JP7255865B2 - Kynurenine monooxygenase and method for measuring kynurenine using the same - Google Patents

Description

The present invention relates to a kynurenine monooxygenase, a method for measuring kynurenine using the same, and a kit for measuring kynurenine containing the kynurenine monooxygenase.

L-kynurenine is known to exist in vivo as a metabolite of L-tryptophan, and the significance of its measurement in the medical field has been investigated in various ways. An example using kynurenine monooxygenase (KMO) is known for measuring L-kynurenine concentration in a sample. For example, examples using human-derived or Pseudomonas fluorescence-derived kynurenine monooxygenase have been reported (Non-Patent Document 1, Patent Document 1). Since kynurenine monooxygenase derived from eukaryotes such as humans is a membrane protein enzyme, it is difficult to mass-produce it in Escherichia coli or the like, and it is necessary to use a surfactant in combination for solubilization. A method of attaching a tag to solubilize kynurenine monooxygenase is also known, but this method has been reported to have a problem of lower activity per enzyme protein (Non-Patent Document 2). On the other hand, kynurenine monooxygenase derived from prokaryotic P.fluorescence is water-soluble and relatively easy to obtain using E. coli (Non-Patent Document 3). However, P.fluorescence-derived kynurenine monooxygenase is unstable against heat, so in Patent Document 1, it is mainly measured at 25°C. Cytophaga is also known as a kynurenine monooxygenase derived from other prokaryotes, but it is insoluble and requires the use of a detergent for solubilization ( Non-Patent Document 4). Thus, a thermostable, water-soluble kynurenine monooxygenase has not been known.

JP 2017-63786 A

2016 Chugoku-Shikoku Branch Medical Laboratory Society of Japan Association of Medical Technologists (49th meeting), 175. Basic study of kynurenine enzymatic measurement method Protein Expression and Purification 95 (2014) 96-103 Protein Expression and Purification 51 (2007) 324-333 Chemistry & Biology 10 (2003) 1195-1204

As described above, it is desired to develop a kynurenine monooxygenase that can be mass-produced and that is useful for measuring kynurenine in biological samples. An object of the present invention is to provide a water-soluble and thermostable kynurenine monooxygenase.

In order to solve the above problems, the present inventors screened prokaryotes, cloned genes, cultured and investigated the properties of purified enzymes. As a result, surprisingly, the prokaryotic enzyme obtained this time has kynurenine monooxygenase activity, is water-soluble, and has thermostability compared to the kynurenine monooxygenase derived from P.fluorescence. I found In addition, they have found that these enzymes are stable in alkaline conditions and have high substrate specificity to L-kynurenine, and completed the present invention.

The present invention relates to the following [1] to [9].
[1] A kynurenine monooxygenase having the following properties (a) to (h).
(a) reacting with NAD(P)H or derivatives thereof and L-kynurenine in the presence of oxygen to produce NAD(P) ⁺ or derivatives thereof and 3-hydroxykynurenine (b) prokaryotic (c) water-soluble (d) at pH 9.0, residual activity after standing at 25°C for 30 minutes is 70% or more (e) at pH 7.5, at 40°C for 60 minutes Residual activity after standing is 60% or more (f) It acts specifically on L-kynurenine and has activity on L-tryptophan of 5% or less (g) Molecular weight is 40 kDa to 60 kDa (h ) The kynurenine monooxygenase according to [1], which uses flavin as a coenzyme [2], wherein the prokaryote is selected from the group belonging to the order Deltaproteobacteria or the order Xanthomonadales.
[3] The kynurenine monooxygenase of [2], wherein the prokaryote is selected from the group belonging to the genus Pseudoxanthomonas, Myxococcus, Dyella, or Rhodanobacter.
[4] The kynurenine monooxygenase of [3], wherein the prokaryote is selected from Pseudoxanthomonas suwonensis, Myxococcus stipitatus, Dyella japonica and Rhodanobacter denitrificans.
[5] Any one of [1] to [4], wherein the amino acid sequence of kynurenine monooxygenase has 90% or more similarity to any one of the sequences shown in SEQ ID NOs: 6 to 9. A kynurenine monooxygenase as described.
[6] A method for measuring L-kynurenine using the kynurenine monooxygenase according to any one of [1] to [5].
[7] NAD(P)H or derivatives thereof and kynurenine monooxygenase are allowed to act to measure changes in one or more selected from the group consisting of the following (i) to (iv), [6 ] The method for measuring kynurenine described.
(i) decreased amount of NAD(P)H or derivatives thereof (ii) increased amount of NAD(P) ⁺ or derivatives thereof (iii) increased amount of 3-hydroxykynurenine (iv) decreased amount of L-kynurenine [ 8] A kit for measuring kynurenine containing the kynurenine monooxygenase according to any one of [1] to [5].
[9] The kynurenine measurement kit according to [8], which contains a liquid composition having a pH of 9.0 or higher or a dried product of the liquid composition.

INDUSTRIAL APPLICABILITY According to the present invention, a water-soluble and thermostable kynurenine monooxygenase was obtained. Since this enzyme is water-soluble, it is not necessary to use a surfactant for the purpose of solubilization. Furthermore, since this enzyme is heat-stable, measurement at 37°C, which is the measurement temperature of an automatic biochemical analyzer, is possible. is possible. When conventional enzymes were used, sensitivity was lowered due to heat inactivation during the reaction, so it was necessary to use an excess amount of enzyme during measurement, but the amount of enzyme can be reduced. Furthermore, since this enzyme has pH stability (especially resistance to alkali), it is possible to easily prepare a kit for measuring kynurenine at pH 9.0 or higher by mixing NADH, NADPH or their derivatives with the enzyme. All of these enzymes have high substrate specificity for L-kynurenine, and as a result, it is possible to measure L-kynurenine in biological samples.

The relationship between absorbance (ΔAbs340) and L-kynurenine (L-Kyn) concentration measurement using each kynurenine monooxygenase is shown.

The present invention will be described in detail below.

1. Kynurenine monooxygenase 1-1. Properties of Kynurenine Monooxygenase (a) Kynurenine Monooxygenase Activity The kynurenine monooxygenase of the present invention (hereinafter sometimes referred to as the present enzyme) is an oxidoreductase that uses L-kynurenine as a substrate. By reacting this enzyme with L-kynurenine in the presence of NADH, NADPH or their derivatives and oxygen (aerobic conditions), an oxidation reaction of L-kynurenine occurs, resulting in NAD ⁺ , NADP ⁺ or NAD ( P) ⁺ derivative and 3-hydroxykynurenine are produced.
In the following specification, NADH and NADPH are collectively referred to as NAD(P)H, and NAD ⁺ and NADP ⁺ are collectively referred to as NAD(P) ⁺ .

The kynurenine monooxygenase-catalyzed reaction of the present invention is carried out in the presence of an electron donor selected from NAD(P)H and derivatives thereof. A derivative of NAD(P)H is a derivative of NADH or NADPH, has the same electron-donating property as NADH or NADPH, and is used to improve the stability of NADH or NADPH, increase the molar extinction coefficient, etc. be able to. In the present invention, the type of derivative of NAD(P)H is not particularly limited as long as the reaction by kynurenine monooxygenase proceeds. Known NAD(P)H derivatives such as H derivatives can be used.

The kynurenine monooxygenase activity can be measured, for example, using the following reagents and conditions (referred to as "activity measurement method 1"). The measurement method is not limited to the following, and reagents and/or conditions can be changed as appropriate within a range that poses no problem for measurement.
0.6 mL of 100 mM potassium phosphate buffer (pH 7.5), 0.1 mL of 7.5 mM NADPH solution, 0.3 mL of 1 mM L-kynurenine solution, and 1.95 mL of ultrapure water were mixed in a quartz cell with an optical path length of 1 cm. , 37°C for 10 minutes, then 0.05 mL of the enzyme sample is added to initiate the reaction at 37°C. After starting the reaction, the change in absorbance at 340 nm is measured, the amount of decrease in absorbance at 340 nm per minute (ΔAbs340/min) is measured, and the kynurenine monooxygenase activity is calculated according to Equation (1). Considering that the substrate kynurenine also absorbs at 340 nm, the molar extinction coefficient of NADPH is calculated at 6.7 [mM ^-1 cm ^-1 ] according to Non-Patent Document 3 mentioned above. do.
In addition, it is also possible to replace the NADPH solution in the activity measurement method 1 with a cheaper NADH solution for measurement.

In formula 1, "3.0" is the volume of the reaction solution [mL], "6.7" is the molar extinction coefficient [mM ^-1 cm ^-1 ], "1.0" is the optical path length [cm], "0.05" is the liquid volume [mL] of the enzyme used.

(b) Origin The kynurenine monooxygenase of the present invention can be mass-produced by recombinant microorganisms using microorganisms such as Escherichia coli, yeast, and fungi as hosts, exhibits water solubility, and is derived from prokaryotes. preferable. The kynurenine monooxygenase of the present invention is not particularly limited as long as it is derived from a prokaryote, but the kynurenine monooxygenase of the present invention is preferably derived from a group whose prokaryotes belong to the Deltaproteobacteria network or the order Xanthomonadales, and belongs to the genus Pseudoxanthomonas, Myxococcus, Dyella, or Rhodanobacter. More preferably, it is derived from the microorganism to which it belongs. Among these, Pseudoxanthomonas suwonensis, Myxococcus stipitatus, Dyella japonica or Rhodanobacter denitrificans are more preferred.

(c) Water solubility The kynurenine monooxygenase of the present invention is water soluble. Specifically, when the enzyme is collected after culturing, it does not require a solubilization step using a surfactant or the like, and the enzyme protein may be present in the soluble fraction. Preferably, the solubility in 1 mL of water is 0.5. It is 1 mg or more, more preferably 1 mg or more. As a method for measuring the protein concentration, BSA (bovine serum albumin) is used as a standard substance, and a colorimetric method based on the Bradford method can be used.

(d) Alkaline stability The kynurenine monooxygenase of the present invention has stability in alkaline. If the residual kynurenine monooxygenase activity after standing at 25° C. for 30 minutes in an alkaline solution is 70% or more, it is judged to have alkali stability. Specifically, the kynurenine monooxygenase of the present invention has a residual activity of and 70% or more, preferably 75% or more, and more preferably 80% or more when the enzyme activity before treatment is 100%.

(e) Thermostability The kynurenine monooxygenase of the present invention is thermostable. If the residual kynurenine monooxygenase activity after standing at 40° C. for 60 minutes is 70% or more, it is judged to have temperature stability. Specifically, the kynurenine monooxygenase of the present invention has a residual activity of 1.0 mg / mL of the enzyme after standing for 60 minutes in a potassium phosphate buffer of pH 7.5 and at 40 ° C. When the enzyme activity is 100%, it is 60% or more, preferably 70% or more, more preferably 75% or more, and still more preferably 80% or more.

(f) Substrate specificity The kynurenine monooxygenase of the present invention has excellent substrate specificity. For example, it is at least less reactive to L-tryptophan than it is to L-kynurenine. In biological samples, L-tryptophan is a precursor of L-kynurenine, the content of L-tryptophan is generally higher than that of L-kynurenine, and the enzyme is highly reactive to tryptophan. and kynurenine can be difficult to measure accurately. Since the kynurenine monooxygenase of the present invention has higher specificity for L-kynurenine than for L-tryptophan, it is possible to accurately measure the amount or concentration of L-kynurenine even when L-tryptophan coexists. is possible. Specifically, the kynurenine monooxygenase of the present invention is measured according to activity measurement method 1 by changing L-kynurenine and L-kynurenine to L-tryptophan or D-kynurenine (an optical isomer of L-kynurenine). , the reactivity to L-tryptophan is 5% or less, preferably 2.5% or less, more preferably 1% or less, relative to the reactivity to L-kynurenine. In addition, the reactivity to D-kynurenine is 20% or less, more preferably 15% or less, and still more preferably 6% or less of the reactivity to L-kynurenine.

(g) Molecular Weight The molecular weight of the polypeptide portion constituting the kynurenine monooxygenase of the present invention is preferably 40-60 kDa, more preferably 45-55 kDa, still more preferably 50-52 kDa.

By "polypeptide portion" is meant kynurenine monooxygenase in a substantially unglycosylated state. The molecular weight of the polypeptide portion can be calculated from the amino acid sequence of the cloned kynurenine monooxygenase. If the non-cloned kynurenine monooxygenase of the present invention produced by a microorganism is of the sugar chain-binding type, the sugar chain is removed by heat treatment or treatment with a glycohydrolase. (ie, "polypeptide portion") and the molecular weight determined by SDS-polyacrylamide gel electrophoresis. The state in which sugar chains are not bound substantially means that the presence of sugar chains that inevitably remain in sugar chain-binding kynurenine monooxygenase treated with heat treatment or glycohydrolase is allowed. Thus, when the kynurenine monooxygenase is not naturally glycosylated, it itself corresponds to a "polypeptide moiety".

The means for removing sugar chains from sugar chain-binding kynurenine monooxygenase is not particularly limited. F (manufactured by Roche Diagnostics) at 37° C. for 6 hours.

When a sugar chain is bound to the kynurenine monooxygenase of the present invention, its molecular weight is not particularly limited as long as it does not negatively affect the kynurenine monooxygenase activity, substrate specificity, affinity for L-kynurenine, and the like. For example, the molecular weight of the kynurenine of the present invention with a sugar chain bound thereto is preferably 50 to 90 kDa. Sugar chain-binding kynurenine monooxygenase is preferable from the viewpoint of making the enzyme more stable and improving water solubility.

(h) Coenzyme The kynurenine monooxygenase of the present invention is preferably a flavin-binding enzyme that uses flavin as a coenzyme.

Other enzymatic properties of the kynurenine monooxygenase of the present invention include optimum pH and optimum temperature. Although it is generally within the following range, the present invention is not limited to this range.

・Optimal pH
The optimum pH for the kynurenine monooxygenase of the present invention is within the range of pH 6.2-8.8. As used herein, the optimum pH is defined as a final enzyme concentration of 0.05 U/mL and buffers exhibiting multiple pHs (acetic acid-sodium acetate buffer (pH 4.0 to 5.0), sodium citrate buffer (pH 5.0-6.0), potassium phosphate buffer (pH 6.0-8.0), TAPS buffer (pH 8.0-9.0) or glycine-NaOH buffer (pH 8.5-9. 5)) is used to determine the enzyme activity by the activity measurement method 2 described later. Taking the highest activity value measured in each buffer as 100%, the pH range in which the activity value is 90% or more is defined as the optimum pH.

- Optimum temperature The optimum temperature for the kynurenine monooxygenase of the present invention is in the range of 35 to 45°C. In this specification, the optimum temperature is 0.25 U/mL at the enzyme final concentration, and the enzyme activity at each temperature in a potassium phosphate buffer (pH 7.5) is measured and compared by the activity measurement method 1 described above. Seek by.

1-2. Polypeptide of kynurenine monooxygenase The kynurenine monooxygenase of the present invention is a polypeptide exhibiting the characteristics of (a) to (h) above, and/or a polypeptide according to any one of (I) to (III) below. If so, it is not particularly limited.
(I) a polypeptide consisting of any one amino acid sequence represented by SEQ ID NOS: 6-9;
(II) any one of the amino acid sequences represented by SEQ ID NOs: 6 to 9, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, added and/or inverted residues, and kynurenine mono a polypeptide having oxygenase activity;
(III) A polypeptide comprising an amino acid sequence homologous to any one of the amino acid sequences shown in SEQ ID NOs: 6 to 9 and having kynurenine monooxygenase activity.

"Several" is not limited as long as the kynurenine monooxygenase activity is maintained, but is, for example, less than 10% of the total number of amino acids, more preferably less than 5%, still more preferably less than 2%. number. For example, preferably at most 45, 40, 30, 20, 10 or 5 can be exemplified.

The type of amino acid substitution is not particularly limited, but conservative amino acid substitutions are preferred from the viewpoint of not significantly affecting the phenotype of kynurenine monooxygenase. In addition, one or several mutations can be obtained by introducing mutations into the DNA encoding the kynurenine monooxygenase of the present invention, which will be described later, using known techniques such as site-directed mutagenesis and random mutagenesis. It is possible to implement.

From the viewpoint of maintaining the activity of kynurenine monooxygenase, addition of a sequence that imparts functionality to the extent that it does not affect the active site or substrate binding site of kynurenine monooxygenase, the N-terminus, C-terminus, or At least one of the arbitrary sequences may be deleted. In addition, when these treatments deviate from the molecular weight range shown in (g), if the molecular weight before addition or deletion is within the range of (g), it is considered equivalent to the "polypeptide portion" of the present invention. can be done.

"Amino acid sequences having homology" are amino acid sequences that are preferably at least 90% or 95% similar, more preferably at least 96%, 97%, 98% or 99% identical, or preferably means at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% or 99% of the amino acid sequence.

The similarity is evaluated using the score table in Table 1 (GENETYX® Instruction Manual p.220 A.18 Homology Score Table A.18.2 Amino Acids, Reference: Atlas of Protein Sequence and Structure Vol.5 (1978)). is a value calculated by dividing the number of characters with a score of 0 or more during alignment by the alignment length. It can be calculated using GENETYX ((registered trademark), manufactured by Genetics Co., Ltd.), which is sequence analysis software. Specifically, the software is used with initial settings, homology analysis between amino acid sequences is performed, and similarity is calculated.
Identity is based on the value of identity calculated by homology analysis between amino acid sequences using GENETYX with default settings.

2. Method for obtaining kynurenine monooxygenase 2-1. DNA encoding kynurenine monooxygenase
The DNA of the present invention is a DNA encoding a polypeptide exhibiting the properties (a) to (h) above, and/or a DNA according to any one of (A) to (E) below.
(A) a DNA encoding the amino acid sequence described in (I) to (III) above;
(B) DNA consisting of the base sequences shown in SEQ ID NOS: 2-5;
(C) a DNA consisting of a nucleotide sequence having 70% or more identity with the nucleotide sequences shown in SEQ ID NOs: 2 to 5 and encoding a polypeptide having kynurenine monooxygenase activity;
(D) a DNA comprising a DNA that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequences shown in SEQ ID NOs: 2 to 5 and encoding a polypeptide having kynurenine monooxygenase activity;
(E) A nucleotide sequence in which one or several nucleotides are substituted, deleted, inserted, added and/or inverted in the nucleotide sequences shown in SEQ ID NOs: 2 to 5, and has kynurenine monooxygenase activity DNA that encodes a polypeptide.

A DNA encoding an enzyme having kynurenine monooxygenase activity herein refers to a DNA having a nucleotide sequence corresponding to the amino acid sequence. Therefore, DNAs that differ due to codon degeneracy are also included. A protein having the amino acid sequence encoded by the DNA of the present invention has kynurenine monooxygenase activity. The DNA of the present invention has 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 98% identity with the nucleotide sequences shown in SEQ ID NOs:2 to 5. % or more.

The identity of base sequences is based on the value of identity calculated by homology analysis between base sequences of GENETYX.

As long as the protein encoded by the DNA has the kynurenine monooxygenase activity, the DNA of the present invention hybridizes under stringent conditions to a nucleotide sequence complementary to any of the nucleotide sequences shown in SEQ ID NOs: 2 to 5. It may be soybean DNA. Here, "hybridization under stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.

Specific conditions include, for example, 50% formamide, 5×SSC (150 mM sodium chloride, 15 mM trisodium citrate, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetic acid, pH 7.2), 5× Denhardt's solution, 42° C. incubation of a solution of 0.1% SDS, 10% dextran sulfate and 100 μg/mL denatured salmon sperm DNA followed by washing at 42° C. in 0.2×SSC for background removal. be able to.

Regarding the DNA of (E) above, "several" has the same meaning as described in the above section "1-2. Polypeptide of kynurenine monooxygenase".

The DNA of the present invention can be obtained by standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. based on the sequence information of SEQ ID NOS: 2-5. can also be obtained by

Specifically, as a standard genetic engineering technique, after selecting appropriate source microorganisms that are thought to produce kynurenine monooxygenase by screening, the chromosomal DNA of these source microorganisms is extracted, and the chromosomal DNA of the source microorganism is extracted. It refers to a method of obtaining a DNA of interest by PCR or the like using suitable primers specific to a DNA sequence (eg, nucleotide sequences of SEQ ID NOs: 2-5).

Isolation and purification of the amplified DNA fragment can be performed by using, for example, gel electrophoresis or a commercially available purification kit.

By using the DNA of the present invention, the kynurenine monooxygenase of the present invention can be easily and stably produced in large amounts.

2-2. Acquisition of kynurenine monooxygenase-producing strains
The DNA obtained in 2-1 is subjected to prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungi (yeast, ascomycetes such as Aspergillus, basidiomycetes, etc.), insect cells, mammalian cells, etc. A production strain can be obtained by introducing it into a cell or the like. In the present invention, the production strain is not particularly limited, but since the kynurenine monooxygenase of the present invention is derived from prokaryotes, it is generally expressed in E. coli host/vector system.

Examples of E. coli host/vector systems include the known pUC system, pBluescriptII, pET expression system, pGEX expression system, pCold expression system, and the like. Moreover, it is also possible to co-express chaperones, lysozymes, etc., as necessary. In the present invention, preferred hosts are E. coli JM109 and E. coli BL21(DE3), and vectors are preferably pUC-based or pET-based plasmids.

Examples of other host/vector systems include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Pichia pastoris, etc. when the host is yeast, and pAUR101, pAUR224, pYES2, etc. as vectors. When the host is a fungus, examples include Aspergillus oryzae, Aspergillus niger and the like.

The means for introducing the DNA of the present invention into a host is not particularly limited. Monooxygenase-producing strains can be obtained. Host cells are not particularly limited as long as they can express the DNA of the present invention and produce kynurenine monooxygenase. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, fungi, insect cells and mammalian cells can be used. E. coli can be easily transformed using commercially available competent cells.

Since the transformant of the present invention has the ability to produce the kynurenine monooxygenase of the present invention, it can be used to efficiently produce the kynurenine monooxygenase of the present invention.

2-3. Method for Producing Kynurenine Monooxygenase The kynurenine monooxygenase of the present invention can be produced by culturing microorganisms or cells capable of producing the kynurenine monooxygenase of the present invention. Microorganisms and cells to be cultured are not particularly limited as long as they have the ability to produce the kynurenine monooxygenase of the present invention. (sequence)) can be preferably used.

The culture method and culture conditions are not particularly limited as long as the kynurenine monooxygenase of the present invention is produced. That is, on condition that kynurenine monooxygenase is produced, a method and conditions suitable for the growth of microorganisms and cells to be used can be appropriately set. Examples of culture conditions for microorganisms, such as culture medium, culture temperature, and culture time, are given below.

As the medium, a normal microbial culture medium can be used, and synthetic medium or natural medium can be used as long as it contains carbon sources, nitrogen sources, vitamins, inorganic substances, and other micronutrients required by the microorganisms to be used. Either can be used. Glucose, sucrose, lactose, dextrin, starch, glycerin, molasses and the like can be used as carbon sources. Nitrogen sources include inorganic salts such as ammonium chloride, ammonium nitrate, ammonium sulfate, and ammonium phosphate, amino acids such as DL-alanine and L-glutamic acid, peptone, meat extract, yeast extract, malt extract, and nitrogen-containing compounds such as corn steep liquor. Natural products can be used. As vitamins, riboflavin, pyridoxine, niacin (nicotinic acid), thiamine and the like can be used. Examples of inorganic substances that can be used include monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, and ferric chloride. Furthermore, compounds required for regulation of protein expression, such as isopropyl-β-thiogalactopyranoside (IPTG) and lactose, may be added as appropriate.

The culture conditions are not particularly limited as long as they are suitable for the production of kynurenine monooxygenase. The culture period is preferably in the range of 1 to 8 days for batch culture, but if the production volume is to be increased by fed-batch culture or continuous culture, the period should be extended as long as the productivity is determined to be optimal. be able to. As the culture method, for example, a shake culture method and an aerobic submerged culture method using a jar fermenter can be used.

As a method for obtaining kynurenine monooxygenase from the culture, a conventional protein purification method can be used. When kynurenine monooxygenase is present in the cells, after culturing the producing strain, the culture solution is centrifuged to obtain the cultured microorganism, then the cultured microorganism is disrupted and/or lysed by an appropriate method, and the treated solution is removed. By obtaining the supernatant by centrifugation, etc., or if kynurenine monooxygenase is secreted in the medium, after culturing the producing bacteria, by centrifuging the culture medium to obtain the supernatant, A crude enzyme solution can be obtained.

The kynurenine monooxygenase contained in these crude enzyme solutions is subjected to appropriate purification procedures such as ultrafiltration, salting out, solvent precipitation, heat treatment, dialysis, ion exchange chromatography, hydrophobic adsorption chromatography, gel filtration, and affinity chromatography. It can be refined by combining. The purified enzyme preparation is preferably purified to the extent that it shows a single band electrophoretically (SDS-PAGE).

When obtaining this enzyme as a recombinant protein, various modifications are possible. For example, if a DNA encoding the present enzyme and other suitable DNA are inserted into the same vector, and the vector is used to produce a recombinant protein, the recombinant protein is composed of any peptide or protein linked thereto. This enzyme can be obtained. In addition, addition of sugar chains and/or lipids, or modification that causes N-terminal or C-terminal processing may be performed. Modifications such as those described above enable the extraction and purification of recombinant proteins to be simplified, and/or the addition of biological functions, and the like.

3. Method for Measuring Kynurenine Using Kynurenine Monooxygenase A method for measuring kynurenine using the kynurenine monooxygenase of the present invention has been established in the art. Therefore, according to known methods, the kynurenine monooxygenase of the present invention can be used to measure the amount or concentration of kynurenine in various samples. By reacting the kynurenine monooxygenase of the invention with L-kynurenine in the presence of NAD(P)H or derivatives thereof, NAD(P) ⁺ or derivatives thereof and 3-hydroxykynurenine are produced. By monitoring the increase or decrease of substances in this reaction, the amount or concentration of L-kynurenine can be measured. Although the aspect is not particularly limited, for example,
(i) a reduced amount of NAD(P)H or derivatives thereof;
(ii) increased amounts of NAD(P) ⁺ or derivatives thereof;
By measuring (iii) the increase in 3-hydroxykynurenine or (iv) the decrease in L-kynurenine, the amount or concentration of kynurenine can be determined.

In the present invention, the amount or concentration of kynurenine can be measured by adding the kynurenine monooxygenase of the present invention to a sample. Examples of samples containing kynurenine include, but are not limited to, body fluids (eg, blood, urine, saliva, etc.), beverages, foods, and the like.

(i) L-kynurenine is oxidized, catalyzed by kynurenine monooxygenase (KMO), with a concomitant decrease in NAD(P)H or derivatives thereof; L-kynurenine can be measured by measuring the decrease in NAD(P)H or derivatives thereof. Specifically, a sample containing L-kynurenine is mixed with a buffer solution (pH 5 to 10) containing NAD(P)H or a derivative thereof, and KMO is added to absorb NAD(P)H. It can be performed by measuring the amount of decrease in absorbance at the maximum wavelength (340 nm). Alternatively, a reaction that further measures NAD(P)H with high sensitivity may be combined, for example, measuring the decrease in absorbance of a substance produced by mixing NAD(P)H with a tetrazolium salt and a charge carrier. method, a method of mixing resazurin and diaphorase with NAD(P)H and measuring the amount of decrease in the substance produced, or a method of measuring by mixing with reagents capable of chemiluminescence or electrochemical detection. can give.

(ii) kynurenine is oxidized catalyzed by kynurenine monooxygenase, and NAD(P) ⁺ or a NAD(P) ⁺ derivative is generated from the electron donor NAD(P)H or a derivative thereof. Kynurenine can be measured by measuring this increase. Specifically, (ii-1) fluorescence measurement derived from NAD(P) ⁺ or NAD(P) ⁺ derivative, or (ii-2) enzymatic cycling method can be used.

(ii-1) Fluorescence measurement derived from NAD(P) ⁺ or NAD(P) ⁺ derivative is performed by the method described in a known document (Chem Commun (Camb). 2013, 49(98):11500-2). be able to. Specifically, NAD(P) ⁺ can be reacted with acetophenone, 2-acetylbenzofuran, or the like, and the resulting fluorescent substance can be measured by fluorescence (Table 2 in the literature).
(ii-2) The enzymatic cycling method can be performed, for example, according to the method described in Example 4 of Patent Document 1.

(iii) kynurenine monooxygenase oxidizes kynurenine to produce 3-hydroxykynurenine; Kynurenine can be measured by measuring the increase in 3-hydroxykynurenine. Specifically, for example, it can be carried out according to the method described in Example 3 of Patent Document 1.

(iv) L-kynurenine is oxidized to produce 3-hydroxykynurenine and the amount of the substrate L-kynurenine is reduced. L-kynurenine can be measured by measuring the amount of decrease in L-kynurenine. The amount of L-kynurenine decreased can be determined by measuring the fluorescence of L-kynurenine (Clinical Chemistry, 1975, Vol. 3, No. 4, 426-434). L-kynurenine is excited at 370 nm and emits fluorescence at 490 nm. On the other hand, since 3-hydroxykynurenine, which is a metabolite of L-kynurenine, does not emit fluorescence, the L-kynurenine concentration can be estimated from the decrease in fluorescence.

The measurement conditions for L-kynurenine are appropriately set according to the stability of reagents used such as pH, temperature, and reaction time, the optimal reaction conditions for kynurenine monooxygenase and enzymes used in combination, and/or the assumed measurement environment. can do. For example, in view of the stability of the kynurenine monooxygenase of the present invention, the temperature is 20 to 50°C, preferably 25 to 45°C, more preferably 30 to 40°C, pH 5.0 to 10.0, preferably pH 6.0. It can be carried out by reacting for 0.5 minutes to 1 hour, preferably 1 minute to 30 minutes within the range of to 9.0.

The measurement of a biological sample may include a pretreatment step of removing at least one of reducing substances, contaminant proteins, metal ions, etc., which may interfere with the measurement, by a known method. Examples of reducing substances include bilirubin, uric acid, and reduced ascorbic acid. For example, a pretreatment step for reducing bilirubin includes treatment with bilirubin oxidase, a pretreatment step for reducing uric acid includes treatment with uricase, and a pretreatment step for reducing ascorbic acid includes ascorbic acid. Examples include treatment with acid oxidase. Furthermore, treatment with hydrogen peroxide or peroxidase can also be performed. Contaminant proteins can be reduced by, for example, deproteinizing agents such as perchloric acid and trichloroacetic acid. Metal ions can be reduced, for example, with metal chelating agents. Although the metal chelating agent is not particularly limited, EDTA (ethylenediaminetetraacetic acid), bicine (N,N-bis(2-hydroxyethyl)glycine), DTPA (diethylenetriaminepentaacetic acid), CyDTA (trans-1,2-diaminocyclohexane- N,N,N,N-tetraacetic acid) and the like are preferably used.

Since the kynurenine monooxygenase of the present invention is highly selective for kynurenine over tryptophan, the effect of contaminants in the sample may optionally be substantially removed for more accurate measurements, although this is not necessary. . For example, since the concentration of L-tryptophan in a biological sample is generally considered to be about 10 to 100 times the concentration of L-kynurenine, it is preferable to suppress the influence of L-tryptophan on L-kynurenine measurement.

If the effect of L-tryptophan is to be reduced, a method for measuring L-kynurenine that includes a step of reducing L-tryptophan in a sample using an enzyme that acts on L-tryptophan is preferred. This step is preferably performed as a pretreatment prior to the reaction with kynurenine monooxygenase. For the L-tryptophan reduction step, it is preferred to use an enzyme that acts on L-tryptophan. Enzymes that act on L-tryptophan are preferably enzymes that do not substantially act on L-kynurenine, such as tryptophan oxidase and tryptophanase. Tryptophan oxidase is preferably used in combination with catalase.

The amount of enzyme used is not particularly limited as long as the amount of L-tryptophan can be reduced, but in the case of tryptophan oxidase, the concentration in the sample is preferably about 0.1 to 100 U/mL, and about 1 to 10 U/mL. is more preferred. When tryptophan oxidase is combined with catalase, the concentration in the sample is preferably about 0.1 to 10,000 U/mL, more preferably about 1 to 1,000 U/mL. In the case of tryptophanase, the concentration in the sample is preferably about 0.01 to 10 U/mL, more preferably about 0.1 to 2 U/mL.

4. Kynurenine measurement kit The present invention also relates to a measurement kit containing the kynurenine monooxygenase of the present invention. Typically, the kit composition comprises, in addition to the kynurenine monooxygenase of the invention, a buffer, an electron donor selected from the group consisting of NAD(P)H and NAD(P)H derivatives. Preferably, the kynurenine monooxygenase is contained in an amount sufficient for at least one measurement, preferably about 0.001 to 200 U, more preferably about 0.05 to 50 U per sample. In addition, the composition of the kynurenine measurement kit of the present invention is in a composition dissolved in a solution (e.g., buffer) suitable for storage or measurement of kynurenine, or in a freeze-dried state (e.g., powder). It is desirable to have

The kynurenine measurement kit of the present invention can be used as a reagent for measuring kynurenine. Since NADH and NADPH are easily decomposed at pH 8.0 or lower, when the composition of the present kit is to be kept in a liquid state, it is preferably weakly alkaline, such as pH 8.5 or higher, and pH 9.0 or higher. is more preferable, and a pH of 9.5 or higher is even more preferable. In addition, the buffer is not particularly limited as long as the pH can be adjusted to a weakly alkaline range. sulfonic acid), glycine-NaOH, carbonate-sodium bicarbonate, and the like can be used.

Compositions other than kynurenine monooxygenase, buffer solution and electron donor include, for example, bovine serum albumin (BSA) or ovalbumin, sugars (eg trehalose) or sugar alcohols (eg maltitol, sorbitol, glycerol) etc.), carboxyl group-containing compounds, alkaline earth metal compounds, ammonium salts, sulfates, amino acids that do not react with enzymes, stabilizers or antioxidants selected from the group consisting of flavins or proteins, etc. By appropriately containing a known stabilizing component, the thermal stability and storage stability of the enzyme and reagent components can be enhanced. In addition, the stabilizer and other substances can be used in the reagent composition for the purpose of enhancing or improving reactivity, specificity, drying property and/or solubility.

The present invention is illustrated by the following examples, but the invention is not limited to the following examples.

Example 1
Acquisition of kynurenine monooxygenase Various strains producing kynurenine monooxygenase were selected by screening against prokaryotes. A plurality of genes (DNA sequences of SEQ ID NOs: 2 to 5) thought to have kynurenine monooxygenase (KMO) activity were obtained by extracting the chromosomal DNA of each strain and performing PCR using the DNA as a template. . For the PCR, primers designed based on the KMO sequences separately elucidated by the present inventors were used. SEQ ID NO: 2 is Pseudoxanthomonas suwonensis (abbreviated as P.suw), SEQ ID NO: 3 is Myxococcus stipitatus (abbreviated as M.sti), SEQ ID NO: 4 is Dyella japonica (abbreviated as D.jap), SEQ ID NO: 5 is Rhodanobacter denitrificans (R den). As a comparative example, according to Patent Document 1, a gene encoding a known amino acid sequence (SEQ ID NO: 1) derived from Pseudomonas fluorescence (abbreviated as P.flu) was also obtained. After inserting each gene into the pET-21a(+) vector, each of the resulting vectors was introduced into Escherichia coli BL21(DE3) competent cells (manufactured by Biodynamics Laboratory) to obtain each recombinant Escherichia coli. The cells obtained by culturing each recombinant E. coli in LB medium were crushed and centrifuged, and the resulting supernatant was subjected to ammonium sulfate fractionation and ion exchange chromatography to obtain water-soluble purified enzymes. Each purified enzyme was subjected to electrophoresis (SDS-PAGE) to confirm that it was purified to almost a single band. Furthermore, it was confirmed by the kynurenine monooxygenase activity assay described in Activity Assay 1 above that all of the purified enzymes have kynurenine monooxygenase activity.

As a result, the E. coli recombinant enzymes of four novel kynurenine monooxygenases derived from P.suw, M.sti, D.jap and R.den, and the E. coli recombinant enzymes of P.flu-derived kynurenine monooxygenase described in Patent Document 1 were found. Recombinant enzymes were obtained (hereinafter referred to as P.suw-derived KMO, M.sti-derived KMO, D.jap-derived KMO, R.den-derived KMO, and P.flu-derived KMO, respectively). The P.suw-derived KMO has the amino acid sequence shown in SEQ ID NO: 6, the M.sti-derived KMO has the amino acid sequence shown in SEQ ID NO: 7, the D.jap-derived KMO has the amino acid sequence shown in SEQ ID NO: 8, and the R.den-derived KMO has the amino acid sequence shown in SEQ ID NO: 9. ing. The final protein concentrations of the new KMO solutions were 7.4, 6.3, 6.0 and 1.7 mg/mL, respectively. When NADH or NADPH is used as an electron donor during activity measurement, the activity ratio (NADH/NADPH) is 1.5 for P.suw-derived KMO and 1.0 for M.sti-derived KMO. , P.flu, D.jap and R.den-derived KMO was 0.5, indicating that NADH or NADPH can be appropriately selected.

Example 2
Characterization of kynurenine monooxygenase 1
(1) Thermostability The above-mentioned P.suw-derived KMO, M.sti-derived KMO, D.jap-derived KMO and R.den-derived KMO, and P.flu-derived KMO were added to 50 mM potassium phosphate buffer (pH 7.5). It was heated at 40° C. for 60 minutes in the presence of the enzyme, and the residual activity of the enzyme solution after heat treatment was evaluated by the kynurenine monooxygenase activity measurement method described in Activity Measurement Method 1 above. The residual activity was obtained as a relative value when the activity of the enzyme after standing at 4° C. for 60 minutes was defined as 100%. The results were as shown in Table 2.

The activity of the P.flu-derived KMO was greatly reduced by heating at 40°C for 1 hour, whereas all the novel KMOs of the present invention retained their activity even after 1 hour.

(2) Substrate specificity The substrate specificity of each KMO was evaluated. The reagents and conditions for measuring kynurenine monooxygenase activity are as described in Activity Assay 1 above.

Of these, the substrate L-kynurenine was changed to L-tryptophan and D-kynurenine, respectively, and measured. Table 3 shows the activity on L-tryptophan and D-kynurenine when the activity on L-kynurenine is defined as 100%.

(3) Molecular Weight The molecular weight of each enzyme was calculated from the amino acid composition of the amino acid sequences shown in SEQ ID NOS:6-9. The results were as shown in Table 4.

(4) pH stability (resistance to alkali)
Each KMO with a protein concentration of 1 mg/mL was treated at 25° C. for 30 minutes in the presence of 200 mM glycine-NaOH buffer (pH 9.0 or pH 9.5). After 30 minutes, the solution was diluted with 1 M potassium phosphate buffer (pH 7.5) to neutralize the pH. Then, the kynurenine monooxygenase activity was measured by the method described in Activity Assay 1 above. The residual activity was obtained as a relative value when the activity of the enzyme at the start of treatment was taken as 100%. The results were as shown in Table 5.

As described above, all of the kynurenine monooxygenases of the present invention are known P. flu-derived It was found to have higher stability than kynurenine monooxygenase.

(5) Coenzyme An absorption spectrum at 200-700 nm was measured with a spectrophotometer for each KMO. As a result, a characteristic spectrum derived from FAD was obtained, and the amino acid sequences described in SEQ ID NOS: 6 to 9 all contain the consensus sequence GXGXXG of the FAD enzyme. determined that FAD is a coenzyme (G for glycine, X for any amino acid).

Example 3
Measurement of L-kynurenine concentration using KMO KMO was applied to a sample containing L-kynurenine, and the L-kynurenine concentration in the sample was measured from the decrease in NADPH (decrease in absorbance at 340 nm). 0.6 mL of 100 mM potassium phosphate buffer (pH 7.5), 0.1 mL of 3 mM NADPH solution, 0.3 mL of L-kynurenine solution, and 1.95 mL of ultrapure water were mixed in a quartz cell with an optical path length of 1 cm. At this time, the concentration of the L-kynurenine solution to be added was changed to 50 μM, 100 μM, 150 μM and 200 μM, and samples were also prepared by adding ultrapure water instead of L-kynurenine. After heating at 37° C. for 10 minutes, the absorbance at 340 nm was measured and used as a blank value. 3 U/mL KMO enzyme or 0.05 mL of ultrapure water was added, the absorbance at 340 nm was measured after 1 minute, and the corresponding blank value was subtracted from the obtained value (measured value A). The measured value A for each L-kynurenine concentration was subtracted from the measured value A for the sample containing no L-kynurenine (measured value B). A calibration curve was prepared by plotting the L-kynurenine (L-Kyn) concentration on the X-axis and the measured value B (ΔAbs340) on the Y-axis.

As a result, as shown in FIG. 1, the slope was higher when the KMO of the present invention was used than when the P.flu-derived KMO of the comparative example was used (for reference, ΔAbs340 Numerical data are shown in Table 6). Although not shown in FIG. 1 or Table 6, the activity of P.flu-derived KMO was similar to that of the KMO of the present invention when double the amount of KMO was used. That is, while the conventional KMO was deactivated during the reaction, the KMO of the present invention has high thermal stability and does not deactivate, resulting in approximately double the reactivity. It was found that the use of KMO enables measurement with a smaller amount of enzyme than conventional KMO.

Example 4
Preparation of Kynurenine Measurement Kit A kynurenine measurement kit was obtained by preparing a solution consisting of KMO final concentration 0.5 U/mL, NADPH final concentration 250 μM, and 50 mM glycine-NaOH buffer (pH 9.0). This kit was mixed with a buffer solution for measurement and a sample, and reacted at 37°C. As a result, a change in absorbance at 340 nm was observed in the reaction solution, indicating that it is possible to measure the kynurenine concentration in the sample. rice field.
Moreover, by freeze-drying this solution, a kynurenine measurement kit could be obtained as a dried product.

Example 5
Characterization of kynurenine monooxygenase 2
For the kynurenine monooxygenase obtained in Example 1, the optimal temperature and optimal pH of the enzyme were measured.

The optimum temperature was set to an enzyme final concentration of 0.25 U/mL, and was measured, compared, and determined by the kynurenine monooxygenase activity measurement method described in Activity Measurement Method 1 above. The measurement temperature was varied, and the temperature at which the activity value was the highest was taken as the optimum temperature. P.flu-derived KMOs were evaluated at 25, 30, 35 and 40°C, and other KMOs were evaluated at 25, 37 and 45°C. Table 7 shows the results. The optimum temperature was within the range of 35-45°C.

The optimum pH was measured using a microplate. 1 M buffers with different pH (acetic acid-sodium acetate buffer (pH 4.0, 4.5, 5.0), sodium citrate buffer (pH 5.0, 5.5, 6.0), potassium phosphate buffer (pH 6.0, 6.5, 7.0, 7.5, 8.0), TAPS buffer (pH 8.0, 8.5, 9.0) or glycine-NaOH buffer (pH 8.5 , 9.0, 9.5)) were used to prepare 80 mM of each buffer solution and used in the enzyme reaction solution. In a microplate, 50 μL of each pH 80 mM buffer solution, 50 μL of a solution consisting of 1 mM NADPH and 0.8 mM L-kynurenine are mixed, then 100 μL of a 0.1 U/mL enzyme solution is mixed and reacted at 25° C. for 5 minutes. let me To measure hydroxykynurenine produced by the enzymatic reaction, 50 μL of the reaction solution was sampled and mixed with 50 μL of a solution of 200 mM potassium phosphate buffer (pH 7.5) and 0.8 mM potassium ferricyanide. Since the color developed rapidly, the absorbance at 450 nm was measured immediately after the color development using a plate reader (SpectraMax Plus 384, manufactured by Molecular Devices). Absorbance at 450 nm of a solution consisting of 200 mM potassium phosphate buffer (pH 7.5) and 0.8 mM potassium ferricyanide was used as a blank, and activity was evaluated by subtracting this value (referred to as "activity measurement method 2"). . The pH of the enzyme reaction solution was measured with a pH meter, and the measured value was taken as the actual pH. In the measurement of each enzyme, when the highest activity value was defined as 100%, the pH range in which the activity value was 90% or more was defined as the optimum pH. The optimum pH of the kynurenine monooxygenase of the present invention was in the range of 6.2 to 8.8, and Table 7 shows the optimum pH of each enzyme. The pH at which each enzyme showed the highest activity value is also shown.

Claims (5)

A kynurenine monooxygenase having an amino acid sequence having 98% or more identity with any one of the amino acid sequences represented by SEQ ID NOs: 6 to 8,
A kynurenine monooxygenase having the following properties (a)-( g ).
(a) reacts with L-kynurenine in the presence of NAD(P) H and oxygen to produce NAD(P) ⁺ and 3-hydroxykynurenine ( b ) is water soluble ( c ) pH 9.0. At pH 7.5, the residual activity after standing at 25°C for 30 minutes is 70% or more ( d ) At pH 7.5, the remaining activity after standing at 40°C for 60 minutes is 60% or more ( e ) L- Acts specifically on kynurenine, and exhibits activity on L-tryptophan of 5% or less when measured at 37°C using a potassium phosphate buffer (pH 7.5) ( f ) with a molecular weight of 40 kDa to 60 kDa a ( g ) flavin coenzyme A method for measuring L-kynurenine using the kynurenine monooxygenase according to claim 1 . The method for measuring kynurenine according to claim 2 , wherein one or more changes selected from the group consisting of the following (i) to (iv) are measured by allowing NAD(P) H and kynurenine monooxygenase to act: .
(i) decrease in NAD(P) H (ii) increase in NAD(P) ⁺ (iii) increase in 3-hydroxykynurenine (iv) decrease in L-kynurenine A kynurenine assay kit comprising the kynurenine monooxygenase according to claim 1 . 5. The kit for measuring kynurenine according to claim 4 , comprising a liquid composition having a pH of 9.0 or higher or a dried product of the liquid composition.

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