptomyces exfoliatus (SeHSD: UniProt Access No. P19992, SEQ ID NO: 1). Figure 1 shows the alignment of the amino acid sequence of ChnA (SEQ ID NO: 2) and the amino acid sequence of SeHSD (SEQ ID NO: 1). The sequence identity between the amino acid sequence of ChnA (SEQ ID NO: 2) and the amino acid sequence of SeHSD (SEQ ID NO: 1) was 37.3%.

[0048] Furthermore, examples include oxidoreductases having high sequence identity (for example, 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 Sequence ID No. 1 or 2, 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.

[0049] For example, the Y189L (ChnA-1M) mutant, in which the tyrosine at position 189 of the ChnA amino acid sequence (SEQ ID NO: 2) is modified to leucine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0050] For example, in the amino acid sequence of ChnA (SEQ ID NO: 2), the Y189L / A146L mutant, in which the alanine at position 146 is modified to leucine, the Y189L / Y244V mutant, in which the tyrosine at position 244 is modified to valine, the Y189L / L95V mutant, in which the leucine at position 95 is modified to valine, the Y189L / I151V mutant, in which the isoleucine at position 151 is modified to valine, the Y189L / F198L mutant, in which the phenylalanine at position 198 is modified to leucine, and the Y189L / Y244F mutant, in which the tyrosine at position 244 is modified to phenylalanine are more preferable because they improve 25-hydroxyvitamin D oxidoreductase activity.

[0051] For example, the M203V mutant, in which the methionine at position 203 of the ChnA amino acid sequence (SEQ ID NO: 2) is modified to valine, is preferred because it exhibits improved 17β-hydroxysteroid oxidoreductase activity.

[0052] Furthermore, oxidoreductase can also be screened by culturing microorganisms of the genus Acinetobacter under specified conditions (see, for example, the Journal of the Japanese Society for Bacteriology, 18(1), 1963), mixing the extracted solution obtained by crushing the bacterial cells with an oxidase reaction reagent or dehydrogenase reaction reagent (details described below) containing a hormone or hormone precursor, and confirming whether or not the reagent reacts with the extract.

[0053] 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 2, or DNA having the base sequence of SeHSD (SEQ ID NO: 13) or ChnA (SEQ ID NO: 14). In one embodiment, the present invention provides DNA that has a nucleotide sequence having 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 Sequence ID No. 13 or 14, and that encodes a protein having oxidoreductase activity.

[0054] The inventors further introduced mutations into PtHSD to improve the reactivity of hormones or hormone precursors, and found that several mutants acted on 25-hydroxyvitamin D or 17β-hydroxysteroids, which are hormones or hormone precursors. In one embodiment, the oxidoreductase of the present invention is an oxidoreductase derived from Pseudomonas testosterone (PtHSD: UniProt Accession No. P80702, SEQ ID NO: 3), but it may also be an oxidoreductase derived from a microorganism classified in the genus Pseudomonas, an oxidoreductase derived from a microorganism classified in the family Pseudomonadaceae, or an oxidoreductase derived from a microorganism classified in the order Pseudomonales. In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from a microorganism classified as belonging to the genus Comamonas sp., and may be an oxidoreductase derived from Comamonas fluminis (CfHSD: NCBI Reference Sequence No. WP_218242984.1, SEQ ID NO: 4), and may be an oxidoreductase derived from Comamonas guangdonggensis (CgHSD: NCBI Reference Sequence No. WP_369340578.1, SEQ ID NO: 5), and Comamonas resistens It may be an oxidoreductase derived from *C. resistance* (CrHSD: NCBI Reference Sequence No. WP_283487925.1, SEQ ID NO. 6), or an oxidoreductase derived from *C. resistance* testosteroni (*C. resistance* (CtHSD: NCBI Reference Sequence No. WP_325801698.1, SEQ ID NO. 7)).In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from a microorganism classified in the genus Diaphorobacter (DcHSD: NCBI Reference Sequence No. WP_194770597.1, SEQ ID NO: 8). In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from a microorganism classified in the genus Sphaerotylus sp., and may be an oxidoreductase derived from Sphaerotylus microaerophilus (SmHSD: NCBI Reference Sequence No. WP_251969518.1, SEQ ID NO: 9). In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from a microorganism classified as belonging to the genus Pseudomonas sp., and may be an oxidoreductase derived from Pseudomonas flexibilis (PfHSD: NCBI Reference Sequence No. WP_039560249.1, SEQ ID NO: 10), or an oxidoreductase derived from Pseudomonas putida (PpHSD: NCBI Reference Sequence No. WP_077185960.1, SEQ ID NO: 12). In one embodiment, the oxidoreductase of the present invention may be an oxidoreductase derived from a microorganism classified in the genus Steroidobactera (Steroidobacteraceae sp.), and may be an oxidoreductase derived from Steroidobacteraceae bacterium (SbHSD: GenBank Access No. MBP6105028.1, SEQ ID NO: 11).Figure 2 shows the alignment of the amino acid sequences of PtHSD (SEQ ID NO: 3), CfHSD (SEQ ID NO: 4), CgHSD (SEQ ID NO: 5), CrHSD (SEQ ID NO: 6), CtHSD (SEQ ID NO: 7), DcHSD (SEQ ID NO: 8), SmHSD (SEQ ID NO: 9), PfHSD (SEQ ID NO: 10), and SbHSD (SEQ ID NO: 11). Figure 3 shows the sequence identity of the amino acid sequences of the following: sequence (SEQ ID NO: 4), CgHSD (SEQ ID NO: 5), CrHSD (SEQ ID NO: 6), CtHSD (SEQ ID NO: 7), DcHSD (SEQ ID NO: 8), SmHSD (SEQ ID NO: 9), PfHSD (SEQ ID NO: 10), SbHSD (SEQ ID NO: 11), PpHSD (SEQ ID NO: 12), ChnA (SEQ ID NO: 2), and SeHSD (SEQ ID NO: 1).

[0055] Furthermore, examples include oxidoreductases having high sequence identity (for example, 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 any one of Sequence IDs 3 to 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.

[0056] Furthermore, examples include oxidoreductases having high sequence identity (for example, 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 any one of Sequence IDs 3 to 11, 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.

[0057] For example, the A187F mutant (PtHSD-1M), in which the alanine at position 187 of the amino acid sequence of PtHSD (SEQ ID NO: 3) is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0058] For example, in the A187F mutant of the amino acid sequence (SEQ ID NO: 3) of PtHSD, there are A187F / Q75L mutants in which glutamine at position 75 is replaced with leucine, A187F / Q75W mutants in which glutamine at position 75 is replaced with tryptophan, A187F / V115F mutants in which valine at position 115 is replaced with phenylalanine, A187F / V115W mutants in which valine at position 115 is replaced with tryptophan, A187F / A116F mutants in which alanine at position 116 is replaced with phenylalanine, A187F / H119F mutants in which histidine at position 119 is replaced with phenylalanine, A187F / H119W mutants in which histidine at position 119 is replaced with tryptophan, and asparagine at position 152 is replaced with phenylalanine. The A187F / N152F mutant, modified with lanin; the A187F / N152L mutant, modified with leucine instead of asparagine at position 152; the A187F / G196F mutant, modified with phenylalanine instead of glycine at position 196; the A187F / G196W mutant, modified with tryptophan instead of glycine at position 196; the A187F / Y202L mutant, modified with leucine instead of tyrosine at position 202; the A187F / S205A mutant, modified with alanine instead of serine at position 205; the A187F / S205F mutant, modified with phenylalanine instead of serine at position 205; and the A187F / S205V mutant, modified with valine instead of serine at position 205, are more preferable because they exhibit improved 25-hydroxyvitamin D oxidoreductase activity.

[0059] For example, among the A187F mutants of the amino acid sequence (SEQ ID NO: 3) of PtHSD, the A187F / N152H mutant, in which asparagine at position 152 is modified to histidine; the A187F / R201H mutant, in which arginine at position 201 is modified to histidine; the A187F / R201N mutant, in which arginine at position 201 is modified to asparagine; the A187F / Y202H mutant, in which tyrosine at position 202 is modified to histidine; the A187F / S205H mutant, in which serine at position 205 is modified to histidine; and the A187F / P74I mutant, in which proline at position 74 is modified to isoleucine, are more preferable because they exhibit improved 25-hydroxyvitamin D oxidoreductase activity.

[0060] For example, among the A187F / V115F mutants of the amino acid sequence (SEQ ID NO: 3) of PtHSD, the A187F / V115F / N152F mutant, in which asparagine at position 152 is modified to phenylalanine; the A187F / V115F / G196F mutant, in which glycine at position 196 is modified to phenylalanine; the A187F / V115F / Y202L mutant, in which tyrosine at position 202 is modified to leucine; and the A187F / V115F / S205A mutant, in which serine at position 205 is modified to alanine, are even more preferable because they show improved 25-hydroxyvitamin D oxidoreductase activity. In the A187F / N152F mutant of the amino acid sequence (SEQ ID NO: 3) of PtHSD, the A187F / N152F / H119W mutant, in which histidine at position 119 is modified to tryptophan; the A187F / N152F / G196F mutant, in which glycine at position 196 is modified to phenylalanine; the A187F / N152F / Y202L mutant, in which tyrosine at position 202 is modified to leucine; and the A187F / N152F / S205A mutant, in which serine at position 205 is modified to alanine, are even more preferable because they exhibit improved 25-hydroxyvitamin D oxidoreductase activity.

[0061] For example, among the A187F / V115F / Y202L mutants of the amino acid sequence (SEQ ID NO: 3) of PtHSD, the A187F / V115F / Y202L / H119W mutant, in which histidine at position 119 is modified to tryptophan; the A187F / V115F / Y202L / N152F mutant, in which asparagine at position 152 is modified to phenylalanine; the A187F / V115F / Y202L / G196F mutant, in which glycine at position 196 is modified to phenylalanine; and the A187F / V115F / Y202L / S205A mutant, in which serine at position 205 is modified to alanine, are even more preferable because they exhibit improved 25-hydroxyvitamin D oxidoreductase activity.

[0062] For example, among the A187F / V115F / Y202L / G196F mutants of the amino acid sequence (SEQ ID NO: 3) of PtHSD, the A187F / V115F / Y202L / G196F / H119W mutant, in which histidine at position 119 is modified to tryptophan; the A187F / V115F / Y202L / G196F / N152F mutant, in which asparagine at position 152 is modified to phenylalanine; and the A187F / V115F / Y202L / G196F / S205A mutant (PtHSD-5M), in which serine at position 205 is modified to alanine, are particularly preferred because they improve 25-hydroxyvitamin D oxidoreductase activity. The V115F / Y202L / G196F / S205A mutant (PtHSD-5M / F187A), which retains alanine at position 187 in the amino acid sequence (SEQ ID NO: 3) of PtHSD, showed reduced 25-hydroxyvitamin D oxidoreductase activity.

[0063] For example, in the A187F / V115F / G196F / S205A mutant of the amino acid sequence of PtHSD (SEQ ID NO: 3), the A187F / V115F / Y202H / G196F / S205A mutant, in which the tyrosine at position 202 is changed to histidine, is particularly preferred because it improves 25-hydroxyvitamin D oxidoreductase activity.

[0064] For example, in the A187F / V115F / Y202L / G196F / S205A mutant (PtHSD-5M) of the amino acid sequence (SEQ ID NO: 3) of PtHSD, the A187F / V115F / Y202L / G196F / S205A / N152H mutant has the asparagine at position 152 replaced with histidine, the A187F / V115F / Y202L / G196F / S205A / N152Q mutant has the asparagine at position 152 replaced with glutamine, and the arginine at position 201 is replaced with histidine. The A187F / V115F / Y202L / G196F / S205A / R201H mutant, the A187F / V115F / Y202L / G196F / S205A / R201N mutant, and the A187F / V115F / Y202L / G196F / S205A / R201Q mutant, which have been modified to include asparagine at position 201, are particularly preferred because they exhibit improved 25-hydroxyvitamin D oxidoreductase activity.

[0065] For example, in the A187F / V115F / Y202L / G196F / S205A / N152H mutant (PtHSD-6M) of the amino acid sequence (SEQ ID NO: 3) of PtHSD, the A187F / V115F / Y202L / G196F / S205A / N152H / R201N mutant (PtHSD-7M), in which the arginine at position 201 is modified to asparagine, is particularly preferred because it improves 25-hydroxyvitamin D oxidoreductase activity.

[0066] For example, in the A187F / V115F / Y202L / G196F / S205A / N152H / R201N mutant (PtHSD-7M) of the amino acid sequence (SEQ ID NO: 3) of PtHSD, the following mutants were found: A187F / V115F / Y202L / G196F / S205A / N152H / R201N / P74A mutant in which the proline at position 74 was changed to alanine; A187F / V115F / Y202L / G196F / S205A / N152H / R201N / P74F mutant in which the proline at position 74 was changed to phenylalanine; and A187F / V115F / Y202L / G196F / S205A mutant in which the proline at position 74 was changed to isoleucine. The / N152H / R201N / P74I mutant, the A187F / V115F / Y202L / G196F / S205A / N152H / R201N / P74L mutant in which proline at position 74 is replaced with leucine, the A187F / V115F / Y202L / G196F / S205A / N152H / R201N / P74V mutant in which proline at position 74 is replaced with valine, and the A187F / V115F / Y202L / G196F / S205A / N152H / R201N / H119D mutant in which histidine at position 119 is replaced with aspartic acid are particularly preferred because they improve 25-hydroxyvitamin D oxidoreductase activity.

[0067] For example, PtHSD A187F mutant, A187F / Q75L mutant, A187F / Q75W mutant, A187F / V115F mutant, A187F / V115W mutant, A187F / A116F mutant, A187F / H119F mutant, A187F / H119W mutant, A187F / N152F mutant, A187F / N152L mutant, A187F / G196F mutant, A187F / G196W mutant Variants, A187F / Y202L variant, A187F / S205A variant, A187F / S205F variant, A187F / S205V variant, A187F / V115F / N152F variant, A187F / V115F / G196F variant, A187F / V115F / Y202L variant, A187F / V115F / S205A variant, A187F / N152F / H119W variant, A187F / N152F / G196F variant, A187F / N152F / Y202L variant, A187F / N152F / S205A variant, A187F / V115F / Y202L / H119W variant, A187F / V115F / Y202L / N152F variant, A187F / V115F / Y202L / G196F variant, A187F / V115F / Y202L / S205A variant, A187F / V115F / Y202L / G The 196F / H119W mutant, A187F / V115F / Y202L / G196F / N152F mutant, and A187F / V115F / Y202L / G196F / S205A mutant may be further modified by changing the proline at position 185 to glycine or alanine, or by changing the threonine at position 188 to alanine or serine, in order to improve 25-hydroxyvitamin D oxidoreductase activity.

[0068] For example, the A187F mutant (CfHSD-1M), in which the alanine at position 187 of the amino acid sequence (SEQ ID NO: 4) of CfHSD is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity. The A187F / V115F / Y202L / G196F / S205A mutant (CfHSD-5M), in which the alanine at position 187 of CfHSD (SEQ ID NO: 4) is modified to phenylalanine, the valine at position 115 to phenylalanine, the tyrosine at position 202 to leucine, the glycine at position 196 to phenylalanine, and the serine at position 205 to alanine, is particularly preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0069] For example, in the A187F / V115F / Y202L / G196F / S205A mutant (CfHSD-5M) of the amino acid sequence (SEQ ID NO: 4) of CfHSD, the A187F / V115F / Y202L / G196F / S205A / N152H / Q201N mutant (CfHSD-7M), in which asparagine at position 152 is modified to histidine and glutamine at position 201 is modified to asparagine, is even more preferable because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0070] For example, the A187F mutant (CgHSD-1M), in which the alanine at position 187 of the amino acid sequence (SEQ ID NO: 5) of CgHSD is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity. The A187F / V115F / Y202L / G196F / S205A mutant (CgHSD-5M), in which the alanine at position 187 of CgHSD (SEQ ID NO: 5) is modified to phenylalanine, the valine at position 115 to phenylalanine, the tyrosine at position 202 to leucine, the glycine at position 196 to phenylalanine, and the serine at position 205 to alanine, is particularly preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0071] For example, in the A187F / V115F / Y202L / G196F / S205A mutant (CgHSD-5M) of the amino acid sequence (SEQ ID NO: 5) of CgHSD, the A187F / V115F / Y202L / G196F / S205A / N152H / R201N mutant (CgHSD-7M), in which asparagine at position 152 is modified to histidine and arginine at position 201 is modified to asparagine, is even more preferable because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0072] For example, the A187F mutant (CrHSD-1M), in which the alanine at position 187 of the amino acid sequence of CrHSD (SEQ ID NO: 6) is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity. The A187F / V115F / Y202L / G196F / S205A mutant (CrHSD-5M), in which the alanine at position 187 of CrHSD (SEQ ID NO: 6) is modified to phenylalanine, the valine at position 115 to phenylalanine, the tyrosine at position 202 to leucine, the glycine at position 196 to phenylalanine, and the serine at position 205 to alanine, is particularly preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0073] For example, the A187F mutant (CtHSD-1M), in which the alanine at position 187 of the amino acid sequence of CtHSD (SEQ ID NO: 7) is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity. The A187F / V115F / Y202L / G196F / S205A mutant (CtHSD-5M), in which the alanine at position 187 of CtHSD (SEQ ID NO: 7) is modified to phenylalanine, the valine at position 115 to phenylalanine, the tyrosine at position 202 to leucine, the glycine at position 196 to phenylalanine, and the serine at position 205 to alanine, is particularly preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0074] For example, in the A187F / V115F / Y202L / G196F / S205A mutant (CtHSD-5M) of the amino acid sequence (SEQ ID NO: 7) of CtHSD, the A187F / V115F / Y202L / G196F / S205A / N152H / R201N mutant (CtHSD-7M), in which the asparagine at position 152 is modified to histidine and the arginine at position 201 is modified to asparagine, is even more preferable because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0075] For example, the A187F mutant (DcHSD-1M), in which the alanine at position 187 of the DcHSD amino acid sequence (SEQ ID NO: 8) is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity. The A187F / V115F / F202L / S196F / S205A mutant (DcHSD-5M), in which the alanine at position 187 of the DcHSD amino acid sequence (SEQ ID NO: 8) is modified to phenylalanine, the valine at position 115 to phenylalanine, the phenylalanine at position 202 to leucine, the serine at position 196 to phenylalanine, and the serine at position 205 to alanine, is particularly preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0076] For example, in the A187F / V115F / F202L / S196F / S205A mutant (DcHSD-5M) of the amino acid sequence (SEQ ID NO: 8) of DcHSD, the A187F / V115F / F202L / S196F / S205A / N152H / R201N mutant (DcHSD-7M), in which asparagine at position 152 is modified to histidine and arginine at position 201 is modified to asparagine, is particularly preferred because it improves 25-hydroxyvitamin D oxidoreductase activity.

[0077] For example, the A187F mutant (SmHSD-1M), in which the alanine at position 187 of the amino acid sequence of SmHSD (SEQ ID NO: 9) is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity. The A187F / V115F / Y202L / G196F mutant (SmHSD-4M), in which the alanine at position 187 of SmHSD (SEQ ID NO: 9) is modified to phenylalanine, the valine at position 115 to phenylalanine, the tyrosine at position 202 to leucine, and the glycine at position 196 to phenylalanine, is particularly preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0078] For example, in the A187F / V115F / Y202L / G196F mutant (SmHSD-4M) of the amino acid sequence (SEQ ID NO: 9) of SmHSD, the A187F / V115F / Y202L / G196F / N152H / R201N mutant (SmHSD-6M), in which the asparagine at position 152 is modified to histidine and the arginine at position 201 is modified to asparagine, is even more preferable because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0079] For example, the A185F mutant (PfHSD-1M), in which the alanine at position 185 of the amino acid sequence (SEQ ID NO: 10) of PfHSD is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity. The A185F / I115F / Y200L / S194F / S203A mutant (PfHSD-5M), in which the alanine at position 185 of the amino acid sequence (SEQ ID NO: 10) of PfHSD is modified to phenylalanine, the isoleucine at position 115 to phenylalanine, the tyrosine at position 200 to leucine, the serine at position 194 to phenylalanine, and the serine at position 203 to alanine, is particularly preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0080] For example, in the A185F / I115F / Y200L / S194F / S203A mutant (PfHSD-5M) of the amino acid sequence (SEQ ID NO: 10) of PfHSD, the A185F / I115F / Y200L / S194F / S203A / R199N mutant (PfHSD-6M), in which the arginine at position 199 is modified to asparagine, is even more preferable because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0081] For example, the M187F mutant (SbHSD-1M), in which the methionine at position 187 of the amino acid sequence of SbHSD (SEQ ID NO: 11) is modified to phenylalanine, is preferred because it exhibits improved 25-hydroxyvitamin D oxidoreductase activity.

[0082] For example, the amino acid sequence of PpHSD (SEQ ID NO: 12) is preferred because it has 25-hydroxyvitamin D oxidoreductase activity.

[0083] For example, the wild-type amino acid sequences of PtHSD (SEQ ID NO: 3), CfHSD (SEQ ID NO: 4), CgHSD (SEQ ID NO: 5), CrHSD (SEQ ID NO: 6), CtHSD (SEQ ID NO: 7), DcHSD (SEQ ID NO: 8), and SmHSD (SEQ ID NO: 9) are preferred because they possess 17β-hydroxysteroid oxidoreductase activity. For example, the Q75F mutant (PtHSD-Q75F), in which the glutamine at position 75 of the amino acid sequence of PtHSD (SEQ ID NO: 3) is modified to phenylalanine, is more preferred because it exhibits improved 17β-hydroxysteroid oxidoreductase activity.

[0084] Furthermore, oxidoreductase can also be screened by culturing Pseudomonas testosterone microorganisms under specified conditions (see, for example, the Journal of the Japanese Society for Bacteriology, 18(1), 1963), mixing the extracted solution obtained by disrupting the bacterial cells with an oxidase reaction reagent or dehydrogenase reaction reagent (details described below) containing a hormone or hormone precursor, and confirming whether or not the reagent reacts with the extract.

[0085] In one embodiment, the present invention provides DNA encoding oxidoreductase. In one embodiment, the present invention provides DNA encoding any one of the amino acid sequences shown in SEQ ID NOs: 3 to 12, or DNA having the nucleotide sequence of PtHSD (SEQ ID NO: 15), CfHSD (SEQ ID NO: 16), CgHSD (SEQ ID NO: 17), CrHSD (SEQ ID NO: 18), CtHSD (SEQ ID NO: 19), DcHSD (SEQ ID NO: 20), SmHSD (SEQ ID NO: 21), PfHSD (SEQ ID NO: 22), PpHSD (SEQ ID NO: 24), or SbHSD (SEQ ID NO: 23). In one embodiment, the present invention provides DNA that has a base sequence having 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 any one of the base sequences shown in Sequence ID No. 15 to 24, and that encodes a protein having oxidoreductase activity.

[0086] In one embodiment, the reaction conditions for oxidoreductase can be any conditions that act on a hormone or hormone precursor and efficiently catalyze an oxidation or reduction reaction. Generally, enzymes have an optimal temperature and pH at which they exhibit the highest activity. Therefore, reaction conditions near the optimal temperature and pH are preferable. In one embodiment, the reaction conditions for oxidoreductase are determined by comprehensively considering factors such as appropriate conditions for components other than the enzyme, such as chromogenic reagents, mediators, enzyme stabilizers, and stabilizers for the measurement sample, as well as compatibility with the measuring device. The measurement method of the present invention also includes a method for quantifying a hormone or hormone precursor under conditions other than the optimal conditions for the enzyme alone.

[0087] [Identity or Similarity of Amino Acid Sequences] The identity or similarity of amino acid sequences can be calculated using programs such as maximum matching and search homology in GENETYX Ver. 11 or Ver. 14 (manufactured by GENETYX Corporation) or programs such as maximum matching and multiple alignment in DNASIS Pro (manufactured by Hitachi Solutions Corporation). To calculate amino acid sequence identity, when two or more oxidoreductases are aligned, the positions of identical amino acids in those two or more oxidoreductases can be examined. Based on this information, identical regions in the amino acid sequences can be determined.

[0088] Furthermore, the positions of similar amino acids can be investigated in two or more oxidoreductases. 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 judged to be similar when multiple amino acid sequences are aligned are sometimes called similar amino acids. In the variants of the present invention, amino acid substitutions may be due to substitutions between such similar amino acids. Through such alignment, it is possible to investigate the regions where the amino acid sequences are identical and the positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, homology regions (conserved regions) in the amino acid sequences can be determined.

[0089] In this specification, "homologous region" refers to a region consisting of amino acids in which, when two or more oxidoreductases are aligned, the amino acids at the corresponding positions of a reference oxidoreductase and a comparison oxidoreductase are identical.

[0090] For example, Figure 1 shows the alignment of the amino acid sequence of SeHSD (SEQ ID NO: 1) and the amino acid sequence of ChnA (SEQ ID NO: 2). The positions of identical amino acids are indicated by squares. In one embodiment, the homology region of oxidoreductase is defined as follows, relative to the oxidoreductase shown in the amino acid sequence of ChnA (SEQ ID NO: 2): positions 3, 8, 12-14, 18, 20, 24, 31, 34-35, 38, 42, 50, 54-55, 58, 69-70, 75, 78, 82-83, 90-94, 102, 105, 107, 110, 112, 114, 116-117, 120-121, 124, 129-130, 1 This region consists of amino acid sequences at positions 34-137, 139-141, 144, 146-148, 150-155, 157, 159, 161, 163-164, 166-167, 169, 171-172, 174, 180-181, 183-188, 192-193, 196-197, 208, 213-214, 216, 219, 221-223, 226, 229-232, 235, 237-239, 244-246, and 249.

[0091] The amino acid sequence in the homology region of the oxidoreductase of the present invention has sequence identity of 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 the amino acid sequence in the homology region of the amino acid sequence of ChnA (SEQ ID NO: 2).

[0092] For example, Figure 2 shows the amino acid sequences of PtHSD (SEQ ID NO: 3), CfHSD (SEQ ID NO: 4), CgHSD (SEQ ID NO: 5), CrHSD (SEQ ID NO: 6), CtHSD (SEQ ID NO: 7), DcHSD (SEQ ID NO: 8), SmHSD (SEQ ID NO: 9), PfHSD (SEQ ID NO: 10), and SbHSD (SEQ ID NO: 11) aligned. The positions of identical amino acids are indicated by squares. In one embodiment, the homology region of oxidoreductase is located at positions 1, 8, 12, 14-16, 21, 25-26, 32, 36, 41-43, 47-48, 59, 64-65, 67, 70-71, 73, 85-89, 95, 101, 104, 107-110, 114, 116, and 126, relative to the oxidoreductase shown in the amino acid sequence of PtHSD (SEQ ID NO: 3). This region consists of the amino acid sequences at positions 130, 134, 147, 154-155, 157-159, 163, 167, 173, 177, 179-181, 184-186, 190-195, 199-200, 209, 212, 214-215, 219, 221, 223, 228-229, 234, 238-239, 245-247, 249-350, 253, and 257.

[0093] The amino acid sequence in the homology region of the oxidoreductase of the present invention has sequence identity of 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 the amino acid sequence in the homology region of the amino acid sequence of PtHSD (SEQ ID NO: 3).

[0094] [Corresponding Position] The position corresponding to position 187 in the amino acid sequence shown in Sequence ID No. 3 refers to the position in the amino acid sequence of an oxidoreductase from another species that corresponds to position 187 in the amino acid sequence of Sequence ID No. 3 when aligned with the amino acid sequence of Sequence ID No. 3.

[0095] One method for identifying "corresponding positions" is to first compare amino acid sequences using a known algorithm such as the Lippmann-Parson method, and then perform multiple alignment to give maximum identity to the conserved amino acid residues present in the amino acid sequences of each oxidoreductase. By aligning the amino acid sequences of oxidoreductases in this way, the positions of homologous amino acid residues in each oxidoreductase sequence can be determined, regardless of insertions or deletions in the amino acid sequences. Subsequently, if necessary, secondary structures such as α-helices, β-sheets, and coils can be predicted using known secondary structure prediction algorithms.

[0096] For example, the secondary structure of an amino acid sequence, such as the one shown in Sequence ID No. 3 or a suitable amino acid sequence of an oxidoreductase, can be predicted using a secondary structure prediction algorithm. Examples of secondary structure prediction tools include Jpred 3 (Cole C et al. The Jpred 3 secondary structure prediction server. Nucleic Acids Res. 2008, W197-201) and Jpred 4 (Drozdetsky A et al. (2015) JPred 4: a protein secondary structure prediction server, Nucleic Acids Res., doi:10.1093 / nar / gkv332), which implement the JNet algorithm.

[0097] For example, Figure 4 shows the alignment of the amino acid sequence of PtHSD (SEQ ID NO: 3) and the amino acid sequence of ChnA (SEQ ID NO: 2). In Figures 2 and 4, the arrows indicate the position corresponding to position 187 in the amino acid sequence of PtHSD (SEQ ID NO: 3).

[0098] [Vectors] Any vector known to those skilled in the art, such as bacteriophages and cosmids, can be used as vectors in the present invention. Specifically, for example, pCold II (manufactured by Takara Bio Inc.), pUC18 (manufactured by Takara Bio Inc.), pBluescriptII SK+ (manufactured by STRATAGENE Inc.), pET-22b(+) (manufactured by Merck Ltd.), pKK223-3 (manufactured by addgene Inc.) are preferred.

[0099] [Construction of Expression Plasmid] The oxidoreductase expression plasmid according to the present invention can be obtained by commonly used methods. 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, the 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 the DNA fragment encoding the oxidoreductase according to the present invention.

[0100] Alternatively, a DNA fragment encoding the oxidoreductase according to the present invention may be chemically synthesized, and the DNA fragment may be ligated to a vector to obtain a plasmid having the DNA encoding the oxidoreductase according to the present invention.

[0101] [Mutation of the Oxidoreductase Gene] Mutation of the oxidoreductase gene can be carried out by any known method depending on the desired mutation. Specifically, methods such as contacting and acting on the oxidoreductase gene or recombinant DNA containing the gene with a mutagenic drug, ultraviolet irradiation, genetic engineering techniques, or protein engineering techniques can be widely used.

[0102] Examples of mutagenic drugs used in the above-mentioned mutagenesis treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrite, sulfite, hydrazine, formic acid, or 5-bromouracil.

[0103] The above contact and action conditions can be adjusted according to the type of drug used, and are not particularly limited as long as the desired mutation can be induced in the oxidoreductase gene. Typically, the desired mutation can be induced by contact and action for 10 minutes or more, preferably 10 to 180 minutes, at a drug concentration of 0.5 to 12 M and a reaction temperature of 20 to 80°C. Even when ultraviolet irradiation is performed, it can be carried out according to the conventional method as described above (Modern Chemistry, pp. 24-30, June 1989).

[0104] As a method utilizing protein engineering techniques, a technique generally known as Site-Specific Mutagenesis (Site-Directed Mutagenesis) can be used. Specific methods for transforming the base sequence in DNA and converting the encoded amino acids include the use of commercially available kits (e.g., Transformer Mutagenesis Kit; Clonetech; EXOIII / Mung Bean Deletion Kit; Stratagene; Quick Change Site Directed Mutagenesis Kit; Stratagene).

[0105] Alternatively, a method known as the general PCR (polymerase chain reaction) can be used. In addition to the gene modification methods described above, the desired modified oxidoreductase gene can also be directly synthesized by organic synthesis or enzymatic synthesis.

[0106] When determining or confirming the DNA base sequence of the oxidoreductase gene obtained by the above method, this can be done using, for example, an Applied Biosystems 3730xl DNA analyzer (manufactured by Thermo Fisher Scientific).

[0107] [Transformation and Transduction] The oxidoreductase gene obtained as described above can be incorporated into a vector such as a bacteriophage, cosmid, or plasmid used for the transformation of prokaryotic or eukaryotic cells by conventional methods, and the host corresponding to each vector can be transformed by conventional methods. For example, the obtained recombinant DNA can be used to transform any host, such as a microorganism belonging to the genus Eschericia, specifically Eschericia coli strain K-12, preferably Eschericia coli strain JM109, Eschericia coli strain DH5α, or Eschericia coli strain B, preferably Eschericia coli strain BL21, to obtain the respective strains.

[0108] Furthermore, yeast can be cited as an example of a eukaryotic host cell. Examples of microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Saccharomyces, Pichia, and Candida. The inserted gene may include a marker gene that enables the selection of transformed cells. Examples of marker genes include genes that complement the host's nutritional requirements, such as URA3 and TRP1. It is also desirable that the inserted gene includes a promoter or other regulatory sequence (e.g., secretory signal sequence, enhancer sequence, terminator sequence, polyadenylation sequence, etc.) that enables the expression of the gene of the present invention in the host cell. Specific examples of promoters include the GAL1 promoter and the ADH1 promoter. As a method for transforming yeast, known methods, such as the lithium acetate method (Methods Mol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003) 481-484), can be suitably used, but the method is not limited to these. Transformation can be carried out using any method, including the spheroplast method and the glass bead method.

[0109] Furthermore, other examples of eukaryotic host cells include filamentous fungi such as those of the genera Aspergillus and Trichoderma. The method for producing transformants of filamentous fungi is not particularly limited, and one example is to insert a gene encoding oxidoreductase into the host filamentous fungus in a manner that expresses it, according to a conventional method. Specifically, a DNA construct is prepared by inserting a gene encoding oxidoreductase between an expression-inducing promoter and a terminator, and then the host filamentous fungus is transformed with the DNA construct containing the gene encoding oxidoreductase to obtain a transformant that overexpresses the gene encoding oxidoreductase. In this specification, a DNA fragment consisting of an expression-inducing promoter, a gene encoding oxidoreductase, and a terminator, and a recombinant vector containing said DNA fragment, prepared for transforming a host filamentous fungus, are collectively referred to as a DNA construct.

[0110] The method for inserting the gene encoding oxidoreductase into a host filamentous fungus in a manner in which it is expressed is not particularly limited, but examples include methods for directly inserting it onto the chromosome of the host organism using homologous recombination, and methods for introducing it into the host filamentous fungus by ligating it onto a plasmid vector.

[0111] In methods utilizing homologous recombination, a DNA construct can be ligated between sequences homologous to the upstream and downstream regions of a recombination site on a chromosome and inserted into the genome of the host fungus. By overexpressing the construct within the host fungus under the control of its own high-expression promoter, a transformant can be obtained through self-cloning. The high-expression promoter is not particularly limited, but examples include the promoter region of the translation elongation factor TEF1 gene (tef1), the promoter region of the α-amylase gene (amy), and the promoter region of the alkaline protease gene (alp).

[0112] In the vector-based method, a DNA construct can be incorporated into a plasmid vector used for filamentous fungal transformation using a standard method, and the corresponding host filamentous fungus can then be transformed using a standard method.

[0113] Such suitable vector-host systems are not particularly limited as long as they are capable of producing oxidoreductase in the host filamentous fungus, and examples include the pUC19 and filamentous fungus system, and the pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and filamentous fungus system.

[0114] While it is preferable to introduce the DNA construct into the chromosome of the host filamentous fungus, another method is to incorporate the DNA construct into an autonomously replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)), thereby enabling its use without chromosome introduction.

[0115] The DNA construct may include marker genes to enable the selection of transformed cells. The marker genes are not particularly limited and include, for example, genes that complement the host's nutritional requirements, such as pyrG, niaD, and adeA, and drug resistance genes to drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably includes promoters, terminators, and other regulatory sequences (e.g., enhancers, polyadenylation sequences) that enable the overexpression of genes encoding oxidoreductase in host cells. The promoters are not particularly limited but include appropriate inductive promoters and constitutive promoters, such as the tef1 promoter, alp promoter, and amy promoter. The terminators are also not particularly limited but include, for example, the alp terminator, amy terminator, and tef1 terminator.

[0116] In DNA constructs, the expression regulatory sequence of the gene encoding oxidoreductase is not necessarily required if the DNA fragment containing the gene encoding oxidoreductase to be inserted contains a sequence with expression regulatory function. Furthermore, when transformation is performed using co-transformation, the DNA construct may not need to contain a marker gene.

[0117] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 promoter, the gene encoding oxidoreductase, the alp terminator, and the pyrG marker gene are ligated to the In-Fusion Cloning Site located at the multi-cloning site of pUC19.

[0118] 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 can be used (see, for example, Mol. Gen. Genet. 218, 99-104, 1989, Japanese Patent Publication No. 2007-222055). The culture medium for regenerating the transformed filamentous fungus should be appropriate depending on the host filamentous fungus and the transformation marker gene used. For example, if Aspergillus soybean 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 Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2 M sorbitol.

[0119] [Method for preparing the enzyme] The method for preparing oxidoreductase according to the present invention will be described below.

[0120] A strain of E. coli or the like is transformed with a plasmid having DNA encoding oxidoreductase according to the present invention to obtain a strain of E. coli or the like having DNA encoding oxidoreductase according to the present invention.

[0121] [Recombinant Expression of Enzymes] A strain of Escherichia coli or the like having DNA encoding the oxidoreductase according to the present invention is cultured in a culture medium. When culturing microbial host cells, it is fine to culture them at a culture temperature of 10 to 42°C, preferably around 25°C for several hours to several days, and more preferably at around 25°C for 1 to 7 days, by aerated stirring deep culture, shaking culture, static culture, etc. Any conventional culture medium for culturing filamentous fungi, i.e., one that contains carbon sources, nitrogen sources, inorganic substances, and other nutrients in appropriate proportions, can be used, whether it is a synthetic medium or a natural medium. Furthermore, as a culture medium for the above-mentioned microbial host cells, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or extract from soybeans or wheat bran are used, to which one or more inorganic salts such as sodium chloride, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate are added, and carbohydrate raw materials, vitamins, etc. are added as needed.

[0122] The culture conditions can be those commonly known to those skilled in the art for culturing filamentous fungi. For example, the initial pH of the culture medium can be adjusted to 5-10, the culture temperature to 20-40°C, and the culture time to several hours to several days, preferably 1-7 days, more preferably 2-5 days, and can be set as appropriate. The culture method is not particularly limited, and deep culture with aeration and stirring, shaking culture, and static culture can be employed, but it is preferable to culture under conditions that ensure sufficient dissolved oxygen. For example, an example of a culture medium and culture conditions for culturing Aspergillus microorganisms is 3-5 days of shaking culture at 30°C and 160 rpm using DPY medium.

[0123] After the culturing is complete, the oxidoreductase of the present invention is collected from the culture. This can be done using conventional, known enzyme collection methods. For example, the supernatant fraction of the culture medium can be collected, or the bacterial cells can be subjected to ultrasonic disruption, grinding, etc., by conventional methods, or the enzyme can be extracted using a lytic enzyme such as lysozyme or yatarase, or the enzyme can be released from the bacterial cells by lysis by shaking or standing in the presence of toluene, etc. Then, the solid portion of this solution is removed by filtration, centrifugation, etc., and if necessary, nucleic acids are removed with streptomycin sulfate, protamine sulfate, or manganese sulfate, etc. Ammonium sulfate, alcohol, acetone, etc. are added to the solution to fractionate it, and the precipitate is collected to obtain the crude enzyme of the oxidoreductase of the present invention.

[0124] [Enzyme Purification] Any method that can purify the enzyme from the crude enzyme solution is acceptable. For example, the purified oxidoreductase enzyme preparation of the present invention can be obtained by appropriately selecting or combining methods such as gel filtration using Sephadex, Ultrogel, or Biogel; adsorption elution using ion exchanger; electrophoresis using polyacrylamide gel; adsorption elution using hydroxyapatite; sedimentation methods such as sucrose density gradient centrifugation; affinity chromatography; or fractionation using molecular sieve membrane or hollow fiber membrane.

[0125] [Measuring Enzyme Activity] Any method is acceptable for measuring enzyme activity, as long as it directly or indirectly measures the products of the oxidation-reduction reaction catalyzed by the enzyme. For example, the change in absorbance over time can be measured to quantify the concentration of the product, and the change in absorbance per unit time (ΔA) can be calculated. ｓａｍｐｌｅ ) is calculated. The same method except that DMSO is used instead of the substrate solution. The change in absorbance over time is measured and the A per unit time is calculated. ５５０ Quantity change (ΔA ｂｌａｎｋ Calculate ΔA. ｓａｍｐｌｅ -ΔA ｂｌａｎｋThe dehydrogenase activity of each enzyme can be evaluated by calculating the ΔA. Furthermore, if the product of a reaction catalyzed by an enzyme is reacted with a reagent that reacts with the product (hereinafter referred to as "product reaction reagent"), and the absorbent substance produced by this reaction is measured, then enzyme activity can be measured by measuring absorbance. For example, to quantify the concentration of the absorbent substance produced, the change in absorbance over time can be measured, and the change in absorbance per unit time (ΔA) can be calculated. ｓａｍｐｌｅ ) is calculated. The same method except that DMSO is used instead of the substrate solution. The change in absorbance over time is measured and the A per unit time is calculated. ５５０ Quantity change (ΔA ｂｌａｎｋ Calculate ΔA. ｓａｍｐｌｅ -ΔA ｂｌａｎｋ By calculating this, the dehydrogenase activity of each enzyme can be evaluated.

[0126] [Method for evaluating mutations that enhance calcifediol dehydrogenase activity] (ΔA) when using androsterone as a substrate ｓａｍｐｌｅ -ΔA ｂｌａｎｋ Let A be the value of (ΔA) when calcifediol is used as the substrate. ｓａｍｐｌｅ -ΔA ｂｌａｎｋ The value of ) was defined as B, and the B / A value for each enzyme was calculated. Mutations in which the B / A value of an enzyme after mutation introduction increased by 0.01 or more, preferably 0.05 or more, more preferably 1.0 or more, even more preferably 5.0 or more, and particularly preferably 10 or more compared to before mutation introduction, were evaluated as mutations that improved reactivity to calcifediol.

[0127] [Method for evaluating mutations that enhance the substrate specificity of calcifediol dehydrogenase] When calcifediol is used as a substrate, (ΔA ｓａｍｐｌｅ -ΔA ｂｌａｎｋ Let B be the value of (ΔA) when cholecalciferol is used as a substrate. ｓａｍｐｌｅ -ΔA ｂｌａｎｋThe value of ) was set as D, and the D / B value for each of the various enzymes was calculated. With the B / A value of the enzyme before mutation introduction set to 1, a mutation in which the relative D / B value of the enzyme after mutation introduction (relative value of cholecalciferol / calcifediol) is 0.01 or more and 0.9 or less, preferably 0.01 or more and 0.8 or less, more preferably 0.01 or more and 0.7 or less, even more preferably 0.01 or more and 0.6 or less, and particularly preferably 0.01 or more and 0.5 or less is evaluated as a mutation with improved substrate specificity for calcifediol.

[0128] [Method for evaluating mutations that enhance β-estradiol dehydrogenase activity] (ΔA) when using androsterone as a substrate ｓａｍｐｌｅ -ΔA ｂｌａｎｋ Let A be the value of (ΔA) when β-estradiol is used as the substrate. ｓａｍｐｌｅ -ΔA ｂｌａｎｋ The value of ) was defined as C, and the C / A value for each enzyme was calculated. Mutations in which the C / A value of an enzyme after mutation introduction increased by 0.01 or more, preferably 0.05 or more, more preferably 0.1 or more, even more preferably 0.2 or more, and particularly preferably 0.3 or more compared to before mutation introduction, were evaluated as mutations that improved reactivity to β-estradiol.

[0129] [Composition containing oxidoreductase and kit for quantitative determination of hormones or hormone precursors] The method for quantitative determination of hormones or hormone precursors using oxidoreductase according to the present invention may be carried out by providing a composition containing oxidoreductase and a product reaction reagent, or by combining oxidoreductase with a commercially available product reaction reagent.

[0130] The present invention provides a method for quantifying hormones or hormone precursors, an oxidoreductase for quantification, a composition for quantification, and a kit for quantification, which, by containing an oxidoreductase, can provide a novel method for quantifying hormones or hormone precursors that serve as indicators of diseases associated with vitamin D deficiency, an enzyme for quantification, a composition for quantification, and a kit for quantification.

[0131] [Sensor Chip and Electrodes] Figure 5(a) is a schematic diagram of a sensor chip 10 according to one embodiment of the present invention, and Figures 5(b) to 5(d) are schematic diagrams showing the components constituting the sensor chip 10. The sensor chip 10 comprises two or more electrodes arranged on a base material 11. The base material 11 is made of an insulating material. In Figures 5(a) and 5(b), as an example, an working electrode 1, a counter electrode 3, and a reference electrode 5 are arranged on the base material 11. Each electrode is electrically connected to a wiring section 7, and the wiring section 7 is electrically connected to a terminal 9 located on the opposite side of the wiring direction from each electrode. The working electrode 1, the counter electrode 3, and the reference electrode 5 are spaced apart from each other. Furthermore, it is preferable that the working electrode 1, the counter electrode 3, and the reference electrode 5 are integrally formed with the wiring section 7 and the terminal 9. Alternatively, the counter electrode 3 and the reference electrode 5 may be an integrated type.

[0132] As shown in Figures 5(a) and 5(c), a spacer 13 is placed at the end of the base material 11 parallel to the wiring section 7, and a cover 15 covering the working electrode 1, counter electrode 3, reference electrode 5, and spacer 13 is placed thereon. The spacer 13 and cover 15 are made of insulating material. The spacer 13 preferably has a thickness approximately equal to that of the working electrode 1, counter electrode 3, and reference electrode 5, and is in close contact with the working electrode 1, counter electrode 3, and reference electrode 5. Alternatively, the spacer 13 and cover 15 may be formed as a single unit. The cover 15 is a protective layer that prevents the wiring section 7 from deteriorating due to exposure to the outside air and prevents short circuits due to seepage of the measurement sample.

[0133] In one embodiment, the oxidoreductase of the present invention may be coated, adsorbed, or immobilized on an electrode. Preferably, the oxidoreductase of the present invention is coated, adsorbed, or immobilized on the working electrode. In another embodiment, a mediator may also be coated, adsorbed, or immobilized on the electrode together with the oxidoreductase. The oxidoreductase, or the oxidoreductase and mediator, may be contained in a reaction layer arranged on the working electrode, counter electrode, and reference electrode. As electrodes, carbon electrodes, metal electrodes such as platinum, gold, silver, nickel, and palladium can be used. In the case of carbon electrodes, materials such as pyrolytic graphite carbon (PG), glassy carbon (GC), carbon paste, and plastic-formed carbon (PFC) can be used. The measurement system may be a two-electrode system or a three-electrode system, and for example, the enzyme can be immobilized on the working electrode. Examples of reference electrodes include standard hydrogen electrodes, reversible hydrogen electrodes, silver-silver chloride electrodes (Ag / AgCl), palladium-hydrogen electrodes, and saturated calomel electrodes. From the viewpoint of stability and reproducibility, it is preferable to use Ag / AgCl.

[0134] The enzyme may be immobilized on the electrode by methods such as crosslinking, coating with a dialysis membrane, encapsulation in a polymer matrix, use of photocrosslinkable polymers, use of electrically conductive polymers, or use of oxidation / reduction polymers. Alternatively, the enzyme may be immobilized in a polymer together with a mediator or adsorbed onto the electrode, and these methods may be combined.

[0135] The mediator (also called 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 accept 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, and examples of phenazine compounds include, but are not limited to, 5-methylphenazinium methosulfate (PMS), methoxy-PMS, 1-methoxy-5-ethylphenazinium ethylsulfate (PES), and methoxy-PES.

[0136] The oxidoreductase of the present invention can be applied to various electrochemical measurement methods by using a potentiostat or galvanostat. Examples of electrochemical measurement methods include amperometry, potentiometry, and coulometry. For example, by amperometry, the concentration of a hormone or hormone precursor in a sample can be calculated by measuring the current value generated when hydrogen peroxide, produced when oxidoreductase reacts with a hormone or hormone precursor, is applied to a hydrogen peroxide electrode at a voltage of +600 mV to +1000 mV (vs. Ag / AgCl). For example, a calibration curve can be created by measuring the current value for known hormone or hormone precursor concentrations (0, 5, 10, 50 μM) and plotting it against the hormone or hormone precursor concentration. The hormone or hormone precursor concentration can be obtained from the calibration curve by measuring the current value for an unknown hormone or hormone precursor. For example, a carbon electrode or platinum electrode can be used as the hydrogen peroxide electrode. Furthermore, instead of a hydrogen peroxide electrode, an electrode immobilized with a reductase such as peroxidase or catalase can be used. By applying -400 mV to +100 mV (vs. Ag / AgCl) and measuring the resulting reduction current, the amount of hydrogen peroxide can be quantified, and the value of the hormone or hormone precursor can be measured.

[0137] Furthermore, by mixing a mediator into the reaction solution and, for example, using the amperometry method, transferring electrons generated when oxidoreductase reacts with a hormone or hormone precursor to an oxidized mediator to generate a reduced mediator, and then measuring the current value generated by applying -1000 mV to +500 mV (vs. Ag / AgCl), the concentration of the hormone or hormone precursor in the sample can be calculated. A carbon electrode or platinum electrode is preferred as the counter electrode. For example, a calibration curve can be created by measuring the current value for known hormone or hormone precursor concentrations (0, 100, 200, 500 μM) and plotting it against the hormone or hormone precursor concentration. The hormone or hormone precursor concentration can be obtained from the calibration curve by measuring the current value for an unknown hormone or hormone precursor.

[0138] Furthermore, printed electrodes (sensor chips) can be used to reduce the amount of solution required for measurement. In this case, it is preferable that the electrodes are formed on a substrate made of an insulating substrate. Specifically, it is desirable that the electrodes be formed on the substrate by printing techniques such as photolithography, screen printing, gravure printing, and flexographic printing. Examples of insulating substrate materials include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, but it is more preferable to use a material that has strong resistance to various solvents and chemicals.

[0139] [Hormone or Hormone Precursor Measurement Sensor] In one embodiment, a hormone or hormone precursor measurement sensor using the oxidoreductase of the present invention is provided. Figure 6(a) is a schematic diagram of a sensor 100 according to one embodiment of the present invention. The sensor is a hormone or hormone precursor measurement device using the oxidoreductase of the present invention, and comprises a sensor chip containing 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 and interruption of hormone or hormone precursor measurement in the sensor 100. The display 33 may, for example, display the measured value of the hormone or hormone precursor, and may include a touch panel as an input unit for controlling the measurement unit 30.

[0140] Figure 6(b) is a block diagram of a 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. In addition, the terminals of the sensor chip 10, which will be 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 that controls the sensor 100, and is composed of, for example, a known central processing unit (CPU) and an operation program that controls the sensor 100. Alternatively, the control unit 110 may include a central processing unit and an operating system (OS), and may include an application program or module for measuring hormones or hormone precursors.

[0141] The display unit 120 may, for example, include a known display 33 and display the measured value of the hormone or hormone precursor, the status of the measurement unit 30, or operation requests to the operator. The input unit 130 is an input device for the operator to operate the sensor 100, and may, for example, be a switch 31 or a touch panel located on the display 33. Multiple switches 31 may be arranged on the measurement unit 30.

[0142] The storage unit 140 consists of a main memory, and an auxiliary storage device (hard disk) may be located externally. The main memory may consist of read-only memory (ROM) and / or random access memory (RAM). Operation programs, operating systems, application programs, or modules are stored in the storage unit 140 and executed by the central processing unit to constitute the control unit 110. Measured values ​​and current values ​​can also be stored in the storage unit 140.

[0143] The communication unit 150 is a known communication device that connects the sensor 100 or the measuring unit 30 to an external device (computer, printer, or network). The communication unit 150 and the external device are connected by wired or wireless communication. The power supply 160 is a known power supply device that supplies power to the sensor 100 or the measuring unit 30.

[0144] As described above, the present invention provides a method for quantifying hormones or hormone precursors, an oxidoreductase for quantification, a composition for quantification, and a kit for quantification, which, by containing an oxidoreductase, can provide a novel method for quantifying hormones or hormone precursors, an enzyme for quantification, a composition for quantification, and a kit for quantification.

[0145] [Method for Quantifying Hormones or Hormone Precursors Using Dehydrogenase Activity] The oxidoreductase used in the present invention is a dehydrogenase that acts on a hormone or hormone precursor as a substrate, oxidizes the hormone or hormone precursor, and transfers the extracted electrons to a coenzyme. Herein, in this specification, an oxidoreductase having dehydrogenase activity may be referred to as a dehydrogenase.

[0146] In one embodiment, the dehydrogenase may be selected from the oxidoreductases described above, or it may be an oxidoreductase among the oxidoreductases described above that has high hormone or hormone precursor dehydrogenase activity.

[0147] In one embodiment, the reaction conditions for the dehydrogenase can be any conditions that allow it to act on a hormone or hormone precursor and efficiently catalyze the dehydrogenation reaction. Generally, enzymes have an optimal temperature and pH at which they exhibit the highest activity. Therefore, reaction conditions near the optimal temperature and pH may be preferable.

[0148] In one embodiment, the reaction process of the dehydrogenase may involve various chemical substances when the dehydrogenase of the present invention acts on a hormone or hormone precursor. For example, when the dehydrogenase acts on a hormone or hormone precursor, electron transfer may be involved in the redox reaction.

[0149] In one embodiment, a dehydrogenase that acts on a hormone or hormone precursor and NAD ＋ This invention provides a method for measuring the hormone or hormone precursor contained in a sample by reacting an oxidized coenzyme, such as [specific example], with a hormone or hormone precursor contained in the sample to oxidize the hormone or hormone precursor to produce an oxidized hormone or oxidized hormone precursor, transferring electrons extracted from the hormone or hormone precursor to the oxidized coenzyme, and then producing a reduced coenzyme from the concentration of the reduced coenzyme. The concentration of the reduced coenzyme is determined by further reacting a mediator with a color-developing or color-degrading reagent to transfer electrons from the reduced coenzyme to the reagent. The mediator transfers electrons extracted from the reduced coenzyme to the color-developing or color-degrading reagent, and the concentration of the hormone or hormone precursor can be determined from the change in the color-developing or color-degrading reagent. By having the mediator oxidize the reduced coenzyme to produce an oxidized first coenzyme, it is possible to achieve high sensitivity in the quantitative determination method of hormones or hormone precursors by enzyme cycling reactions. In addition to WST (Water soluble tetrazolium)-5, other examples of color-developing or color-fading substrates used in the present invention include DCIP (2,6-Dichlorophenolindophenol), tetrazolium compounds (Tetrazolium blue, Nitro-tetrazolium blue, WST-1, WST-3, WST-4, WST-8, WST-9), and the like.

[0150] Furthermore, in one embodiment, a dehydrogenase that acts on a hormone or hormone precursor and Thio-NAD ＋ This invention provides a method for measuring the hormone or hormone precursor contained in a sample by acting an oxidized coenzyme I such as Thio-NAD on a hormone or hormone precursor contained in the sample to oxidize the hormone or hormone precursor to produce an oxidized hormone or oxidized hormone precursor, transferring electrons extracted from the hormone or hormone precursor to the oxidized coenzyme I to produce a reduced coenzyme I, and measuring the hormone or hormone precursor contained in the sample from the concentration of the reduced coenzyme I.Here, by adding an excess amount of reduced coenzyme II such as NADH to the reaction system, the dehydrogenase acts on the oxidized hormone or oxidized hormone precursor to reduce the hormone or hormone precursor.Here, an excess amount of Thio-NAD ＋ By adding an oxidized form of coenzyme I to the reaction system, the reduced form of coenzyme I can be amplified, thereby enabling higher sensitivity in the quantitative determination of hormones or hormone precursors by enzyme cycling reactions. The amount of oxidized coenzyme I is preferably greater than the amount of oxidized coenzyme II, and the amount of oxidized coenzyme I may be approximately the same as the amount of reduced coenzyme II. NAD ＋ As for NADH, β-NAD ＋ / β-NADH is preferred.

[0151] In one embodiment, when quantifying a hormone or hormone precursor using blood as a sample, the sample can be arbitrarily selected from whole blood, plasma, or serum, depending on the hormone or hormone precursor to be measured. Alternatively, the dehydrogenase or a composition for quantifying hormones or hormone precursors containing dehydrogenase may be directly mixed with the sample, or the sample may be pre-treated before mixing with the dehydrogenase or the composition for quantifying hormones or hormone precursors containing dehydrogenase. For example, the hormone or hormone precursor binding protein may be degraded with a protease to release the hormone or hormone precursor before being mixed with the dehydrogenase or the composition for quantifying hormones or hormone precursors containing dehydrogenase.

[0152] [Composition containing dehydrogenase and kit for quantitative determination of hormones or hormone precursors] In one embodiment, a hormone or hormone precursor may be quantified using the dehydrogenase according to the present invention. The method for quantitative determination of a hormone or hormone precursor using the dehydrogenase according to the present invention may be carried out by providing a composition containing dehydrogenase and a product reaction reagent, or by combining dehydrogenase with a commercially available product reaction reagent. For example, it may be provided as a composition for quantitative determination of a hormone or hormone precursor containing dehydrogenase, or as a composition for quantitative determination of a hormone or hormone precursor further comprising a mediator that is reduced by the addition of dehydrogenase and a reagent that reacts with the reduced mediator. Alternatively, it may be provided as a kit for quantitative determination of a hormone or hormone precursor containing dehydrogenase, a mediator that is reduced by the addition of dehydrogenase, and a reagent that reacts with the reduced mediator.

[0153] The mediator (also called an artificial electron mediator, artificial electron acceptor, or electron mediator) used in the measurement method or quantitative kit of the present invention is not particularly limited as long as it can accept electrons from a dehydrogenase. Examples of mediators include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides, such as potassium ferricyanide, ferredoxins, ferrocene, osmium complexes and their derivatives, and examples of phenazine compounds include, but are not limited to, PMS, methoxy-PMS, 5-Methylphenazinium ethylsulfate (PES), methoxy-PES, 1-Methoxy-5-ethylphenazinium ethylsulfate (PES), and methoxy-PES.

[0154] [Sensor Chip and Electrode] In one embodiment, the dehydrogenase of the present invention may be coated, adsorbed, or immobilized on the electrode. Preferably, the dehydrogenase of the present invention is coated, adsorbed, or immobilized on the working electrode. The electrode configuration can be the same as the configuration described for electrodes using oxidoreductase, so a detailed explanation is omitted. Furthermore, 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, etc.

[0155] The dehydrogenase of the present invention can be applied to various electrochemical measurement methods by using a potentiostat or galvanostat. Examples of electrochemical measurement methods include amperometry, potentiometry, and coulometry. Furthermore, by mixing a mediator into the reaction solution, and, for example, using amperometry, transferring electrons generated when the dehydrogenase reacts with a hormone or hormone precursor to an oxidized mediator to produce a reduced mediator, and then applying -1000 mV to +500 mV (vs. Ag / AgCl), the current value generated can be measured to calculate the concentration of the hormone or hormone precursor in the sample. Carbon electrodes or platinum electrodes are preferred as counter electrodes. For example, a calibration curve can be created by measuring the current value for known hormone or hormone precursor concentrations (0, 100, 200, 500 μM) and plotting it against the hormone or hormone precursor concentration. The hormone or hormone precursor concentration can be obtained from the calibration curve by measuring the current value for an unknown hormone or hormone precursor.

[0156] Furthermore, to reduce the amount of solution required for measurement, printed electrodes (sensor chips) can be used. In this case, it is preferable that the electrodes are formed on a substrate made of an insulating substrate. The configuration of the sensor chip using dehydrogenase may be the same as that of the sensor chip using oxidoreductase, and a detailed explanation is omitted.

[0157] [Hormone or Hormone Precursor Measurement Sensor] In one embodiment, a hormone or hormone precursor measurement sensor using the dehydrogenase of the present invention is provided. The sensor is a hormone or hormone precursor measurement device using the dehydrogenase of the present invention, and comprises a sensor chip containing the dehydrogenase and a measurement unit. The configuration of the hormone or hormone precursor measurement sensor using dehydrogenase may be the same as the configuration of the hormone or hormone precursor measurement sensor using oxidoreductase, and a detailed explanation is omitted.

[0158] As described above, the present invention provides a method for quantifying hormones or hormone precursors, a dehydrogenase for quantification, a composition for quantification, and a kit for quantification, which, by containing a dehydrogenase, can provide a new method for quantifying hormones or hormone precursors, a dehydrogenase for quantification, a dehydrogenase for quantification, a composition for quantification, a kit for quantification, an electrode, a sensor chip, and a sensor.

[0159] 1. Construction of Plasmids for Oxidoreductase Expression All enzymes used in this example have a His tag added to the N-terminus. More precisely, the amino acid residue numbers will be shifted compared to the wild-type enzyme, but in this specification, the amino acid numbers will be expressed based on the wild-type amino acid sequence starting with Met.

[0160] Fifteen base pairs (SEQ ID NO: 33) were added upstream of the 5' end of the PtHSD-wt gene (SEQ ID NO: 15), excluding the first atg, and fifteen base pairs (SEQ ID NO: 34) were added downstream of the 3' end. The resulting genes were then synthesized by Integrated DNA Technologies.

[0161] Plasmid fragments were prepared using the pCold II DNA plasmid as a template, and the reaction mixture was prepared with the composition shown in Table 1 using primers SEQ ID NO: 35 and SEQ ID NO: 36. PCR was performed using a thermal cycler (Bio-Rad). Specifically, the mixture was incubated at 94°C for 2 minutes, followed by 30 cycles of "98°C, 10 seconds" - "55°C, 5 seconds" - "68°C, 40 seconds". After PCR, 1.0 μL of DpnI (New England BioLabs) was added to the solution and treated at 37°C for 1 hour. The amplified fragments were then purified using the GFX PCR DNA and Gel Band Purification Kit (Cytiva).

[0162]

[0163] The purified plasmid fragments and gene fragments were conjugated using In-Fusion Snap Assemblely Master Mix (Takara Bio). Specifically, the reaction was carried out at 50°C for 15 minutes using the composition shown in Table 2. E. coli JM109 strain was transformed using the reaction mixture and spread on LB-100 μg / mL ampicillin (hereinafter referred to as Amp) agar medium. The grown colonies were inoculated into 2.5 mL of LB-Amp medium [1% (w / v) bactotryptone, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, 100 μg / mL Amp] and incubated with shaking at 37°C for 8 hours to obtain the culture. This culture was collected by centrifugation at 15,000 rpm for 5 minutes to obtain bacterial cells. Next, recombinant plasmids were extracted and purified from these bacterial cells using the FastGene Plasmid Mini Kit (manufactured by Nippon Genetics Co., Ltd.) to obtain a PtHSD-wt expression plasmid (pCold_PtHSD-wt). The nucleotide sequence inserted into the plasmid was confirmed to be the desired sequence by commissioning DNA sequencing analysis to FASMAC.

[0164]

[0165] Similarly, PtHSD-5M gene (SEQ ID NO: 25), CfHSD-wt gene (SEQ ID NO: 16), CfHSD-5M gene (SEQ ID NO: 26), CgHSD-wt gene (SEQ ID NO: 17), CgHSD-5M gene (SEQ ID NO: 27), CrHSD-wt gene (SEQ ID NO: 18), CrHSD-5M gene (SEQ ID NO: 28), CtHSD-wt gene (SEQ ID NO: 19), CtHSD-5M gene (SEQ ID NO: 29), DcHSD-wt gene (SEQ ID NO: 20), DcHSD-5M gene (SEQ ID NO: 30), SmHS For the D-wt gene (SEQ ID NO: 21), SmHSD-4M gene (SEQ ID NO: 31), ChnA-wt gene (SEQ ID NO: 14), PfHSD-wt gene (SEQ ID NO: 22), PfHSD-5M gene (SEQ ID NO: 32), PPHSD-wt gene (SEQ ID NO: 24), SbHSD-wt gene (SEQ ID NO: 23), and SeHSD-wt gene (SEQ ID NO: 13), 15 bases (SEQ ID NO: 33) were added upstream of the 5' end (excluding the initial atg) and 15 bases (SEQ ID NO: 34) were added downstream of the 3' end, and synthesis was commissioned to Integrated DNA Technologies. Each oxidoreductase expression plasmid (pCold_PtHSD-5M, pCold_CfHSD-wt, pCold_CfHSD-5M, pCold_CgHSD-wt, pCold_CgHSD-5M, pCold_CrHSD-wt, pCold_CrHSD-5M, pCold_CtHSD-wt, pCold_CtHSD-5M, pCold_DcHSD-wt, pCold_DcHSD-5M, pCold_SmHSD-wt, pCold_SmHSD-4M, pCold_ChnA-wt, pCold_PfHSD-wt, pCold_PfHSD-5M, pCold_PpHSD-wt, pCold_SbHSD-wt, and pCold_SeHSD-wt) were obtained using the same procedure as when constructing pCold_PtHSD-wt, and their sequences were confirmed to be as intended by DNA sequencing analysis.

[0166] 2. Preparation of plasmids for mutant oxidoreductase expression When introducing point mutations into the wild-type or mutant oxidoreductase gene, PCR was performed using the obtained wild-type or mutant oxidoreductase expression plasmid as a template, with the composition shown in Table 1. The primer sets used to prepare the mutant oxidoreductase expression plasmids are summarized in the sequence listing and Figures 9-11. The prepared reaction mixture was incubated at 94°C for 2 minutes using a thermal cycler (Bio-Rad), followed by 30 cycles of "98°C, 10 seconds" - "55°C, 5 seconds" - "68°C, 40 seconds". The DNA obtained in this way was treated with the restriction enzyme DpnI (NEW ENGLAND BIOLABS) to cleave the remaining template DNA. This reaction mixture was then used to transform E. coli JM109, and the transformed cells were spread on LB-100 μg / mL Amp agar. The grown colonies were inoculated into 2.5 mL of LB-Amp medium and incubated with shaking at 37°C for 8 hours to obtain a culture. This culture was collected by centrifugation at 15,000 rpm for 5 minutes to obtain bacterial cells.Next, recombinant plasmids were extracted and purified from these bacterial cells using the FastGene Plasmid Mini Kit (manufactured by Genetics Japan Co., Ltd.), and each mutant expression plasmid was created (pCold_SeHSD-1M with the SeHSD-1M gene, pCold_ChnA-1M with the ChnA-1M gene, pCold_PtHSD-1M with the PtHSD-1M gene, pCold_CfHSD-1M with the CfHSD-1M gene, pCold_CgHSD-1M with the CgHSD-1M gene, pCold_CrHSD-1M with the CrHSD-1M gene, and pC with the CtHSD-1M gene). We obtained pCold_DcHSD-1M (possessing the old_CtHSD-1M and DcHSD-1M genes), pCold_SmHSD-1M (possessing the SmHSD-1M gene), pCold_PfHSD-1M (possessing the PfHSD-1M gene), pCold_SbHSD-1M (possessing the SbHSD-1M gene), pCold_ChnA-M203V (possessing the ChnA-M203V gene), pCold_PtHSD-Q75F (possessing the PtHSD-Q75F gene), and pCold_PtHSD-5M / F187A (possessing the PtHSD-5M / F187A gene). We confirmed that the sequences were as intended by DNA sequencing analysis.

[0167] Using a similar method, pCold_PtHSD-5M / N152H, which has the PtHSD-5M / N152H gene; pCold_PtHSD-5M / N152Q, which has the PtHSD-5M / N152Q gene; pCold_PtHSD-5M / R201N, which has the PtHSD-5M / R201Q gene; and pCold_PtHSD-5M / Y202H, which has the PtHSD-5M / Y202H gene, were obtained using pCold_PtHSD-5M as the template plasmid. The primer sets used for preparation are summarized in the sequence listing and Figure 13.

[0168] Using a similar method, pCold_PtHSD-5M / N152H / R201N (PtHSD-7M) containing the PtHSD-5M / N152H / R201N (PtHSD-7M) gene was obtained using the pCold_PtHSD-5M / N152H / R201N (PtHSD-7M) template plasmid and the primer set of Sequence ID No. 67 and 68. Furthermore, pCold_PtHSD-5M / N152H / Y202H containing the PtHSD-5M / N152H / Y202H gene was obtained using the primer set of Sequence ID No. 73 and 74.

[0169] Using a similar method, pCold_PtHSD-7M (PtHSD-5M / N152H / R201N) was used as a template plasmid to obtain the PtHSD-7M / P74A gene, pCold_PtHSD-7M / P74A, the PtHSD-7M / P74F gene, and the PtHSD-7M / P74I gene. We obtained pCold_PtHSD-7M / P74I and PtHSD-7M / P74L genes, pCold_PtHSD-7M / P74L genes, pCold_PtHSD-7M / P74V genes, and pCold_PtHSD-7M / H119D genes. The primer sets used for preparation are summarized in the sequence listing, Figure 14, and Figure 16.

[0170] Using a similar method, pCold_PtHSD-1M (using PtHSD-A187F as a template plasmid) is obtained, containing the PtHSD-A187F / N152H gene, pCold_PtHSD-A187F / R201H gene, and pCold_PtHSD-A187F / R201N gene. We obtained pCold_PtHSD-A187F / Y202H, which has the R201N and PtHSD-A187F / Y202H genes; pCold_PtHSD-A187F / S205H, which has the PtHSD-A187F / S205H genes; and pCold_PtHSD-A187F / P74I, which has the PtHSD-A187F / P74I gene. The primer sets used for preparation are summarized in the sequence listing and Figure 15.

[0171] Using a similar method, pCold_PfHSD-6M was obtained, which contains the PfHSD-6M gene with an additional R199N amino acid substitution, using the template plasmid pCold_PfHSD-5M (PfHSD-A185F / I115F / Y200L / S194F / S203A) and the primer set of Sequence ID No. 97 and 98.

[0172] In-fusion reactions were used to introduce double mutations corresponding to N152H and R201N of PtHSD into each oxidoreductase gene. PCR was performed using the template plasmid and primer set shown in Table 3, with the composition described in Table 1. The extension reaction conditions were 68°C for 45 seconds. After PCR, 1.0 μL of DpnI (New England BioLabs) was added to the solution and treated at 37°C for 1 hour. The amplified fragments were then purified using the GFX PCR DNA and Gel Band Purification Kit (Cytiva).

[0173]

[0174] The purified plasmid fragments and gene fragments were conjugated using In-Fusion Snap Assemblely Master Mix (Takara Bio). Specifically, the reaction was carried out at 50°C for 15 minutes using the composition shown in Table 2. E. coli JM109 strain was transformed using the reaction mixture and spread on LB-100 μg / mL ampicillin (hereinafter referred to as Amp) agar medium. The grown colonies were inoculated into 2.5 mL of LB-Amp medium [1% (w / v) bactotryptone, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, 100 μg / mL Amp] and incubated with shaking at 37°C for 8 hours to obtain the culture. This culture was collected by centrifugation at 15,000 rpm for 5 minutes to obtain bacterial cells. Next, recombinant plasmids were extracted and purified from these bacterial cells using the FastGene Plasmid Mini Kit (manufactured by Genetics Japan Co., Ltd.) to obtain plasmids for CfHSD-7M expression (pCold_CfHSD-7M), CgHSD-7M expression (pCold_CgHSD-7M), CtHSD-7M expression (pCold_CtHSD-7M), DcHSD-7M expression (pCold_DcHSD-7M), and SmHSD-6M expression (pCold_SmHSD-6M). The nucleotide sequences inserted into the plasmids were confirmed to be as intended by commissioning DNA sequencing analysis to FASMAC.

[0175] 3. Recombinant Production of Oxidoreductases E. coli BL21 strain was transformed with plasmids for the expression of various oxidoreductases to obtain strains producing various oxidoreductases. The strains producing various oxidoreductases were spread on LB-100 μg / mL Amp agar medium, and the grown colonies were inoculated into 5 mL of LB-100 μg / mL Amp in a test tube and cultured at 37°C and 180 rpm for 3-4 hours. The culture medium was placed in a water bath adjusted to 15°C and allowed to stand for 30 minutes, after which IPTG was added to a final concentration of 0.1 mM, and the culture was cultured at 15°C and 180 rpm for 24 hours. The pellet obtained by centrifugation of the culture medium at 15,000 × g for 10 minutes was mixed with 600 μL of Lysis buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH). ２ PO４ The cells were resuspended at pH 8.0. After sonication of the bacterial suspension, the supernatant was collected from centrifugation at 15,000 rpm for 10 minutes and used as the crude enzyme solution.

[0176] For the preparation of large quantities of enzymes, various oxidoreductase-producing strains were developed on LB-100 μg / mL Amp agar medium, and the grown colonies were inoculated into 2.5 mL of LB-100 μg / mL Amp in a test tube and cultured at 37°C and 180 rpm for 3-4 hours. The culture solution was inoculated into 250 mL of LB-100 μg / mL Amp in a Sakaguchi flask and cultured at 37°C and 130 rpm for 3-4 hours. The culture solution was placed in a water bath adjusted to 15°C and allowed to stand for 30 minutes, after which IPTG was added to a final concentration of 0.1 mM, and cultured at 15°C and 130 rpm for 24 hours. The culture solution was centrifuged at 8,000 × g for 10 minutes, and the resulting pellet was resuspended in 20 mL of Lysis buffer. After ultrasonically disrupting the bacterial cell suspension, the supernatant obtained by centrifugation at 10,000 rpm for 10 minutes was collected and used as the crude enzyme solution.

[0177] 4. Purification of Oxidoreductase The crude oxidoreductase enzyme solution was equilibrated, washed, and eluted using Ni-NTA Spin Columns (QIAGEN) according to the attached protocol. Lysis buffer was used for column equilibration, and Wash buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH) was used for washing. ２ PO ４ (pH 8.0), Elution buffer (500 mM imidata, 300 mM NaCl, 50 mM NaH) ２ PO ４ (pH 8.0) was used.

[0178] When purifying enzymes in large quantities, use an imidazole-free A buffer (300 mM NaCl, 50 mM NaH). ２ PO ４The enzymes were bound, washed, and eluted using a B buffer and HisTrapHP column (Cytiva) containing 1 M imidazole (pH 8.0). Specifically, the crude enzyme solution was applied to a HisTrapHP column equilibrated with 2% B buffer / 98% A buffer, washed with 10% B buffer / 90% A buffer, and eluted with 30% B buffer / 70% A buffer. The eluted fraction was concentrated to approximately 20-30 mg / mL using Amicon Ultra Ultracel-10K (Merck) to obtain the enzyme solution.

[0179] The purity of each eluted fraction was evaluated by SDS-PAGE, and fractions free of contaminating proteins were collected and used as purified oxidoreductase standards. As an example, Figure 7 shows the results of SDS-PAGE analysis of the purity of the purified PtHSD-1M mutant (PtHSD A187F mutant). The molecular weight of the PtHSD-1M mutant (PtHSD A187F mutant) was approximately 26 kDa, which was at the same level as the molecular weight (approximately 26 kDa) estimated from its amino acid sequence.

[0180] 5. Measurement of dehydrogenase activity for various substrates. The dehydrogenase activity measurement reagents include calcifediol monohydrate (Tokyo Chemical Industries, Ltd.), β-estradiol (Tokyo Chemical Industries, Ltd.), androsterone (Merck), and β-nicotinamide adenine dinucleotide (oxidized form, β-NAD ＋ The following were used: (manufactured by Oriental Yeast Co., Ltd.), 1-methoxy PMS (mPMS, manufactured by Dojin Chemical Laboratories Co., Ltd.) or 1-methox (mPES, manufactured by Dojin Chemical Laboratories Co., Ltd.), WST-5 (manufactured by Dojin Chemical Laboratories Co., Ltd.), Triton X-100 (manufactured by Merck), and dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0181] 6. Principle of the Activity Measurement Method Diagram 8 illustrates the principle of the activity measurement method. When oxidoreductase oxidizes the substrate, WST-5 receives electrons from the β-NADH produced simultaneously via electron mediators such as mPMS and mPES, thereby generating water-soluble WST-5 formazan (maximum absorption wavelength 550 nm).

[0182] Activity Measurement Method 1: 5 μL of purified oxidoreductase solutions were added to a 96-well plate and mixed with 145 μL of the activity measurement reagents shown in Table 4. After standing at room temperature for 30 minutes, the absorbance of light at a wavelength of 550 nm (A) was measured using a microplate reader SH-9000 (Corona Electric Co., Ltd.). ５５０ ) is measured, and A per unit time ５５０ Quantity change (ΔA ｓａｍｐｌｅ The following was calculated: Measurements were also performed using an activity measurement reagent with the same composition as the substrate solution, but with DMSO instead, and the A per unit time was calculated. ５５０ Quantity change (ΔA ｂｌａｎｋ ) was calculated. ΔA ｓａｍｐｌｅ -ΔA ｂｌａｎｋ The dehydrogenase activity of each enzyme was evaluated by calculating the following. The dehydrogenase activity of ChnA wild type (wt), M203V mutant, and Y189L mutant is shown in Figure 9. The dehydrogenase activity of PtHSD wild type (wt), Q75F mutant, and A187F mutant is shown in Figure 10.

[0183]

[0184] Activity Measurement Method 2: 5 μL of purified oxidoreductase solutions were added to a 96-well plate and mixed with 145 μL of the activity measurement reagents shown in Table 5. Every 6 minutes after mixing, the absorbance of light at a wavelength of 550 nm (A) was measured using a microplate reader SH-9000 (Corona Electric Co., Ltd.). ５５０ ) is measured, and A per unit time ５５０ Quantity change (ΔA ｓａｍｐｌｅ The following was calculated: The substrate was dissolved and diluted with DMSO. Measurements were also performed using an activity assay reagent with the same composition as the substrate solution, but using DMSO instead, and the A per unit time was calculated. ５５０ Quantity change (ΔA ｂｌａｎｋ ) was calculated. ΔA ｓａｍｐｌｅ -ΔA ｂｌａｎｋ The dehydrogenase activity of each enzyme was evaluated by calculating the θ. Figure 11 shows the dehydrogenase activity of wild-type and mutant oxidoreductases.

[0185]

[0186] Activity Measurement Method 3: 5 μL of purified oxidoreductase solutions were added to a 96-well plate and mixed with 145 μL of the activity measurement reagents shown in Table 6. Every 6 minutes after mixing, the absorbance of light at a wavelength of 550 nm (A) was measured using a microplate reader SH-9000 (Corona Electric Co., Ltd.). ５５０ ) is measured, and A per unit time ５５０ Quantity change (ΔA ｓａｍｐｌｅ The following was calculated: The substrate was dissolved and diluted with DMSO. Measurements were also performed using an activity assay reagent with the same composition as the substrate solution, but using DMSO instead, and the A per unit time was calculated. ５５０ Quantity change (ΔA ｂｌａｎｋ ) was calculated. ΔA ｓａｍｐｌｅ -ΔA ｂｌａｎｋ The dehydrogenase activity of each enzyme was evaluated by calculating the following. The dehydrogenase activity of wild-type and mutant oxidoreductases is shown in Figures 13-15 and 17-22.

[0187]

[0188] Activity Measurement Method 4: 5 μL of purified oxidoreductase solutions were added to a 96-well plate and mixed with 145 μL of the activity measurement reagents shown in Table 7. Every 6 minutes after mixing, the absorbance of light at a wavelength of 550 nm (A) was measured using a microplate reader SH-9000 (Corona Electric Co., Ltd.). ５５０ ) is measured, and A per unit time ５５０ Quantity change (ΔA ｓａｍｐｌｅ The following was calculated: The substrate was dissolved and diluted with DMSO. Measurements were also performed using an activity assay reagent with the same composition as the substrate solution, but using DMSO instead, and the A per unit time was calculated. ５５０ Quantity change (ΔA ｂｌａｎｋ ) was calculated. ΔA ｓａｍｐｌｅ -ΔA ｂｌａｎｋ The dehydrogenase activity of each enzyme was evaluated by calculating the θ. Figure 16 shows the dehydrogenase activity of wild-type and mutant oxidoreductases.

[0189]

[0190] 7-1. Evaluation method for mutations that enhance calcifediol dehydrogenase activity when using androsterone as a substrate (ΔA ｓａｍｐｌｅ -ΔA ｂｌａｎｋ Let A be the value of (ΔA) when calcifediol is used as the substrate. ｓａｍｐｌｅ -ΔA ｂｌａｎｋ The value of ) was defined as B, and the B / A value for each enzyme was calculated. Mutations in which the B / A value of the enzyme after mutation introduction increased by 0.01 or more compared to before mutation introduction were evaluated as mutations that improved reactivity to calcifediol. As shown in Figures 9 to 11, the 1M and 5M mutants of each oxidoreductase were shown to have improved reactivity to calcifediol.

[0191] 7-2. Evaluation method for mutations that enhance calcifediol dehydrogenase activity. When calcifediol is used as a substrate, (ΔA ｓａｍｐｌｅ -ΔA ｂｌａｎｋ Let A be the value of (ΔA) when cholecalciferol is used as a substrate. ｓａｍｐｌｅ -ΔA ｂｌａｎｋ The value of () was set as B, and the B / A value for each enzyme was calculated. The B / A value of the enzyme before mutation was set to 1, and mutations in which the relative B / A value of the enzyme after mutation (relative value of cholecalciferol / calcifediol) was 0.9 or less compared to the enzyme before mutation were evaluated as mutations with improved substrate specificity for calcifediol. The mutants of various oxidoreductases shown in Figures 13 to 22 were shown to have improved substrate specificity for calcifediol.

[0192] 8. Method for evaluating mutations that enhance β-estradiol dehydrogenase activity when using androsterone as a substrate (ΔA ｓａｍｐｌｅ -ΔA ｂｌａｎｋ Let A be the value of (ΔA) when β-estradiol is used as the substrate. ｓａｍｐｌｅ -ΔA ｂｌａｎｋLet C be the value of ), and the C / A value of each of various enzymes was calculated. A mutant enzyme whose C / A value after mutation introduction increased by 0.01 or more compared to that before mutation introduction was evaluated as a mutation having improved reactivity to β-estradiol. As shown in Figures 9 and 10, it was revealed that the ChnA-M203V mutant and the PtHSD-Q75F mutant had improved reactivity to β-estradiol.

[0193] 9. Reagents used in the method for quantifying calcifediol The calcifediol quantification reagent included calcifediol monohydrate (manufactured by Tokyo Chemical Industry Co., Ltd.), β-nicotinamide adenine dinucleotide (reduced form, β-NADH, manufactured by Oriental Yeast Co., Ltd.), thionicotinamide adenine dinucleotide (oxidized form, Thio-NAD ＋ ), Triton X-100 (manufactured by Merck), and dimethyl sulfoxide (DMSO, manufactured by FUJIFILM Wako Pure Chemical Corporation) were used. The purified PtHSD-5M mutant (A187F / V115F / Y202L / G196F / S205A) was used as the enzyme.

[0194] 10. Principle of calcifediol measurement by enzyme cycling reaction Calcifediol is reacted with mutant oxidoreductase and Thio-NAD ＋ is oxidized in the presence of the above components to produce oxidized calcifediol and Thio-NADH. The reaction also acts reversibly, and the produced oxidized calcifediol is converted into calcifediol and β-NAD in the presence of oxidoreductase and β-NADH ＋ is produced. Under conditions where β-NADH is present in excess, enzyme cycling efficiently occurs, and the absorbance of light at a wavelength of 400 nm due to the produced Thio-NADH (A ４００ ) is measured, and A per minute ４００ Quantification can be performed by calculating the change in mass.

[0195] 11. Method for Quantifying Calcifediol After pre-warming 1485 μL of the calcifediol quantification reagent shown in Table 8 at 37°C for 5 minutes, the reagent was mixed with 15 μL of a substrate solution (32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 0 μM). After mixing, the reaction solution was transferred to a disposable cell made of PMMA, and the absorbance of light at a wavelength of 400 nm (A ４００ ) was measured for 10 minutes, and A per minute ４００ increase (ΔA ４００ ) was calculated. The measurement was performed three times, and the average value was plotted. The relationship between substrate concentration and absorbance (A ４００ ) is shown in Figure 12.

[0196]

[0197] As shown in FIG. 12, the PtHSD-5M mutant (A187F / V115F / Y202L / G196F / S205A) has a coefficient of determination (R), which is an indicator of the correlation between substrate concentration and absorbance in the range of 0 nM to 320 nM ２ ) was 0.9968, so a result indicating that there is a correlation between substrate concentration and absorbance was obtained. Therefore, it was shown that calcifediol can be quantified using the dehydrogenase activity of the PtHSD-5M mutant.

[0198] 11. Method for Quantifying Calcifediol After pre-warming 1485 μL of the calcifediol quantification reagent shown in Table 9 at 37°C for 5 minutes, the reagent was mixed with 15 μL of a substrate solution (8 μM, 4 μM, 2 μM, 1 μM, 0.4 μM, 0.2 μM, 0 μM). After pre-warming 1485 μL of the calcifediol quantification reagent shown in Table 10 at 37°C for 5 minutes, the reagent was mixed with 15 μL of a substrate solution (8 μM, 4 μM, 2 μM, 1 μM, 0.4 μM, 0 μM). After mixing, the reaction solution was transferred to a disposable cell made of PMMA, and the absorbance of light at a wavelength of 400 nm (A ４００ ) was measured for 10 minutes, and A per minute ４００ increase (ΔA ４００ ) was calculated. The measurement was performed three times, and the average value was plotted. The relationship between substrate concentration and absorbance (A ４００The relationship between the two is shown in Figure 23.

[0199]

[0200]

[0201] As shown in Figure 23, PtHSD-5M, PtHSD-7M, or PtHSD-5M / N152H / Y202H have a coefficient of determination (R) which is an indicator of the correlation between substrate concentration and absorbance in the range of 0 nM to 80 nM. ２ The coefficients of determination (R) were 0.9998, 0.9997, and 0.9994, respectively, indicating a correlation between substrate concentration and absorbance. CfHSD-7M has a coefficient of determination (R) that is an indicator of the correlation between substrate concentration and absorbance in the range of 0 nM to 80 nM. ２ The result showed a correlation between substrate concentration and absorbance, as the value was 0.9977. Therefore, it was shown that calcifediol can be quantified using the dehydrogenase activity of PtHSD-5M, PtHSD-7M, PtHSD-5M / N152H / Y202H, or CfHSD-7M.

[0202] 1 Working electrode, 3 Counter electrode, 5 Reference electrode, 7 Wiring section, 9 Terminal, 10 Sensor chip, 11 Substrate, 13 Spacer, 15 Cover, 19 Reaction layer, 30 Measurement section, 31 Switch, 33 Display, 100 Sensor, 110 Control section, 120 Display section, 130 Input section, 140 Memory section, 150 Communication section, 160 Power supply, 190 Wiring

Claims

1. A method for quantifying a hormone or hormone precursor, comprising adding oxidoreductase and oxidized coenzyme I to a sample, wherein the oxidoreductase oxidizes the hormone or hormone precursor contained in the sample to produce an oxidized hormone or oxidized hormone precursor, transfers electrons extracted from the hormone or hormone precursor to the oxidized coenzyme I to produce a reduced coenzyme I, and determines the concentration of the hormone or hormone precursor from the concentration of the reduced coenzyme I, wherein the oxidoreductase has (1) sequence identity of 70% or more with respect to the full-length amino acid sequence and 90% or more with respect to the amino acid sequence of the homology region, or (2) sequence identity of 90% or more with respect to the full-length amino acid sequence, and has hormone or hormone precursor oxidoreductase activity.

2. The oxidized form of coenzyme 1 is NAD ＋ A method for quantifying a hormone or hormone precursor according to claim 1, including the following:

3. A method for quantifying a hormone or hormone precursor according to claim 1, wherein by further adding a mediator and a reagent to the sample, the mediator transfers electrons from the reduced first coenzyme to the reagent, and the concentration of the hormone or hormone precursor is determined from the change in the amount of the reagent.

4. The method for quantifying a hormone or hormone precursor according to claim 3, wherein the mediator oxidizes the reduced coenzyme-1 to produce the oxidized coenzyme-1.

5. The oxidized first coenzyme is Thio-NAD ＋ A method for quantifying a hormone or hormone precursor according to claim 1, wherein the reduced second coenzyme contains NADH.

6. A method for quantifying a hormone or hormone precursor according to claim 5, wherein by further adding a reduced coenzyme II in an amount greater than that of the reduced coenzyme I to the sample, the oxidoreductase oxidizes the reduced coenzyme II to produce an oxidized coenzyme II, and transfers the electrons extracted from the reduced coenzyme II to the oxidized hormone or oxidized hormone precursor to produce the hormone or hormone precursor.

7. The method for quantifying a hormone or hormone precursor according to any one of claims 1 to 6, wherein the hormone or hormone precursor comprises 25-hydroxyvitamin D or 17β-hydroxysteroid.

8. A quantitative enzyme having (1) 70% or more sequence identity with respect to the full-length amino acid sequence and 90% or more sequence identity with respect to the amino acid sequence of the homologous region, or (2) 90% or more sequence identity with respect to the full-length amino acid sequence, and possessing hormone or hormone precursor oxidoreductase activity.

9. The quantitative enzyme according to claim 8, wherein the hormone or hormone precursor comprises 25-hydroxyvitamin D or 17β-hydroxysteroid.

10. A composition for quantifying a hormone or hormone precursor, comprising the quantitative enzyme according to claim 8 or 9 and an oxidized first coenzyme, wherein the oxidoreductase oxidizes a hormone or hormone precursor contained in a sample to produce an oxidized hormone or oxidized hormone precursor, and transfers electrons extracted from the hormone or hormone precursor to the oxidized first coenzyme to produce a reduced first coenzyme.

11. A kit for quantifying hormones or hormone precursors, comprising the quantitative enzyme described in claim 8 or 9 and an oxidized coenzyme I, wherein the oxidoreductase oxidizes a hormone or hormone precursor contained in a sample to produce an oxidized hormone or oxidized hormone precursor, and transfers electrons extracted from the hormone or hormone precursor to the oxidized coenzyme I to produce a reduced coenzyme I.

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