Method for quantifying citrulline, composition for quantifying citrulline, kit for quantifying citrulline, sensor chip, sensor, and method for evaluating peptidylarginine deiminase activity

Abstract

Provided are: a new quantification method for measuring citrulline, which is associated with various diseases and is an especially useful biomarker for early diagnosis of rheumatoid arthritis; an enzyme for quantification; a composition for quantification; and a kit for quantification. Also provided is a method for evaluating the activity of PAD using a method for measuring citrulline. A method for quantifying citrulline, the method including adding an oxidoreductase to a sample, is provided. The oxidoreductase is an oxidase, and the concentration of citrulline may be determined by quantifying the hydrogen peroxide produced by adding the oxidase. Alternatively, the hydrogen peroxide produced by adding the oxidase may be reacted with a reagent to determine the concentration of citrulline.

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying citrulline, an oxidoreductase for the quantification, a composition for the quantification, a kit for the quantification, and a method for evaluating the activity of peptidylarginine deiminase. [Background technology]

[0002] Citrulline is not a codon-specified amino acid, but rather can be produced by conversion of arginine residues in proteins to citrulline by peptidylarginine deiminase (PAD) or by conversion from ornithine by ornithine transcarbamoylase in the ornithine cycle. Abnormal citrullination of proteins in vivo has been reported to be associated with diseases such as multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, psoriasis, prion disease, liver fibrosis, chronic obstructive pulmonary disease, and cancer.

[0003] For example, rheumatoid arthritis is considered an autoimmune disease that causes chronic inflammation in the joints, which, as the disease progresses, destroys the joints and causes various functional disorders. Therefore, early detection and treatment of rheumatoid arthritis are important, but the conventional test item, rheumatoid factor (RF), can sometimes give false positives even in healthy individuals.

[0004] Anti-cyclic citrullinated peptide (anti-CCP) antibodies are considered a useful indicator for the early diagnosis of rheumatoid arthritis because they show positivity earlier than RF in patients with rheumatoid arthritis and have higher specificity than RF. However, it has been pointed out that in early rheumatoid arthritis, anti-CCP antibody tests produce false negatives in approximately 30% of cases, delaying a definitive diagnosis. In addition, because the test is based on an immunological assay, it has the drawbacks of long measurement times and high testing costs.

[0005] Therefore, a method for measuring citrullinated peptides themselves is desired, rather than measuring anti-CCP antibodies. Specifically, a method is considered in which citrullinated peptides are decomposed with protease or the like, and the liberated citrulline is measured. For example, an enzymatic measurement system can be used to measure citrulline. For example, Patent Document 1 describes a method in which argininosuccinate synthase acts on citrulline to generate pyrophosphate, pyrophosphate pyruvate dikinase acts on the resulting pyrophosphate to generate pyruvic acid, and citrulline is quantified based on the amount of pyruvic acid obtained.

[0006] Five types of PAD are known: PAD1 to PAD4 and PAD6. PAD2 is thought to be involved in the development of Alzheimer's disease, and PAD4 is thought to be involved in the development of rheumatoid arthritis (Non-Patent Document 1). From the perspective of treating these diseases, not only is there a need for a method for quantifying citrullinated peptides, but there is also a need for a method for measuring the activity of PADs that convert arginine peptides into citrullinated peptides. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 5303715 [Non-patent literature]

[0008] [Non-Patent Document 1] Akito Ishigami, "Citrullination Molecules and Geriatric Diseases," Journal of the Japan Geriatrics Society, Japan Geriatrics Society, July 2014, Vol. 51, No. 4, pp. 314-320 Summary of the Invention [Problem to be solved by the invention]

[0009] However, the method disclosed in Patent Document 1 requires three-step enzyme reactions, which involve the action of argininosuccinate synthase, pyrophosphate pyruvate dikinase, and pyruvate oxidase for quantifying pyruvic acid, making the operation complicated.In addition, the complicated operation causes errors in measurement values, reducing the accuracy of the test.Therefore, a method is required for easily quantifying citrulline by directly oxidizing or reducing citrulline.

[0010] An object of the present invention is to provide a novel method for measuring citrulline, a biomarker that is associated with various diseases and is particularly useful for the early diagnosis of rheumatoid arthritis, as well as a novel enzyme for the measurement, a composition for the measurement, and a kit for the measurement. Another object of the present invention is to provide a method for evaluating PAD activity using the citrulline measurement method. [Means for solving the problem]

[0011] According to one embodiment of the present invention, there is provided a method for quantifying citrulline by adding citrulline oxidoreductase to a sample.

[0012] The citrulline oxidoreductase is citrulline oxidase, and the concentration of citrulline may be determined by quantifying the hydrogen peroxide produced by adding citrulline oxidase.

[0013] The citrulline oxidoreductase is citrulline oxidase, and the concentration of citrulline may be determined by reacting the hydrogen peroxide generated by adding the citrulline oxidase with a reagent.

[0014] The citrulline oxidoreductase is citrulline dehydrogenase, and the concentration of citrulline may be determined by adding citrulline dehydrogenase to reduce the mediator.

[0015] According to one embodiment of the present invention, there is provided a composition for quantifying citrulline, which comprises citrulline oxidoreductase for use in a method for quantifying citrulline.

[0016] The citrulline oxidoreductase is citrulline oxidase and may include a reagent that reacts with hydrogen peroxide generated by adding citrulline oxidase.

[0017] According to one embodiment of the present invention, there is provided a kit for quantifying citrulline, comprising citrulline oxidoreductase and a reagent that reacts with hydrogen peroxide.

[0018] According to one embodiment of the present invention, there is provided a composition for quantifying citrulline, which comprises a mediator that is reduced by adding citrulline oxidoreductase.

[0019] According to one embodiment of the present invention, there is provided a kit for quantifying citrulline, which comprises citrulline oxidoreductase and a mediator that is reduced by the addition of citrulline oxidoreductase.

[0020] According to one embodiment of the present invention, there is provided a sensor chip containing citrulline oxidoreductase for use in the above-described method for quantifying citrulline.

[0021] According to one embodiment of the present invention, there is provided a sensor including the above sensor chip.

[0022] According to one embodiment of the present invention, there is provided a method for evaluating the activity of peptidylarginine deiminase using any of the above methods for quantifying citrulline.

[0023] According to one embodiment of the present invention, there is provided a method for evaluating the activity of peptidylarginine deiminase, which comprises adding a peptide that can be a substrate for peptidylarginine deiminase to a sample, adding a protease or peptidase to the sample, and adding citrulline oxidoreductase to the sample.

[0024] The peptidyl arginine deiminase may be PAD2 or PAD4. [Effects of the Invention]

[0025] The present invention provides a novel method for measuring citrulline, a biomarker associated with various diseases and particularly useful for the early diagnosis of rheumatoid arthritis, as well as an enzyme for the measurement, a composition for the measurement, and a kit for the measurement. The present invention also provides a method for evaluating PAD activity using the method for measuring citrulline. [Brief explanation of the drawings]

[0026] [Figure 1] 1A is a schematic diagram of a sensor 100 according to an embodiment of the present invention, and FIG. 1B is a block diagram of the sensor 100 according to an embodiment of the present invention. [Figure 2] FIG. 1(a) is a schematic diagram of a sensor chip 10 according to one embodiment of the present invention, and FIGS. 1(b) to 1(d) are schematic diagrams showing components constituting the sensor chip 10. FIG. [Figure 3] FIG. 1(a) is a graph showing the correlation between the citrulline concentration (mM) and enzyme activity (U / ml) of citrulline oxidase (WT) treated with a buffer solution at pH 6.0 according to an embodiment of the present invention, and FIG. 1(b) is a graph showing the correlation between the citrulline concentration (mM) and enzyme activity (U / ml) of citrulline oxidase variant E486Q treated with a buffer solution at pH 6.0 according to an embodiment of the present invention. [Figure 4] FIG. 1(a) is a graph showing the correlation between the citrulline concentration (mM) and enzyme activity (U / ml) measured using the dehydrogenase reaction of citrulline oxidase (WT) treated with a buffer solution at pH 6.0 according to an example of the present invention, and FIG. 1(b) is a graph showing the correlation between the citrulline concentration (mM) and enzyme activity (U / ml) measured using the dehydrogenase reaction of citrulline oxidase mutant E486Q treated with a buffer solution at pH 6.0 according to an example of the present invention. [Figure 5] FIG. 1 is a graph showing the correlation between citrulline concentration and oxidation current value at +0.4 V in an example of the present invention. [Figure 6]FIG. 1 is a graph showing the relationship between the citrullinated peptide concentration and the amount of change in absorbance per minute for an example of the present invention. [Figure 7] FIG. 1 is a partial alignment of citrulline oxidoreductases whose full-length amino acid sequences share 67% or more sequence identity. [Figure 8] FIG. 1 is a partial alignment of citrulline oxidoreductases whose full-length amino acid sequences share 67% or more sequence identity. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following describes a novel quantitative method for measuring citrulline, a biomarker of the present invention, an enzyme for the quantitative determination, a composition for the quantitative determination, and a kit for the quantitative determination. However, the novel quantitative method for measuring citrulline, a biomarker of the present invention, an enzyme for the quantitative determination, a composition for the quantitative determination, and a kit for the quantitative determination are not to be construed as being limited to the description of the following embodiments and examples.

[0028] In one embodiment, the oxidoreductase used in the present invention is a dehydrogenase that acts on the substrate citrulline. An enzyme capable of directly oxidizing or reducing citrulline had not been identified at the time of filing the present application. As a result of investigations, the present inventors discovered for the first time a citrulline oxidoreductase derived from a bacterium of the genus Pseudomonas. Note that, in this specification, citrulline oxidoreductase derived from the genus Pseudomonas and its variants are shown and described as examples of citrulline oxidoreductase, but the present invention is not limited thereto and may include those having a certain level of reactivity to citrulline.

[0029] For example, the citrulline oxidoreductase may be an enzyme that recognizes citrulline as a substrate and has citrulline oxidoreductase activity among oxidoreductases belonging to EC number 1.4 or EC number 1.5. That is, an oxidase that recognizes citrulline as a substrate and has citrulline oxidase activity, or a dehydrogenase that recognizes citrulline as a substrate and has citrulline dehydrogenase activity, may be used.

[0030] In one embodiment, the citrulline oxidoreductase may be an oxidoreductase produced by a naturally occurring microorganism or may be an oxidoreductase produced by a transformed microorganism. From the viewpoint of efficient over-expression of the enzyme, the enzyme can be efficiently over-expressed by using a transformed microorganism.

[0031] In one embodiment, the citrulline oxidoreductase may be a polymer or a monomer. For example, when only a certain subunit (monomer) among several subunits constituting the polymeric oxidoreductase catalyzes a dehydrogenation reaction in which hydrogen is taken from a substrate and transferred to a hydrogen acceptor, the oxidoreductase used in the present invention may be a polymer or the subunit (monomer). Furthermore, the oxidoreductase may be composed of a partial structure of an enzyme as long as it has citrulline oxidoreductase activity.

[0032] As described above, the present inventors have discovered for the first time a citrulline oxidoreductase derived from the genus Pseudomonas. In one embodiment, the citrulline oxidoreductase of the present invention is a citrulline oxidoreductase derived from Pseudomonas sp. strain BYC41-1, but also includes citrulline oxidoreductases derived from microorganisms classified into the genus Pseudomonas. In one embodiment, the citrulline oxidoreductase may be derived from Pseudomonas japonica, Pseudomonas putida, or Pseudomonas mosselii. Further examples include citrulline oxidoreductases having high sequence identity (e.g., 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) to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 1, and citrulline oxidoreductases having an amino acid sequence in which one to several amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 1. Furthermore, citrulline oxidoreductase can also be screened by culturing a microorganism of the genus Pseudomonas under specified conditions (see, for example, Journal of the Japanese Society for Bacteriology, 18(1), 1963), disrupting the cells, mixing the extract with a citrulline-containing oxidase or dehydrogenase reaction reagent (described in detail below), and confirming whether or not there is reactivity with the reagent.

[0033] In one embodiment, the present invention provides a DNA encoding a citrulline oxidoreductase. In one embodiment, the present invention provides a DNA encoding the amino acid sequence set forth in SEQ ID NO: 1 or a DNA having the nucleotide sequence set forth in SEQ ID NO: 2. In one embodiment, the present invention provides a DNA having a nucleotide sequence that has 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2, and encoding a protein having citrulline oxidoreductase activity.

[0034] In one embodiment, when arginine is contained in a citrulline-containing measurement solution, it is preferable that citrulline oxidoreductase has high substrate specificity for citrulline and low substrate specificity for arginine. In other words, it is preferable that the ratio of reactivity for citrulline to reactivity for arginine (Cit / Arg) is high. For example, Cit / Arg is 0.1% or more, preferably 1% or more, more preferably 3% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. It is even more preferable that Cit / Arg is 100% or more. Alternatively, it is preferable that it does not react with arginine.

[0035] In one embodiment, the citrulline oxidoreductase of the present invention may be a citrulline oxidoreductase derived from Pseudomonas sp. strain BYC41-1, or may be a citrulline oxidoreductase produced by Escherichia coli transformed with a plasmid containing the citrulline oxidoreductase gene derived from Pseudomonas sp. strain BYC41-1. However, by using Escherichia coli transformed with a plasmid containing the citrulline oxidoreductase gene derived from Pseudomonas sp. strain BYC41-1, citrulline oxidoreductase can be efficiently expressed in large quantities.

[0036] In one embodiment, the reaction conditions for citrulline oxidoreductase may be any conditions as long as they act on citrulline and efficiently catalyze an oxidation or reduction reaction. Generally, enzymes have optimal temperatures and pHs at which they exhibit the highest activity. Therefore, reaction conditions near the optimal temperatures and pHs are preferred. In one embodiment, the reaction conditions for citrulline oxidoreductase are comprehensively considered, taking into account the conditions suitable for components other than the enzyme, such as color reagents, mediators, enzyme stabilizers, and stabilizers for measurement samples, as well as compatibility with measurement devices, and the measurement method of the present invention also includes a method for quantifying citrulline under conditions other than the optimal conditions for the enzyme alone. Specifically, the reaction time of citrulline oxidoreductase can be set to a certain time, for example, 5 seconds or more, 10 seconds or more, or 20 seconds or more, and less than 180 minutes or less than 150 minutes, for example, 0.5 to 120 minutes, preferably 0.5 to 60 minutes, more preferably 1 to 30 minutes, after mixing citrulline oxidoreductase with a citrulline-containing sample. The active temperature of citrulline oxidoreductase varies depending on the optimal temperature of the enzyme used, but is, for example, 20 to 45°C, and can be appropriately selected from temperatures used in ordinary enzyme reactions.

[0037] In one embodiment, the citrulline oxidoreductase of the present invention is a citrulline oxidoreductase derived from a Pseudomonas japonica strain. Examples of citrulline oxidoreductases include those having a high sequence identity (e.g., 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) to the amino acid sequence of citrulline oxidoreductase set forth in SEQ ID NO: 31, and citrulline oxidoreductases having an amino acid sequence in which one to several amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 31.

[0038] In one embodiment, the present invention provides DNA encoding the amino acid sequence set forth in SEQ ID NO: 31 or DNA having the nucleotide sequence set forth in SEQ ID NO: 32. In one embodiment, the present invention provides DNA having a nucleotide sequence that has 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 32, and encoding a protein having citrulline oxidoreductase activity.

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

[0040] In one embodiment, the citrulline oxidoreductase of the present invention is citrulline oxidoreductase derived from Pseudomonas sp. strain WCHPs060044. Examples of citrulline oxidoreductases include those having a high sequence identity (e.g., 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) to the amino acid sequence of citrulline oxidoreductase set forth in SEQ ID NO: 60, and citrulline oxidoreductases having an amino acid sequence in which one to several amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 60.

[0041] In one embodiment, the present invention provides DNA encoding the amino acid sequence set forth in SEQ ID NO: 60 or DNA having the nucleotide sequence set forth in SEQ ID NO: 61. In one embodiment, the present invention provides DNA having a nucleotide sequence that has 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 61, and encoding a protein having citrulline oxidoreductase activity.

[0042] In one embodiment, the citrulline oxidoreductase of the present invention may be a citrulline oxidoreductase derived from Pseudomonas sp. strain WCHPs060044, or may be a citrulline oxidoreductase produced by Escherichia coli transformed with a plasmid containing the citrulline oxidoreductase gene derived from Pseudomonas sp. strain WCHPs060044. However, by using Escherichia coli transformed with a plasmid containing the citrulline oxidoreductase gene derived from Pseudomonas sp. strain WCHPs060044, citrulline oxidoreductase can be efficiently expressed in large amounts.

[0043] In one embodiment, the citrulline oxidoreductase of the present invention is AncARODn2, which is one of the amino acid sequences deduced based on the Pseudomonas sp. TPU 7192 strain described in S. Nakano et al., Appl. Environ. Microbiol. 2019 85(12) e00459-19. Examples of citrulline oxidoreductases include those having high sequence identity (e.g., 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) to the amino acid sequence of citrulline oxidoreductase set forth in SEQ ID NO: 81, and citrulline oxidoreductases having an amino acid sequence in which one to several amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 81.

[0044] In one embodiment, the present invention provides DNA encoding the amino acid sequence set forth in SEQ ID NO: 81 or DNA having the nucleotide sequence set forth in SEQ ID NO: 82. In one embodiment, the present invention provides DNA having a nucleotide sequence that has 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 61, and encoding a protein having citrulline oxidoreductase activity.

[0045] In one embodiment, the citrulline oxidoreductase of the present invention may be one derived from computational science, such as the citrulline oxidoreductase set forth in SEQ ID NO: 81, or a citrulline oxidoreductase produced by Escherichia coli transformed with a plasmid containing the citrulline oxidoreductase gene set forth in SEQ ID NO: 82. However, by using Escherichia coli transformed with a plasmid containing the citrulline oxidoreductase gene set forth in SEQ ID NO: 82, citrulline oxidoreductase can be efficiently expressed in large quantities.

[0046] Suitable examples of microorganisms from which the citrulline oxidoreductase of the present invention is derived include microorganisms classified into the phylum Proteobacteria, preferably the class Gammaproteobacteria, more preferably the order Pseudomonadales or the order Oceanospirillales, and even more preferably the family Pseudomonadaceae, family Alteromonadaceae, or family Oceanospirillales. Specific examples include citrulline oxidoreductases derived from the genus Pseudomonas, Oceanobacter, or Pseudoalteromonas.

[0047] [vector] The vectors that can be used in the present invention are not limited to the above-mentioned plasmids, and any other vectors known to those skilled in the art, such as bacteriophages, cosmids, etc. Specifically, pBluescriptII SK+ (STRATAGENE) and pET-22b(+) (Merck) are preferred.

[0048] [Mutation of the citrulline oxidoreductase gene] The citrulline oxidoreductase gene can be mutated by any known method depending on the intended mutation type, including a method of contacting and reacting the citrulline oxidoreductase gene or a recombinant DNA incorporating the gene with a mutagenic agent, ultraviolet irradiation, genetic engineering techniques, or protein engineering techniques.

[0049] Examples of mutagenic agents used in the above mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil.

[0050] The above-mentioned contact and reaction conditions can be adjusted depending on the type of drug used, and are not particularly limited as long as the desired mutation can be actually induced in the citrulline oxidoreductase gene. Generally, the desired mutation can be induced by contact and reaction at a drug concentration of preferably 0.5 to 12 M, at a reaction temperature of 20 to 80°C, for 10 minutes or more, preferably 10 to 180 minutes. Ultraviolet irradiation can also be performed according to the standard method described above (Gendai Kagaku, pp. 24-30, June 1989 issue).

[0051] Protein engineering techniques can be used, generally known as site-specific mutagenesis, such as the Kramer method (Nucleic Acids Res., 12, 9441 (1984); Methods Enzymol., 154, 350 (1987); Gene, 37, 73 (1985)), the Eckstein method (Nucleic Acids Res., 13, 8749 (1985); Nucleic Acids Res., 13, 8765 (1985); Nucleic Acids Res., 14, 9679 (1986)), and the Kunkel method (Proc. Natl. Acid. Sci. USA, 82, 488 (1985); Methods Enzymol., 154, 367 (1987)). Specific methods for converting the base sequence in DNA include, for example, using commercially available kits (Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Deletion Kit; Stratagene, Quick Change Site Directed Mutagenesis Kit; Stratagene, etc.).

[0052] Alternatively, a technique known as the general PCR (Polymerase Chain Reaction) method can be used (Technique, 1, 11 (1989)). In addition to the above gene modification methods, the desired modified citrulline oxidoreductase gene can also be directly synthesized by organic synthesis or enzymatic synthesis.

[0053] The DNA base sequence of the citrulline oxidoreductase gene obtained by the above method can be determined or confirmed using, for example, a multi-capillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter).

[0054] [Transformation and transduction] The citrulline oxidoreductase gene obtained as described above can be incorporated into a vector such as a bacteriophage, cosmid, or a plasmid used for transforming prokaryotic or eukaryotic cells by conventional methods, and a host corresponding to each vector can be transformed or transduced by conventional methods. For example, the obtained recombinant DNA can be used to transform or transduce any host, such as a microorganism belonging to the genus Escherichia, specifically, E. coli K-12, preferably E. coli JM109 or E. coli DH5α (both manufactured by Takara Bio Inc.), or E. coli B, preferably E. coli BL21 (manufactured by Nippon Gene Co., Ltd.), to obtain the respective strains.

[0055] An example of a eukaryotic host cell is yeast. Microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Saccharomyces, Pichia, and Candida. The inserted gene may contain a marker gene that enables the selection of transformed cells. Examples of marker genes include genes that complement the auxotrophy of the host, such as URA3 and TRP1. The inserted gene also preferably contains a promoter or other regulatory sequence (e.g., secretory signal sequence, enhancer sequence, terminator sequence, polyadenylation sequence, etc.) that can express the gene of the present invention in the host cell. Specific examples of promoters include the GAL1 promoter and the ADH1 promoter. Methods for transforming yeast include well-known methods, such as a method using lithium acetate (Methods Mol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003) 481-484), but are not limited thereto. Transformation can be performed using any of a variety of methods, including the spheroplast method and the glass bead method.

[0056] Other examples of eukaryotic host cells include filamentous fungi such as those of the genera Aspergillus and Trichoderma. Methods for producing filamentous fungal transformants are not particularly limited, and include, for example, conventional methods for inserting a gene encoding citrulline oxidoreductase into a host filamentous fungus in a manner that allows the gene to be expressed. Specifically, a DNA construct is prepared in which the gene encoding citrulline oxidoreductase is inserted between an expression-inducible promoter and a terminator, and then the host filamentous fungus is transformed with the DNA construct containing the gene encoding citrulline oxidoreductase to obtain a transformant that overexpresses the gene encoding citrulline oxidoreductase. In this specification, a DNA fragment consisting of an expression-inducible promoter-citrulline oxidoreductase-encoding gene-terminator, and a recombinant vector containing the DNA fragment, prepared for transforming a host filamentous fungus, are collectively referred to as a DNA construct.

[0057] The method for inserting the gene encoding citrulline oxidoreductase into a host filamentous fungus in such a manner that the gene is expressed is not particularly limited, and examples include a method of directly inserting the gene into the chromosome of the host organism by utilizing homologous recombination; and a method of introducing the gene into the host filamentous fungus by linking it to a plasmid vector.

[0058] In methods using homologous recombination, a DNA construct is ligated between sequences homologous to the upstream and downstream regions of a recombination site on a chromosome and inserted into the genome of a host filamentous fungus. A transformant can be obtained by self-cloning by overexpression within the host filamentous fungus under the control of its own high-expression promoter. 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).

[0059] In the method using a vector, the DNA construct can be inserted into a plasmid vector used for transforming filamentous fungi by a conventional method, and the corresponding host filamentous fungus can be transformed by a conventional method.

[0060] Such a suitable vector-host system is not particularly limited as long as it is a system that allows citrulline oxidoreductase to be produced in a host filamentous fungus, and examples thereof include a system of pUC19 and a filamentous fungus, and a system of pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and a filamentous fungus.

[0061] The DNA construct is preferably introduced into the chromosome of the host filamentous fungus for use; however, as an alternative, the DNA construct can be incorporated into an autonomously replicating vector (Ozeki et al., Biosci. Biotechnol. Biochem. 59, 1133 (1995)) for use without being introduced into the chromosome.

[0062] The DNA construct may contain a marker gene to enable the selection of transformed cells. The marker gene is not particularly limited, but examples include genes that complement auxotrophy of the host, such as pyrG, niaD, and adeA; and drug resistance genes for drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably contains a promoter, terminator, or other regulatory sequence (e.g., enhancer, polyadenylation sequence, etc.) that enables overexpression of the gene encoding citrulline oxidoreductase in the host cell. The promoter is not particularly limited, but examples include appropriate inducible promoters and constitutive promoters, such as the tef1 promoter, alp promoter, and amy promoter. The terminator is also not particularly limited, but examples include the alp terminator, amy terminator, and tef1 terminator.

[0063] In the DNA construct, an expression control sequence for the gene encoding citrulline oxidoreductase is not necessarily required if the DNA fragment containing the gene encoding citrulline oxidoreductase to be inserted contains a sequence with an expression control function. Furthermore, when transformation is performed by cotransformation, the DNA construct may not necessarily have a marker gene.

[0064] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, a gene encoding citrulline oxidoreductase, the alp gene terminator, and the pyrG marker gene are ligated to an In-Fusion Cloning Site in the multiple cloning site of pUC19.

[0065] Methods for transforming filamentous fungi can be selected from those known to those skilled in the art. For example, the protoplast PEG method, which involves preparing protoplasts of the host filamentous fungus and then using polyethylene glycol and calcium chloride (see, for example, Mol. Gen. Genet. 218, 99-104, 1989; JP 2007-222055 A), can be used. The medium for regenerating the transformed filamentous fungus is appropriate depending on the host filamentous fungus and transformation marker gene used. For example, when Aspergillus sojae is used as the host filamentous fungus and the pyrG gene as the transformation marker gene, the transformed filamentous fungus can be regenerated in, for example, Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2 M sorbitol.

[0066] [Amino acid sequence identity or similarity] Amino acid sequence identity or similarity can be calculated using programs such as maximum matching and search homology in GENETYX Ver. 11 (Genetyx Corporation) or programs such as maximum matching and multiple alignment in DNASIS Pro (Hitachi Solutions, Ltd.). To calculate amino acid sequence identity, two or more citrulline oxidoreductases can be aligned and the positions of identical amino acids in the two or more citrulline oxidoreductases can be examined. Based on this information, identical regions in the amino acid sequences can be determined.

[0067] It is also possible to examine the positions of similar amino acids in two or more citrulline oxidoreductases. For example, multiple amino acid sequences can be aligned using CLUSTALW. In this case, the Blosum62 algorithm is used, and amino acids that are determined to be similar when multiple amino acid sequences are aligned are sometimes referred to as similar amino acids. In the mutants of the present invention, amino acid substitutions may be due to substitutions between such similar amino acids. Such alignments allow for the examination of regions of identical amino acid sequences and positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, homologous regions (conserved regions) in the amino acid sequences can be determined.

[0068] As used herein, the term "homologous region" refers to a region in which, when two or more citrulline oxidoreductases are aligned, the amino acids at corresponding positions in a reference citrulline oxidoreductase and a comparison citrulline oxidoreductase are identical or similar, and the region is composed of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more consecutive amino acids. For example, in Figures 7 and 8, citrulline oxidoreductases with full-length amino acid sequences having a sequence identity of 67% or more are aligned. Among these, the 10th to 18th positions are composed of identical amino acids relative to the citrulline oxidoreductase shown in SEQ ID NO: 1, and therefore correspond to a homologous region. Similarly, based on the citrulline oxidoreductase shown in SEQ ID NO: 1, the following amino acids are present: positions 20 to 26, 34 to 40, 43 to 50, 53 to 67, 96 to 99, 113 to 119, 125 to 127, 134 to 136, 142 to 144, 147 to 149, 151 to 155, 160 to 162, 183 to 186, 192 to 196, 198 to 214, 216 to 225, 237 to 239 241~245th, 312~314th, 316~323rd, 339~341st, 345~347th, 350~353rd, 355~358th, 361~363rd, 365~375th, 377~3 Positions 83, 393-407, 409-413, 467-472, 481-489, 500-504, 511-518, 520-527, and 530-532 may correspond to homologous regions.

[0069] In one embodiment, the homologous regions of the citrulline oxidoreductase include positions 10 to 18, 20 to 26, 33 to 35, 37 to 40, 43 to 48, 53 to 62, 64 to 67, 96 to 99, 113 to 119, 125 to 127, 134 to 136, 140 to 144, 146 to 149, 151 to 155, 160 to 162, 170 to 186, 192 to 196, 197 to 198, 200 to 201, 201 to 202, 202 to 203, 203 to 204, 204 to 205, 205 to 206, 206 to 207, 207 to 208, 208 to 209, 210 to 219, 219 to 220, 221 to 222, 222 to 223, 223 to 224, 224 to 225, 225 to 226, 226 to 227, 227 to 228, 230 to 231, 232 to 233, 234 to 235, 236 to 237, 238 to 239, 240 to 244, 246 to 249, 251 to 255, 251 to 255, 252 to 253, 254 to 255, 256 to 257, 258 to 259, 260 to 261, 262 This region consists of the amino acid sequence of positions 98 to 214, 216 to 227, 231 to 235, 237 to 239, 241 to 245, 311 to 314, 316 to 323, 330 to 334, 339 to 341, 345 to 347, 352 to 360, 361 to 375, 377 to 383, 393 to 407, 409 to 413, 467 to 472, 481 to 490, 500 to 504, 510 to 518, 520 to 527, and 529 to 534.

[0070] The citrulline oxidoreductase of the present invention has a full-length amino acid sequence identity of 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more when aligned with a citrulline oxidoreductase having the amino acid sequence set forth in SEQ ID NO: 1, and has citrulline oxidoreductase activity. Furthermore, the amino acid sequence in the homology region of the citrulline oxidoreductase of the present invention has 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 sequence identity to the amino acid sequence of the homology region in SEQ ID NO:1.

[0071] Similarly, positions 8 to 16 of the citrulline oxidoreductase shown in SEQ ID NO: 81 consist of the same amino acids and therefore correspond to homologous regions. Similarly, positions 18 to 24, 32 to 38, 41 to 48, 51 to 65, 88 to 91, 115 to 121, 127 to 129, 136 to 138, 144 to 146, 149 to 151, 153 to 157, 162 to 164, 185 to 188, 194 to 198, 200 to 216, 218 to 227, 239 to 241, 242 to 243, 250 to 251, 252 to 253, 254 to 255, 256 to 257, 260 to 261, 262 to 264, 264 to 265, 266 to 267, 268 to 269, 270 to 271, 272 to 273, 274 to 275, 276 to 277, 278 to 279, 280 to 281, 282 to 283, 284 to 285, 286 to 287, 288 to 289, 290 to 291, 300 to 301, 302 to 303, 304 to 305, 306 to 307, 308 to 309, 310 to 311, 312 to 313, 314 to 315, 316 to 317, 3 243-247, 308-310, 312-319, 335-337, 341-343, 346-349, 351-354, 357-359, 361-371, 373-3 Positions 79, 389-403, 405-409, 463-468, 477-485, 496-500, 507-514, 516-523, and 526-528 may correspond to homologous regions.

[0072] The citrulline oxidoreductase of the present invention has a full-length amino acid sequence identity of 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more when aligned with a citrulline oxidoreductase having the amino acid sequence set forth in SEQ ID NO: 81, and has citrulline oxidoreductase activity. Furthermore, the amino acid sequence in the homology region of the citrulline oxidoreductase of the present invention has 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 sequence identity to the amino acid sequence of the homology region in SEQ ID NO: 81.

[0073] Corresponding Location The position corresponding to position 486 in the amino acid sequence shown in SEQ ID NO: 1 refers to the position in the amino acid sequence of a citrulline oxidoreductase derived from another organism species that corresponds to position 486 in the amino acid sequence of SEQ ID NO: 1 when aligned with the amino acid sequence of SEQ ID NO: 1.

[0074] For example, a method for identifying "corresponding positions" involves first comparing amino acid sequences using a known algorithm such as the Lippmann-Parson method, followed by multiple alignment to maximize identity for conserved amino acid residues present in the amino acid sequences of each citrulline oxidoreductase. By aligning the amino acid sequences of citrulline oxidoreductases in this manner, the positions of homologous amino acid residues in each citrulline oxidoreductase sequence can be determined, regardless of insertions or deletions in the amino acid sequences. Then, in some cases, secondary structures such as α-helices, β-sheets, and coils can be predicted using known secondary structure prediction algorithms.

[0075] For example, the secondary structure of the amino acid sequence shown in SEQ ID NO: 1 or an appropriate citrulline 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 Jpred4 (Drozdetskiy A et al. (2015) JPred4: a protein secondary structure prediction server, Nucleic Acids Res., doi:10.1093 / nar / gkv332), which implement the JNet algorithm.

[0076] [Enzyme preparation method] The method for preparing citrulline oxidoreductase according to the present invention will now be described.

[0077] [Construction of expression plasmid] Plasmids for expressing the citrulline oxidoreductase of the present invention can be obtained by commonly used methods. For example, DNA is extracted from a microorganism that produces the citrulline oxidoreductase of the present invention, and a DNA library is prepared. From the prepared DNA library, DNA fragments encoding the citrulline oxidoreductase of the present invention are identified and isolated. The isolated DNA fragments are amplified by polymerase chain reaction (PCR) using complementary primers that use the isolated DNA fragments as templates, and the gene encoding the citrulline oxidoreductase of the present invention is cloned. The amplified DNA fragments are ligated into a vector to obtain plasmids containing DNA fragments encoding the citrulline oxidoreductase of the present invention.

[0078] Alternatively, a DNA fragment encoding the citrulline oxidoreductase of the present invention is chemically synthesized and ligated into a vector to obtain a plasmid carrying DNA encoding the citrulline oxidoreductase of the present invention.

[0079] A strain of bacteria such as E. coli is transformed with the obtained plasmid to obtain a strain of bacteria such as E. coli having a DNA encoding the citrulline oxidoreductase of the present invention.

[0080] [Recombinant expression of enzymes] A strain such as Escherichia coli carrying DNA encoding the citrulline oxidoreductase of the present invention is cultured in a medium. The microbial host cells can be cultured at a temperature of 10 to 42°C, preferably around 25°C, for several hours to several days, more preferably around 25°C, for 1 to 7 days, using methods such as submerged culture with aeration and agitation, shaking culture, or static culture. Conventional culture media for culturing filamentous fungi, i.e., synthetic or natural media containing appropriate proportions of carbon sources, nitrogen sources, inorganic substances, and other nutrients, can be used. The medium for culturing the microbial host cells may contain, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran infusion, supplemented with one or more inorganic salts such as sodium chloride, potassium diphosphate, potassium diphosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate, and may further contain, as needed, carbohydrate raw materials, vitamins, and the like.

[0081] Culture conditions may be those commonly known to those skilled in the art for culturing filamentous fungi. For example, the initial pH of the medium may be adjusted to 5 to 10, the culture temperature may be 20 to 40°C, and the culture time may be set appropriately to several hours to several days, preferably 1 to 7 days, and more preferably 2 to 5 days. The culture method is not particularly limited, and submerged culture with aeration and stirring, shaking culture, static culture, etc. may be used, but culture is preferably performed under conditions that ensure sufficient dissolved oxygen. For example, an example of a medium and culture conditions for culturing an Aspergillus microorganism is shaking culture at 30°C and 160 rpm for 3 to 5 days using DPY medium, as described in the Examples below.

[0082] After the culture is completed, the citrulline oxidoreductase of the present invention is collected from the culture. This can be done using a conventional enzyme collection method. For example, the supernatant fraction can be collected, or the cells can be subjected to ultrasonic disruption, trituration, or other conventional methods, or the enzyme can be extracted using a lytic enzyme such as lysozyme or yatalase, or the cells can be lysed by shaking or standing in the presence of toluene or the like, thereby excreting the enzyme outside the cells. The solution can then be filtered, centrifuged, or the like to remove solids, and, if necessary, nucleic acids can be removed using streptomycin sulfate, protamine sulfate, manganese sulfate, or the like. After this, the solution is fractionated by adding ammonium sulfate, alcohol, acetone, or the like, and the precipitate is collected to obtain a crude citrulline oxidoreductase of the present invention.

[0083] [Enzyme purification] The method for purifying the enzyme may be any method that can purify the enzyme from a crude enzyme solution. For example, a purified citrulline oxidoreductase enzyme preparation of the present invention can be obtained by appropriately selecting from gel filtration using Sephadex, Ultrogel, Biogel, etc., adsorption / elution using an ion exchanger, electrophoresis using polyacrylamide gel, etc., adsorption / elution using hydroxyapatite, sedimentation methods such as sucrose density gradient centrifugation, affinity chromatography, fractionation methods using molecular sieve membranes or hollow fiber membranes, etc., or by combining these methods.

[0084] [Enzyme activity measurement] The method for measuring enzyme activity may be any method that directly or indirectly measures the product of an enzyme-catalyzed oxidation-reduction reaction. For example, enzyme activity can be measured by measuring the current value generated when a reduced product is produced by catalyzing an enzyme-catalyzed oxidation-reduction reaction and the reduced product transfers electrons to an electrode. Preferably, enzyme activity can be measured by reacting the reduced product of the enzyme-catalyzed oxidation-reduction reaction with a reagent containing a light-absorbing substance that reacts with the reduced product (hereinafter referred to as the "light-absorbing reagent") and measuring the absorbance.

[0085] [Composition containing citrulline oxidoreductase and kit for quantifying citrulline] The method for quantifying citrulline using citrulline oxidoreductase according to the present invention may be carried out by providing a composition containing citrulline oxidoreductase and a product-reaction reagent, or by combining citrulline oxidoreductase with a commercially available product-reaction reagent.

[0086] The method for quantifying citrulline, citrulline oxidoreductase for quantification, composition for quantification, and kit for quantification according to the present invention contain citrulline oxidoreductase, and thereby can provide a new method, enzyme for quantification, composition for quantification, and kit for quantifying the concentration of citrullinated peptides, which are biomarkers for diseases associated with abnormal citrullination of proteins, such as multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, psoriasis, prion disease, liver fibrosis, chronic obstructive pulmonary disease, and cancer, or the concentration of citrulline released from citrullinated peptides.

[0087] [Composition containing citrulline oxidase and kit for quantifying citrulline] In one embodiment, citrulline may be quantified using the citrulline oxidase of the present invention. The method for quantifying citrulline using the citrulline oxidase of the present invention may be carried out by providing a composition containing citrulline oxidase and a product reaction reagent, or by combining citrulline oxidase with a commercially available product reaction reagent. For example, the method may be provided as a composition for quantifying citrulline containing citrulline oxidase, or as a composition for quantifying citrulline further containing a reagent that reacts with hydrogen peroxide generated by adding citrulline oxidase. Alternatively, the method may be provided as a kit for quantifying citrulline containing citrulline oxidase and a reagent that reacts with hydrogen peroxide generated by adding citrulline oxidase.

[0088] [Citrulline measurement sensor] In one embodiment, a citrulline measurement sensor using citrulline oxidase of the present invention is provided. FIG. 1(a) is a schematic diagram of a sensor 100 according to one embodiment of the present invention. The sensor 100 is a citrulline measurement device using citrulline oxidase and includes a sensor chip 10 containing citrulline oxidase and a measurement unit 30. The measurement unit 30 may include, for example, a switch 31 serving as an input unit and a display 33 serving as a display unit. The switch 31 may be used, for example, to control the ON / OFF of the power supply to the measurement unit 30, or to control the start and stop of citrulline generation in the sensor 100. The display 33 may, for example, display the measured citrulline value, and may include a touch panel as an input unit for controlling the measurement unit 30.

[0089] FIG. 1(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 memory unit 140, a communication unit 150, and a power supply 160 in a measurement unit 30, which may be electrically connected to each other by wiring 190. Terminals of the sensor chip 10 (described below) and 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 citrulline.

[0090] The display unit 120 may include, for example, a known display 33, and may display the measured value of citrulline, the status of the measurement unit 30, and operation requests to the person taking the measurement. The input unit 130 is an input device that allows the person taking the measurement to operate the sensor 100, and may be, for example, a switch 31 or a touch panel arranged on the display 33. The measurement unit 30 may be provided with multiple switches 31.

[0091] The storage unit 140 is configured with a main storage device (memory), and an auxiliary storage device (hard disk) may be arranged externally. The main storage device (memory) may be configured with a read-only memory (ROM) and / or a random access memory (RAM). Operation programs, operating systems, application programs, or modules are stored in the storage unit 140 and executed by the central processing unit to configure the control unit 110. Measured values ​​and current values ​​can also be stored in the storage unit 140.

[0092] The communication unit 150 is a known communication device that connects the sensor 100 or the measurement unit 30 to an external device (a computer, printer, or network). The communication unit 150 and the external device are connected via wired or wireless communication. The power supply 160 is a known power supply device that supplies power to the sensor 100 or the measurement unit 30.

[0093] [Sensor chip] FIG. 2(a) is a schematic diagram of a sensor chip 10 according to one embodiment of the present invention, and FIGS. 2(b) to 2(d) are schematic diagrams showing components constituting the sensor chip 10. The sensor chip 10 includes two or more electrodes disposed on a substrate 11. The substrate 11 is made of an insulating material. In FIGS. 2(a) and 2(b), as an example, a working electrode 1, a counter electrode 3, and a reference electrode 5 are disposed on the substrate 11. Each electrode is electrically connected to a wiring portion 7, and the wiring portion 7 is electrically connected to a terminal 9 located on the opposite side of the electrode in the wiring direction. The working electrode 1, the counter electrode 3, and the reference electrode 5 are disposed spaced apart from one another. Preferably, the working electrode 1, the counter electrode 3, and the reference electrode 5 are integrally formed with the wiring portion 7 and the terminal 9. Alternatively, the counter electrode 3 and the reference electrode 5 may be integrally formed.

[0094] As shown in Figures 2(a) and 2(c), a spacer 13 is placed on the end of the substrate 11 parallel to the wiring portion 7, and a cover 15 is placed to cover the working electrode 1, counter electrode 3, reference electrode 5, and spacer 13. The spacer 13 and cover 15 are made of an insulating material. The spacer 13 preferably has approximately the same thickness as 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 integrally formed. The cover 15 is a protective layer that prevents deterioration of the wiring portion 7 due to exposure to the outside air and prevents short circuits due to seepage of the measurement sample.

[0095] As shown in Figures 2(a) and 2(d), a reaction layer 19 is disposed on the working electrode 1, counter electrode 3, and reference electrode 5. The reaction layer 19 provides a site for the reaction between citrulline and citrulline oxidase. In one embodiment, the citrulline oxidase of the present invention may be coated, adsorbed, or immobilized on these electrodes. Preferably, the citrulline oxidase of the present invention is coated, adsorbed, or immobilized on the working electrode. In another embodiment, a mediator may be coated, adsorbed, or immobilized on the electrode together with citrulline oxidase. Examples of electrodes that can be used include carbon electrodes and metal electrodes such as platinum, gold, silver, nickel, and palladium. In the case of carbon electrodes, examples of materials include pyrolytic graphitic carbon (PG), glassy carbon (GC), carbon paste, and plastic-formed carbon (PFC). The measurement system may be a two-electrode system or a three-electrode system, and the enzyme may be immobilized on the working electrode, for example. Examples of reference electrodes include a standard hydrogen electrode, a reversible hydrogen electrode, a silver-silver chloride electrode (Ag / AgCl), a palladium-hydrogen electrode, and a saturated calomel electrode, and from the viewpoints of stability and reproducibility, it is preferable to use Ag / AgCl.

[0096] Enzymes can be immobilized on electrodes 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. Enzymes may also be immobilized in a polymer together with a mediator or adsorbed onto an electrode, or these methods may be combined.

[0097] The citrulline oxidase of the present invention can be applied to various electrochemical measurement methods using a potentiostat, galvanostat, or the like. Electrochemical measurement methods include various techniques such as amperometry, potentiometry, and coulometry. For example, by amperometry, hydrogen peroxide generated when citrulline oxidase reacts with citrulline is measured using a hydrogen peroxide electrode. A voltage of +600 to +1000 mV (vs. Ag / AgCl) is applied from a power source 160 to the hydrogen peroxide electrode, and the resulting current is measured by the control unit 110, thereby calculating the citrulline concentration in a sample. For example, a calibration curve can be created by measuring current values ​​for known citrulline concentrations (5, 10, 20, 30, 40, 50 mM) and plotting them against the citrulline concentration. The citrulline concentration can be obtained from the calibration curve by measuring the current value of unknown citrulline. For example, a platinum electrode can be used as the hydrogen peroxide electrode. Alternatively, instead of a hydrogen peroxide electrode, an electrode with immobilized reductase such as peroxidase or catalase can be used, and the amount of hydrogen peroxide can be quantified by measuring the reduction current value generated by applying -400 to +100 mV (vs. Ag / AgCl), and the citrulline value can be measured.

[0098] Furthermore, printed electrodes can be used to reduce the amount of solution required for measurement. In this case, the electrodes are preferably formed on a base material 11 made of an insulating substrate. Specifically, it is desirable to form the electrodes on the base material 11 by photolithography or a printing technique such as screen printing, gravure printing, or flexographic printing. In addition, examples of materials for the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, but it is more preferable to use a material that is highly resistant to various solvents and chemicals.

[0099] As described above, the method for quantifying citrulline, the citrulline oxidase for quantification, the composition for quantification, and the kit for quantification according to the present invention contain citrulline oxidase, and thereby can provide a new method for quantifying the concentration of citrulline, which is a biomarker for diseases associated with abnormal citrullination of proteins, such as multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, psoriasis, prion disease, liver fibrosis, chronic obstructive pulmonary disease, and cancer.

[0100] [Method for quantifying citrulline using the citrulline oxidase reaction] The citrulline oxidase used in the present invention is an oxidase that acts on citrulline as a substrate. However, as of the time of filing of the present application, citrulline oxidase has not yet been identified.

[0101] In one embodiment, the citrulline oxidase is selected from the above-mentioned citrulline oxidoreductases or may be a variant of the above-mentioned citrulline oxidoreductases having high citrulline oxidase activity. In one embodiment, the citrulline oxidase is a citrulline oxidase derived from the genus Pseudomonas. Further examples include citrulline oxidases that have high sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 (e.g., 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). Also included are citrulline oxidases that have an amino acid sequence in which one or several amino acids in the amino acid sequence of SEQ ID NO: 1 have been modified or mutated, or deleted, substituted, added, and / or inserted.

[0102] In one embodiment, the citrulline oxidase may be citrulline oxidase derived from Pseudomonas sp. strain BYC41-1, or may be citrulline oxidase produced by Escherichia coli transformed with a plasmid containing the citrulline oxidase gene derived from Pseudomonas sp. strain BYC41-1. However, by using Escherichia coli transformed with a plasmid containing the citrulline oxidase gene derived from Pseudomonas sp. strain BYC41-1, citrulline oxidase can be efficiently expressed in large quantities.

[0103] Furthermore, in one embodiment, the citrulline oxidase may be citrulline oxidase derived from Pseudomonas japonica strain, or may be citrulline oxidase produced by Escherichia coli transformed with a plasmid containing the citrulline oxidase gene derived from Pseudomonas japonica strain. However, by using Escherichia coli transformed with a plasmid containing the citrulline oxidase gene derived from Pseudomonas japonica strain, citrulline oxidase can be efficiently expressed in large quantities.

[0104] Furthermore, in one embodiment, the citrulline oxidase may be citrulline oxidase derived from Pseudomonas sp. WCHPs060044 strain, or may be citrulline oxidase produced by Escherichia coli transformed with a plasmid containing the citrulline oxidase gene derived from Pseudomonas sp. WCHPs060044 strain. However, by using Escherichia coli transformed with a plasmid containing the citrulline oxidase gene derived from Pseudomonas sp. WCHPs060044 strain, citrulline oxidase can be efficiently expressed in large quantities.

[0105] Furthermore, in one embodiment, the citrulline oxidase may be the citrulline oxidase set forth in SEQ ID NO: 81, or may be a citrulline oxidase produced by Escherichia coli transformed with a plasmid containing the citrulline oxidase gene set forth in SEQ ID NO: 82. However, by using Escherichia coli transformed with a plasmid containing the citrulline oxidase gene set forth in SEQ ID NO: 82, citrulline oxidase can be efficiently expressed in large quantities.

[0106] Suitable examples of microorganisms from which the citrulline oxidase of the present invention is derived include microorganisms classified into the phylum Proteobacteria, preferably the class Gammaproteobacteria, more preferably the order Pseudomonadales or the order Oceanospirillales, and even more preferably the family Pseudomonadaceae, family Alteromonadaceae, or family Oceanospirillales. Specific examples include citrulline oxidases derived from the genus Pseudomonas, Oceanobacter, or Pseudoalteromonas.

[0107] In one embodiment, the reaction conditions for citrulline oxidase may be any conditions that act on citrulline and efficiently catalyze the oxidation reaction. Generally, enzymes have optimal temperatures and pHs at which they exhibit the highest activity. Therefore, reaction conditions near the optimal temperatures and pHs may be preferable.

[0108] In one embodiment, various chemical substances may be involved in the reaction process of citrulline oxidase when the citrulline oxidase of the present invention acts on citrulline. For example, when citrulline oxidase acts on citrulline, oxygen may be involved as an electron acceptor in the oxidation-reduction reaction.

[0109] Known citrullinated proteins include myelin basic protein, filaggrin, histone proteins, fibrin, vimentin, and fibrinogen. In one embodiment, when quantifying citrulline using blood as a sample, the sample can be selected from whole blood, plasma, or serum depending on the citrullinated protein to be measured. Alternatively, citrulline oxidase or a composition for quantifying citrulline containing citrulline oxidase may be directly mixed with the sample, or the sample may be pretreated before being mixed with citrulline oxidase or a composition for quantifying citrulline containing citrulline oxidase. For example, citrullinated proteins may be degraded with protease and / or peptidase to release citrulline, and then mixed with citrulline oxidase or a composition for quantifying citrulline containing citrulline oxidase.

[0110] The quantitative range of citrulline is not particularly limited, but may be, for example, 0.001 mM to 1000 mM, 0.01 mM to 500 mM, 0.01 mM to 300 mM, 0.01 mM to 100 mM, 0.01 mM to 50 mM, 0.01 mM to 30 mM, or 0.01 mM to 20 mM.

[0111] [Enzyme preparation method] The citrulline oxidase according to the present invention can be prepared by the same preparation method as the above-mentioned preparation method for citrulline oxidoreductase.

[0112] [Construction of expression plasmid] For example, similar to the above-mentioned method for preparing citrulline oxidoreductase, an expression plasmid can be constructed, or a DNA fragment encoding the citrulline oxidase of the present invention can be chemically synthesized and ligated into a vector to obtain a plasmid carrying the DNA fragment encoding the citrulline oxidase of the present invention. A strain of E. coli or the like can be transformed with the obtained plasmid to obtain a strain of E. coli or the like carrying DNA encoding the citrulline oxidase of the present invention.

[0113] [Recombinant expression and purification of the enzyme] Citrulline oxidase may be expressed and purified by the same method as the above-mentioned expression and purification of oxidoreductase, and detailed explanation will be omitted.

[0114] [Enzyme activity measurement] The method for measuring enzyme activity may be any method that directly or indirectly measures the product of an enzyme-catalyzed reaction. For example, if the product of an enzyme-catalyzed reaction is reacted with a reagent that reacts with the product (hereinafter referred to as a "product reaction reagent") and the light-absorbing substance produced by the reaction is measured, the enzyme activity can be measured by measuring absorbance.

[0115] [Composition containing citrulline dehydrogenase and kit for quantifying citrulline] In one embodiment, citrulline may be quantified using the citrulline dehydrogenase of the present invention. The method for quantifying citrulline using the citrulline dehydrogenase of the present invention may be carried out by providing a composition containing citrulline dehydrogenase and a product reaction reagent, or by combining citrulline dehydrogenase with a commercially available product reaction reagent. For example, it may be provided as a composition for quantifying citrulline containing citrulline dehydrogenase, or as a composition for quantifying citrulline further containing a mediator that is reduced by adding citrulline dehydrogenase and a reagent that reacts with the reduced mediator. It may also be provided as a kit for quantifying citrulline containing citrulline dehydrogenase, a mediator that is reduced by adding citrulline dehydrogenase, and a reagent that reacts with the reduced mediator.

[0116] The mediator (also referred to as an artificial electron mediator, artificial electron acceptor, or electron mediator) used in the measurement method or quantification kit of the present invention is not particularly limited as long as it can accept electrons from citrulline dehydrogenase. Examples of mediators include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, phenylenediamines, ferricyanides such as potassium ferricyanide, ferredoxins, ferrocene, ruthenium complexes, osmium complexes, and derivatives thereof. Examples of phenazine compounds include, but are not limited to, PMS and methoxy PMS.

[0117] [Citrulline measurement sensor] In one embodiment, a citrulline measurement sensor using citrulline dehydrogenase of the present invention is provided. The citrulline measurement sensor using citrulline dehydrogenase may have the same basic configuration as the sensor 100 described above, except that citrulline dehydrogenase is used as the enzyme, and detailed description thereof will be omitted.

[0118] [Sensor chip] A sensor chip using citrulline dehydrogenase may have the same basic configuration as the sensor chip 10 described above, except that citrulline dehydrogenase is used as the enzyme. Electrodes such as carbon electrodes, gold electrodes, and platinum electrodes can be used, and the citrulline dehydrogenase of the present invention can be coated or immobilized on the electrodes. Immobilization methods include using a cross-linking reagent, encapsulating it in a polymer matrix, or covering it with a dialysis membrane. The enzyme can also be immobilized in a polymer such as a photocrosslinkable polymer, a conductive polymer, or a redox polymer, or adsorbed and immobilized on the electrode, or a combination of these methods can be used. Typically, the citrulline dehydrogenase of the present invention can be immobilized on a carbon electrode using glutaraldehyde, and then blocked by treating it with a reagent having an amine group.

[0119] The citrulline dehydrogenase of the present invention can be applied to various electrochemical measurement methods using a potentiostat, galvanostat, or the like. Electrochemical measurement methods include various techniques such as amperometry, voltammetry, potentiometry, and coulometry. For example, by using amperometry, the control unit 110 measures the current generated when citrulline is reduced, thereby calculating the citrulline concentration in the sample. The applied voltage varies depending on the conditions and device settings, but can be, for example, −1000 mV to +1000 mV (vs. Ag / AgCl).

[0120] Citrulline concentration can be measured as follows: A buffer solution is placed in a thermostatic cell and maintained at a constant temperature. An electrode on which the citrulline dehydrogenase of the present invention is immobilized is used as the working electrode, and a counter electrode (e.g., a platinum electrode) and a reference electrode (e.g., an Ag / AgCl electrode) are used. The control unit 110 applies a constant voltage from the power supply 160 to the carbon electrode. After the current becomes steady, a sample containing citrulline is added and the increase in current is measured. The citrulline concentration in the sample can be calculated according to a calibration curve prepared using citrulline solutions of standard concentrations.

[0121] As a specific example, 0.2 U to 150 U, preferably 0.5 U to 100 U, of the citrulline dehydrogenase of the present invention is immobilized on a glassy carbon (GC) electrode, and the response current versus citrulline concentration is measured. 10.0 ml of 100 mM potassium phosphate buffer (pH 6.0) containing 300 mM potassium ferricyanide is added to an electrolysis cell. The GC electrode is connected to a potentiostat BAS100B / W (manufactured by BAS), the solution is stirred at 37°C, and +500 mV is applied relative to a silver-silver chloride reference electrode. 1 M citrulline solution is added to the system to final concentrations of 5, 10, 20, 30, 40, and 50 mM, and the steady-state current is measured after each addition. This current is plotted against known citrulline concentrations (5, 10, 20, 30, 40, and 50 mM) to create a calibration curve. By measuring the current value of unknown citrulline, the citrulline concentration can be obtained from the calibration curve. In this way, the enzyme-immobilized electrode using citrulline dehydrogenase of the present invention makes it possible to quantify citrulline.

[0122] Furthermore, printed electrodes can be used for electrochemical measurements. This reduces the amount of solution required for measurement. In this case, the electrodes are preferably formed on a substrate 11 made of an insulating substrate. Specifically, it is desirable to form the electrodes on the substrate 11 by photolithography or printing techniques such as screen printing, gravure printing, and flexographic printing. In addition, examples of materials for the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, but it is more preferable to use a material that is highly resistant to various solvents and chemicals.

[0123] As described above, the method for quantifying citrulline, citrulline dehydrogenase for quantification, composition for quantification, and kit for quantification according to the present invention contain citrulline dehydrogenase, and thereby can provide a new method for quantifying the concentration of citrulline, which is a biomarker for diseases associated with abnormal citrullination of proteins, such as multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, psoriasis, prion disease, liver fibrosis, chronic obstructive pulmonary disease, and cancer.

[0124] [Quantitative method for citrulline using citrulline dehydrogenase reaction] The citrulline dehydrogenase used in the present invention is a dehydrogenase that acts on citrulline as a substrate. However, as of the time of filing this application, citrulline dehydrogenase has not been identified.

[0125] In one embodiment, the citrulline dehydrogenase is selected from the above-mentioned citrulline oxidoreductases or may be a mutant of the above-mentioned citrulline oxidoreductases having high citrulline dehydrogenase activity. In one embodiment, the citrulline dehydrogenase is a citrulline dehydrogenase derived from the genus Pseudomonas. Further examples include citrulline dehydrogenases that have high sequence identity (e.g., 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) to the amino acid sequence set forth in SEQ ID NO: 1, and citrulline dehydrogenases that have an amino acid sequence in which one or several amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 1.

[0126] In one embodiment, the citrulline dehydrogenase may be citrulline dehydrogenase derived from Pseudomonas sp. strain BYC41-1, or may be citrulline dehydrogenase produced by Escherichia coli transformed with a plasmid containing the citrulline dehydrogenase gene derived from Pseudomonas sp. strain BYC41-1. However, by using Escherichia coli transformed with a plasmid containing the citrulline dehydrogenase gene derived from Pseudomonas sp. strain BYC41-1, citrulline dehydrogenase can be efficiently expressed in large amounts.

[0127] Furthermore, in one embodiment, the citrulline dehydrogenase may be citrulline dehydrogenase derived from a Pseudomonas japonica strain, or may be citrulline dehydrogenase produced by Escherichia coli transformed with a plasmid containing the citrulline dehydrogenase gene derived from a Pseudomonas japonica strain. However, by using Escherichia coli transformed with a plasmid containing the citrulline dehydrogenase gene derived from a Pseudomonas japonica strain, citrulline dehydrogenase can be efficiently expressed in large quantities.

[0128] Furthermore, in one embodiment, the citrulline dehydrogenase may be citrulline dehydrogenase derived from Pseudomonas sp. WCHPs060044 strain, or may be citrulline dehydrogenase produced by Escherichia coli transformed with a plasmid containing the citrulline dehydrogenase gene derived from Pseudomonas sp. WCHPs060044 strain. However, by using Escherichia coli transformed with a plasmid containing the citrulline dehydrogenase gene derived from Pseudomonas sp. WCHPs060044 strain, citrulline dehydrogenase can be efficiently expressed in large quantities.

[0129] Furthermore, in one embodiment, the citrulline dehydrogenase may be one derived from computational science, such as the citrulline dehydrogenase set forth in SEQ ID NO: 81, or may be a citrulline dehydrogenase produced by Escherichia coli transformed with a plasmid containing the citrulline dehydrogenase gene set forth in SEQ ID NO: 82. However, by using Escherichia coli transformed with a plasmid containing the citrulline dehydrogenase gene set forth in SEQ ID NO: 82, citrulline dehydrogenase can be efficiently expressed in large quantities.

[0130] Suitable examples of microorganisms from which the citrulline dehydrogenase of the present invention is derived include microorganisms classified into the phylum Proteobacteria, preferably the class Gammaproteobacteria, more preferably the order Pseudomonadales or the order Oceanospirillales, and even more preferably the family Pseudomonadaceae, the family Alteromonadaceae, or the family Oceanospirillales. Specific examples include citrulline dehydrogenases derived from the genus Pseudomonas, Oceanobacter, or Pseudoalteromonas.

[0131] In one embodiment, the reaction conditions for citrulline dehydrogenase may be any conditions that act on citrulline and efficiently catalyze the oxidation reaction. Generally, enzymes have optimal temperature and pH values ​​at which they exhibit the highest activity. Therefore, reaction conditions near the optimal temperature and pH values ​​may be suitable.

[0132] In one embodiment, when blood is used as specimen for citrulline quantification, sample can be selected from whole blood, plasma or serum according to the citrullinated protein to be measured.In addition, citrulline dehydrogenase or the composition for citrulline quantification that contains citrulline dehydrogenase can be directly mixed with sample, or before being mixed with citrulline dehydrogenase or the composition for citrulline quantification that contains citrulline dehydrogenase, sample can be pretreated.For example, citrullinated protein can be decomposed with protease to release citrulline, and then mixed with citrulline dehydrogenase or the composition for citrulline quantification that contains citrulline dehydrogenase.

[0133] The quantitative range of citrulline is not particularly limited, but may be, for example, 0.001 mM to 1000 mM, 0.01 mM to 500 mM, 0.01 mM to 300 mM, 0.01 mM to 100 mM, 0.01 mM to 50 mM, 0.01 mM to 30 mM, or 0.01 mM to 20 mM.

[0134] [Enzyme preparation method] The citrulline dehydrogenase of the present invention can be prepared by the same preparation method as the above-mentioned preparation method for citrulline oxidoreductase.

[0135] [Construction of expression plasmid] The expression plasmid for the citrulline dehydrogenase of the present invention can be obtained, for example, by constructing an expression plasmid in the same manner as in the above-mentioned method for preparing citrulline oxidoreductase, or by chemically synthesizing a DNA fragment encoding the citrulline oxidase of the present invention and ligating the DNA fragment to a vector, and then transforming a strain of E. coli or the like with the obtained plasmid to obtain a strain of E. coli or the like having a DNA encoding the citrulline dehydrogenase of the present invention.

[0136] [Recombinant expression and purification of the enzyme] Citrulline dehydrogenase may be expressed and purified by the same method as the above-mentioned expression and purification of oxidoreductase, and detailed explanation will be omitted.

[0137] [Enzyme activity measurement] The method for measuring enzyme activity may be any method that directly or indirectly measures the product of an enzyme-catalyzed reaction. For example, if the product of an enzyme-catalyzed reaction is reacted with a reagent that reacts with the product (hereinafter referred to as a "product reaction reagent") and the light-absorbing substance produced by the reaction is measured, the enzyme activity can be measured by measuring absorbance.

[0138] [Measurement of PAD activity] It is possible to consider applying PAD activity measurement to the early diagnosis of specific cases such as Alzheimer's disease and rheumatoid arthritis. It is also possible to consider applying PAD activity measurement to evaluate the effectiveness of treatment for these specific cases. Furthermore, it is possible to consider applying PAD activity measurement to the search for inhibitors of PADs, such as PAD2 and PAD4, and for factors involved in the regulation of PAD expression and their inhibitors.

[0139] Currently, it is possible to quantify the amount of PAD in blood using antibodies, but this method is insufficient for evaluating PAD contribution to citrullination because it quantifies inactive PAD. PAD activity can be evaluated by reacting PAD in blood with peptides that can serve as PAD substrates (see, for example, C. Assohou-Luty et al., "The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities," Biochi. Biophys. Acta 1844 (2014) 829-836) for a certain period of time and quantifying the resulting citrullinated peptides. In one embodiment, PAD activity can be evaluated by quantifying citrullinated peptides using the above-described method for quantifying citrulline using citrulline oxidoreductase, citrulline oxidase, or citrulline dehydrogenase according to the present invention. The sample for measuring PAD activity can be selected from whole blood, plasma, or serum.

[0140] Specifically, an arginine peptide appropriate for the type of PAD whose activity is to be evaluated and a PAD-containing sample can be incubated for a certain period of time, e.g., 5 seconds or more, 10 seconds or more, or 20 seconds or more, and less than 180 minutes or less than 150 minutes, e.g., 0.5 to 120 minutes, preferably 0.5 to 60 minutes, and more preferably 1 to 30 minutes. The PAD activity temperature, which depends on the optimal temperature of the enzyme used, is, for example, 20 to 45°C, and can be appropriately selected from temperatures used in conventional enzymatic reactions. If necessary, the reaction can be terminated by any means.

[0141] The citrullinated peptide in the reaction solution is quantified by the method described below to measure the amount of citrullination per minute, and the number of micromoles of arginine converted to citrulline per minute is defined as an activity unit (U) in the enzyme solution, which can be calculated. Thus, in one embodiment of the present invention, PAD activity can be calculated, whereas conventional antibody methods can measure the amount of PAD but cannot calculate PAD activity.

[0142] To quantify citrullinated peptides, the citrullinated peptides are treated with a protease or peptidase. The reaction time between the citrullinated peptide and the protease or peptidase is preferably within 1 day, more preferably within 14 hours, 5 hours, or 1 hour, and even more preferably within 30 minutes, 10 minutes, or 5 minutes. The protease or peptidase is not particularly limited as long as it liberates citrulline from the citrullinated peptide. Either a single type of protease or peptidase or a combination of multiple types of proteases and / or peptidases may be used. The temperature at which the protease or peptidase acts depends on the optimal temperature of the enzyme used, but is, for example, 20 to 95°C, and can be appropriately selected from temperatures used in conventional enzymatic reactions. If necessary, the reaction can be terminated using any means.

[0143] The liberated citrulline is reacted with citrulline oxidoreductase, citrulline oxidase, or citrulline dehydrogenase according to the present invention, and citrulline is quantified according to the above-mentioned method for quantifying citrulline. Since the content of arginine residues contained in the peptides that can be used as PAD substrates is known, the amount of peptides citrullinated by PAD in the sample can be calculated. The activity of PAD in the sample can be evaluated from the amount of citrullinated peptides.

[0144] Although the citrullinated peptide can be quantitatively determined by high-speed chromatography or antibody-based ELISA, it is preferable to use the enzymatic method described above, which is based on the citrulline quantitative determination method of the present invention, because the measurement takes a long time.In addition, the enzymatic method is preferable from the viewpoint of detection sensitivity. [Example]

[0145] The above-mentioned quantitative determination method, citrulline oxidoreductase for quantitative determination, quantitative determination composition, and quantitative determination kit according to the present invention will be described in more detail with specific examples and test results.

[0146] [Preparation of recombinant plasmid pET-22b(+)-CitOX] The citrulline oxidoreductase gene (hereinafter also referred to as CitOX or CitOX(WT)) derived from Pseudomonas sp. strain BYC41-1, which has the nucleotide sequence of SEQ ID NO: 2 and contains the restriction enzyme sites NdeI and BamHI at both ends, was totally synthesized. First, the CitOX(WT) gene was inserted between the restriction enzyme sites NdeI and BamHI of pET-22b(+), and Escherichia coli JM109 was transformed with this.

[0147] The E. coli JM109 (pET-22b(+)-CitOX(WT)) strain carrying the recombinant plasmid was inoculated into 2.5 ml of LB-amp medium [1% (W / V) bactotryptone, 0.5% (W / V) peptone, 0.5% (W / V) NaCl, 50 μg / ml ampicilin] and cultured at 37°C for 24 hours with shaking to obtain a culture.

[0148] The culture was centrifuged at 7,000 rpm for 5 minutes to collect the cells, and the recombinant plasmid pET-22b(+)-CitOX(WT) was extracted and purified from the cells using ISOSPIN Plasmid (Nippon Gene Co., Ltd.) to obtain 2.5 μg of recombinant plasmid pET-22b(+)-CitOX(WT) DNA.

[0149] [Construction of the plasmid for the E62Q mutant] The vector fragment was prepared by PCR using pET-22b(+)-CitOX(WT) as a template and primers E62Q-Fw (SEQ ID NO: 4) and E62X-Rv (SEQ ID NO: 3). Specifically, 5 μl of 10× KOD-Plus buffer, 5 μl of a dNTP mixture (prepared so that each dNTP was 2 mM), 2 μl of 25 mM MgSO solution, 50 ng of a DNA construct containing the CitOX(WT) gene as a template, 15 pmol of each of the synthetic oligonucleotides, and 1 unit of KOD-Plus were added, and the total volume was adjusted to 50 μl with sterile water. The prepared reaction solution was incubated at 94°C for 2 minutes using a thermal cycler (Eppendorf), followed by 30 cycles of 94°C for 15 seconds, 50°C for 30 seconds, and 68°C for 8 minutes.

[0150] A portion of the reaction mixture was electrophoresed on a 1.0% agarose gel, confirming that a DNA fragment of approximately 8,000 bp had been specifically amplified. The resulting DNA was digested with the restriction enzyme DpnI (NEW ENGLAND BIOLABS) to cleave the remaining template DNA, and then transformed into E. coli JM109 and spread on LB-amp agar medium.

[0151] E. coli JM109 was cultured in the same manner as above, and the recombinant plasmid was extracted to obtain a plasmid (pET-22b(+)-CitOX(E62Q)) for expressing CitOX(E62Q).

[0152] [Construction of the plasmid for mutant D74N] A plasmid for expressing CitOX(D74N) (pET-22b(+)-CitOX(D74N)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D74N-Fw (sequence number 6) and D74X-Rv (sequence number 5) were used as primers.

[0153] [Construction of a plasmid for the D74R mutant] A plasmid for expressing CitOX(D74R) (pET-22b(+)-CitOX(D74R)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D74R-Fw (sequence number 7) and D74X-Rv (sequence number 5) were used as primers.

[0154] [Construction of the plasmid for the E92Q mutant] A plasmid for expressing CitOX(E92Q) (pET-22b(+)-CitOX(E92Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E92Q-Fw (sequence number 9) and E92X-Rv (sequence number 8) were used as primers.

[0155] [Construction of the plasmid for the E92R mutant] A plasmid for expressing CitOX(E92R) (pET-22b(+)-CitOX(E92R)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E92R-Fw (sequence number 10) and E92X-Rv (sequence number 8) were used as primers.

[0156] [Construction of a plasmid for the E208R mutant] A plasmid for expressing CitOX(E208R) (pET-22b(+)-CitOX(E208R)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E208R-Fw (sequence number 12) and E208X-Rv (sequence number 11) were used as primers.

[0157] [Construction of the plasmid for the E224Q mutant] A plasmid for expressing CitOX(E224Q) (pET-22b(+)-CitOX(E224Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E224Q-Fw (sequence number 14) and E224X-Rv (sequence number 13) were used as primers.

[0158] [Construction of the plasmid for the E224R mutant] A plasmid for expressing CitOX(E224R) (pET-22b(+)-CitOX(E224R)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E224R-Fw (sequence number 15) and E224X-Rv (sequence number 13) were used as primers.

[0159] [Preparation of the plasmid for the D402N mutant] A plasmid for expressing CitOX(D402N) (pET-22b(+)-CitOX(D402N)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D402N-Fw (sequence number 17) and D402X-Rv (sequence number 16) were used as primers.

[0160] [Construction of the plasmid for mutant D402Q] A plasmid for expressing CitOX(D402Q) (pET-22b(+)-CitOX(D402Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D402Q-Fw (sequence number 18) and D402X-Rv (sequence number 16) were used as primers.

[0161] [Construction of the plasmid for the D402R mutant] A plasmid for expressing CitOX(D402R) (pET-22b(+)-CitOX(D402R)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D402R-Fw (sequence number 19) and D402X-Rv (sequence number 16) were used as primers.

[0162] [Construction of the plasmid for the E486M mutant] A plasmid for expressing CitOX(E486M) (pET-22b(+)-CitOX(E486M)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E486M-Fw (sequence number 21) and E486X-Rv (sequence number 20) were used as primers.

[0163] [Construction of the plasmid for the E486Q mutant] A plasmid for expressing CitOX(E486Q) (pET-22b(+)-CitOX(E486Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E486Q-Fw (sequence number 22) and E486X-Rv (sequence number 20) were used as primers.

[0164] [Construction of the plasmid for the E486H mutant] A plasmid for expressing CitOX(E486H) (pET-22b(+)-CitOX(E486H)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E486H-Fw (sequence number 23) and E486X-Rv (sequence number 20) were used as primers.

[0165] [Construction of the plasmid for the D402Q / E486Q mutant] A plasmid for expressing the double mutant CitOX(D402Q / E486Q) (pET-22b(+)-CitOX(D402Q / E486Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D402Q-Fw (sequence number 18) and D402X-Rv (sequence number 16) were used as primers and pET-22b(+)-CitOX(E486Q) was used as a template.

[0166] [Construction of the plasmid for the D402R / E486Q mutant] A plasmid for expressing the double mutant CitOX(D402R / E486Q) (pET-22b(+)-CitOX(D402R / E486Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D402R-Fw (sequence number 19) and D402X-Rv (sequence number 16) were used as primers and pET-22b(+)-CitOX(E486Q) was used as a template.

[0167] [Construction of the plasmid for mutant D402H] A plasmid for expressing CitOX(D402H) (pET-22b(+)-CitOX(D402H)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D402H-Fw (sequence number 24) and D402X-Rv (sequence number 16) were used as primers.

[0168] [Construction of the plasmid for the D476R mutant] A plasmid for expressing CitOX(D476R) (pET-22b(+)-CitOX(D476R)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D476R-Fw (sequence number 26) and D476X-Rv (sequence number 25) were used as primers.

[0169] [Construction of the plasmid for the E514Q mutant] A plasmid for expressing CitOX(E514Q) (pET-22b(+)-CitOX(E514Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E514Q-Fw (sequence number 28) and E514X-Rv (sequence number 27) were used as primers.

[0170] [Construction of the plasmid for the E524Q mutant] A plasmid for expressing CitOX(E524Q) (pET-22b(+)-CitOX(E524Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that E524Q-Fw (sequence number 30) and E524X-Rv (sequence number 29) were used as primers.

[0171] [Construction of the plasmid for the D74R / E486Q mutant] A plasmid for expressing the double mutant CitOX(D74R / E486Q) (pET-22b(+)-CitOX(D74R / E486Q)) was obtained using the same method as for preparing the plasmid for mutant E62Q, except that D74R-Fw (sequence number 7) and D74X-Rv (sequence number 5) were used as primers and pET-22b(+)-CitOX(E486Q) was used as a template.

[0172] [Preparation of recombinant plasmid pET-22b(+)-PjCitOX] A citrulline oxidoreductase gene derived from a Pseudomonas japonica strain (hereinafter referred to as PjCitOX or PjCitOX(WT)) having the nucleotide sequence of SEQ ID NO: 32 containing the restriction enzyme sites NdeI and BamHI at both ends was totally synthesized. First, the PjCitOX(WT) gene was inserted between the restriction enzyme sites NdeI and BamHI of pET-22b(+), and Escherichia coli JM109 was transformed with this.

[0173] The E. coli JM109 (pET-22b(+)-PjCitOX(WT)) strain carrying the recombinant plasmid was inoculated into 2.5 ml of LB-amp medium [1% (W / V) bactotryptone, 0.5% (W / V) peptone, 0.5% (W / V) NaCl, 50 μg / ml ampicilin] and cultured at 37°C for 24 hours with shaking to obtain a culture.

[0174] The culture was centrifuged at 7,000 rpm for 5 minutes to collect the cells, and the recombinant plasmid pET-22b(+)-PjCitOX(WT) was extracted and purified from the cells using ISOSPIN Plasmid (Nippon Gene Co., Ltd.) to obtain 2.5 μg of recombinant plasmid pET-22b(+)-PjCitOX(WT) DNA.

[0175] [Construction of the plasmid for the mutant PjCitOX(D8Q)] The vector fragment was prepared by the same method as that for preparing the plasmid for mutant E62Q, except that pET-22b(+)-PjCitOX(WT) was used as a template and D8Q-Fw (sequence number 34) and D8X-Rv (sequence number 33) were used as primers, and a plasmid for expressing PjCitOX(D8Q) (pET-22b(+)-PjCitOX(D8Q)) was obtained using this method.

[0176] [Construction of the plasmid for the mutant PjCitOX(D8S)] A plasmid for expressing PjCitOX(D8S) (pET-22b(+)-PjCitOX(D8S)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that D8S-Fw (sequence number 35) and D8X-Rv (sequence number 33) were used as primers.

[0177] [Construction of the plasmid for the mutant PjCitOX(E39N)] A plasmid for expressing PjCitOX(E39N) (pET-22b(+)-PjCitOX(E39N)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E39N-Fw (sequence number 37) and E39X-Rv (sequence number 36) were used as primers.

[0178] [Construction of the plasmid for the mutant PjCitOX(E39S)] A plasmid for expressing PjCitOX(E39S) (pET-22b(+)-PjCitOX(E39S)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E39S-Fw (sequence number 38) and E39X-Rv (sequence number 36) were used as primers.

[0179] [Construction of the plasmid for the mutant PjCitOX(E62Q)] A plasmid for expressing PjCitOX(E62Q) (pET-22b(+)-PjCitOX(E62Q)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E62Q-Fw (sequence number 40) and E62X-Rv (sequence number 39) were used as primers.

[0180] [Construction of the plasmid for the mutant PjCitOX(E148H)] A plasmid for expressing PjCitOX(E148H) (pET-22b(+)-PjCitOX(E148H)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E148H-Fw (sequence number 42) and E148X-Rv (sequence number 41) were used as primers.

[0181] [Construction of the plasmid for the mutant PjCitOX(E148Q)] A plasmid for expressing PjCitOX(E148Q) (pET-22b(+)-PjCitOX(E148Q)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E148Q-Fw (sequence number 43) and E148X-Rv (sequence number 41) were used as primers.

[0182] [Construction of the plasmid for the mutant PjCitOX(E148R)] A plasmid for expressing PjCitOX(E148R) (pET-22b(+)-PjCitOX(E148R)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E148R-Fw (sequence number 44) and E148X-Rv (sequence number 41) were used as primers.

[0183] [Construction of the plasmid for the mutant PjCitOX(E148N)] A plasmid for expressing PjCitOX(E148N) (pET-22b(+)-PjCitOX(E148N)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E148N-Fw (sequence number 45) and E148X-Rv (sequence number 41) were used as primers.

[0184] [Construction of the plasmid for the mutant PjCitOX(E148S)] A plasmid for expressing PjCitOX(E148S) (pET-22b(+)-PjCitOX(E148S)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E148S-Fw (sequence number 46) and E148X-Rv (sequence number 41) were used as primers.

[0185] [Construction of the plasmid for the mutant PjCitOX(E353N)] A plasmid for expressing PjCitOX(E353N) (pET-22b(+)-PjCitOX(E353N)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E353N-Fw (sequence number 48) and E353X-Rv (sequence number 47) were used as primers.

[0186] [Construction of the plasmid for the mutant PjCitOX(E353Q)] A plasmid for expressing PjCitOX(E353Q) (pET-22b(+)-PjCitOX(E353Q)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E353Q-Fw (sequence number 49) and E353X-Rv (sequence number 47) were used as primers.

[0187] [Construction of the plasmid for the mutant PjCitOX(E382S)] A plasmid for expressing PjCitOX(E382S) (pET-22b(+)-PjCitOX(E382S)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E382S-Fw (sequence number 51) and E382X-Rv (sequence number 50) were used as primers.

[0188] [Construction of the plasmid for the mutant PjCitOX(E382H)] A plasmid for expressing PjCitOX(E382H) (pET-22b(+)-PjCitOX(E382H)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E382H-Fw (sequence number 52) and E382X-Rv (sequence number 50) were used as primers.

[0189] [Construction of the plasmid for the mutant PjCitOX(D402R)] A plasmid for expressing PjCitOX(D402R) (pET-22b(+)-PjCitOX(D402R)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that D402R-Fw (sequence number 54) and D402X-Rv (sequence number 53) were used as primers.

[0190] [Construction of the plasmid for the mutant PjCitOX(D402N)] A plasmid for expressing PjCitOX(D402N) (pET-22b(+)-PjCitOX(D402N)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that D402N-Fw (sequence number 55) and D402X-Rv (sequence number 53) were used as primers.

[0191] [Construction of the plasmid for the mutant PjCitOX(D402Q)] A plasmid for expressing PjCitOX(D402Q) (pET-22b(+)-PjCitOX(D402Q)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that D402Q-Fw (sequence number 56) and D402X-Rv (sequence number 53) were used as primers.

[0192] [Construction of the plasmid for the mutant PjCitOX(E486Q)] A plasmid for expressing PjCitOX(E486Q) (pET-22b(+)-PjCitOX(E486Q)) was obtained using the same method as for preparing the plasmid for mutant PjCitOX(D8Q), except that E486Q-Fw (sequence number 58) and E486X-Rv (sequence number 57) were used as primers.

[0193] [Construction of the plasmid for the mutant PjCitOX(E486H)] A plasmid for expressing PjCitOX(E486H) (pET-22b(+)-PjCitOX(E486H)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E486H-Fw (sequence number 59) and E486X-Rv (sequence number 57) were used as primers.

[0194] [Construction of the plasmid for the mutant PjCitOX(E148Q / E486Q)] A plasmid for expressing the double mutant PjCitOX(E148Q / E486Q) (pET-22b(+)-PjCitOX(E148Q / E486Q)) was obtained using the same method as for preparing the plasmid for the mutant PjCitOX(D8Q), except that E148Q-Fw (sequence number 43) and E148X-Rv (sequence number 41) were used as primers and pET-22b(+)-PjCitOX(E486Q) was used as a template.

[0195] [Preparation of recombinant plasmid pET-22b(+)-PWCitOX] The citrulline oxidoreductase gene (hereinafter also referred to as PWCitOX or PWCitOX(WT)) derived from Pseudomonas sp. WCHPs060044 strain, which has the nucleotide sequence of SEQ ID NO: 61 and contains the restriction enzyme sites NdeI and BamHI at both ends, was totally synthesized. First, the PWCitOX(WT) gene was inserted between the restriction enzyme sites NdeI and BamHI of pET-22b(+), and Escherichia coli JM109 was transformed with the gene.

[0196] The E. coli JM109 (pET-22b(+)-PWCitOX(WT)) strain carrying the recombinant plasmid was inoculated into 2.5 ml of LB-amp medium [1% (W / V) bactotryptone, 0.5% (W / V) peptone, 0.5% (W / V) NaCl, 50 μg / ml ampicilin] and cultured at 37°C for 24 hours with shaking to obtain a culture.

[0197] The culture was centrifuged at 7,000 rpm for 5 minutes to collect the cells, and the recombinant plasmid pET-22b(+)-PWCitOX(WT) was extracted and purified from the cells using ISOSPIN Plasmid (Nippon Gene Co., Ltd.) to obtain 2.5 μg of recombinant plasmid pET-22b(+)-PWCitOX(WT) DNA.

[0198] [Construction of the plasmid for the mutant PWCitOX(E39Q)] The vector fragment was prepared by the same method as that for preparing the plasmid for mutant E62Q, except that pET-22b(+)-PWCitOX(WT) was used as a template and E39Q-Fw (SEQ ID NO: 63) and E39X-Rv (SEQ ID NO: 62) were used as primers, to obtain a plasmid (pET-22b(+)-PWCitOX(E39Q)) for expressing PWCitOX(E39Q).

[0199] [Construction of the plasmid for the mutant PWCitOX(D74R)] A plasmid for expressing PWCitOX(D74R) (pET-22b(+)-PWCitOX(D74R)) was obtained by the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that D74R-Fw (SEQ ID NO: 65) and D74X-Rv (SEQ ID NO: 64) were used as primers.

[0200] [Construction of the plasmid for the mutant PWCitOX(D74N)] A plasmid for expressing PWCitOX(D74N) (pET-22b(+)-PWCitOX(D74N)) was obtained by the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that D74N-Fw (SEQ ID NO: 66) and D74X-Rv (SEQ ID NO: 64) were used as primers.

[0201] [Construction of the plasmid for the mutant PWCitOX(E224R)] A plasmid for expressing PWCitOX(E224R) (pET-22b(+)-PWCitOX(E224R)) was obtained using the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that E224R-Fw (SEQ ID NO: 68) and E224X-Rv (SEQ ID NO: 67) were used as primers.

[0202] [Construction of the plasmid for the mutant PWCitOX(E354Q)] A plasmid for expressing PWCitOX(E354Q) (pET-22b(+)-PWCitOX(E354Q)) was obtained by the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that E354Q-Fw (SEQ ID NO: 70) and E354X-Rv (SEQ ID NO: 69) were used as primers.

[0203] [Construction of the plasmid for the mutant PWCitOX(E383Q)] A plasmid for expressing PWCitOX(E383Q) (pET-22b(+)-PWCitOX(E383Q)) was obtained by the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that E383Q-Fw (SEQ ID NO: 72) and E383X-Rv (SEQ ID NO: 71) were used as primers.

[0204] [Construction of the plasmid for the mutant PWCitOX(D403R)] A plasmid for expressing PWCitOX(D403R) (pET-22b(+)-PWCitOX(D403R)) was obtained by the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that D403R-Fw (SEQ ID NO: 74) and D403X-Rv (SEQ ID NO: 73) were used as primers.

[0205] [Construction of the plasmid for the mutant PWCitOX(D403N)] A plasmid for expressing PWCitOX(D403N) (pET-22b(+)-PWCitOX(D403N)) was obtained using the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that D403N-Fw (SEQ ID NO: 75) and D403X-Rv (SEQ ID NO: 73) were used as primers.

[0206] [Construction of the plasmid for the mutant PWCitOX(E487H)] A plasmid for expressing PWCitOX(E487H) (pET-22b(+)-PWCitOX(E487H)) was obtained by the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that E487H-Fw (SEQ ID NO: 77) and E487X-Rv (SEQ ID NO: 76) were used as primers.

[0207] [Construction of the plasmid for the mutant PWCitOX(E487Q)] A plasmid for expressing PWCitOX(E487Q) (pET-22b(+)-PWCitOX(E487Q)) was obtained by the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that E487Q-Fw (SEQ ID NO: 78) and E487X-Rv (SEQ ID NO: 76) were used as primers.

[0208] [Construction of the plasmid for the mutant PWCitOX(E532Q)] A plasmid for expressing PWCitOX(E532Q) (pET-22b(+)-PWCitOX(E532Q)) was obtained by the same method as for preparing the plasmid for mutant PWCitOX(E39Q), except that E532Q-Fw (SEQ ID NO: 80) and E532X-Fw (SEQ ID NO: 79) were used as primers.

[0209] [Preparation of recombinant plasmid pET-22b(+)-Pn2CitOX] The citrulline dehydrogenase gene derived from computational science was the citrulline dehydrogenase gene set forth in SEQ ID NO: 82, which encodes AncARODn2 (the amino acid sequence predicted based on the Pseudomonas sp. TPU 7192 strain described in S. Nakano et al., Appl. Environ. Microbiol. 2019 85(12) e00459-19). A citrulline oxidoreductase gene having the nucleotide sequence of SEQ ID NO: 82, including the NdeI and BamHI restriction enzyme sites at both ends (hereinafter referred to as the Pn2CitOX gene or Pn2CitOX(WT) gene) was totally synthesized. The Pn2CitOX(WT) gene was first inserted between the NdeI and BamHI restriction enzyme sites of pET-22b(+), and used to transform Escherichia coli JM109.

[0210] E. coli JM109 (pET-22b(+)-Pn2CitOX(WT)) strain harboring the recombinant plasmid was inoculated into 2.5 ml of LB-amp medium [1% (W / V) bactotryptone, 0.5% (W / V) peptone, 0.5% (W / V) NaCl, 50 μg / ml ampicilin] and cultured with shaking at 37°C for 24 hours to obtain a culture.

[0211] The culture was centrifuged at 7,000 rpm for 5 minutes to collect the cells, and the recombinant plasmid pET-22b(+)-Pn2CitOX(WT) was extracted and purified from the cells using ISOSPIN Plasmid (Nippon Gene Co., Ltd.) to obtain 2.5 μg of recombinant plasmid pET-22b(+)-Pn2CitOX(WT) DNA.

[0212] [Citrulline oxidoreductase production] The recombinant plasmids of each mutant obtained by the above procedure were used to transform E. coli BL21(DE3) strain, which was then cultured for 24 hours at 30°C in 2.5 ml of ZYP-5052 medium (0.5% glycerol, 0.05% glucose, 0.2% lactose, 50 mM (NH4)2SO4, 50 mM KH2PO4, 50 mM Na2HPO4, 1 mM MgSO4).

[0213] Three ml of the culture medium was collected and centrifuged at 12,000 rpm for 5 minutes to collect the bacterial cells, which were then washed with 0.01 M potassium phosphate buffer (pH 7.0), sonicated, and centrifuged at 15,000 rpm for 10 minutes to obtain crude enzyme solutions containing citrulline oxidoreductases having the amino acid sequences of the mutants.

[0214] Similarly, Escherichia coli BL21(DE3) transformed with only the pET-22b(+) vector was also cultured and subjected to ultrasonic disruption to prepare 1.5 ml of crude enzyme solution.

[0215] [Confirmation of citrulline oxidoreductase expression] The expression level of citrulline oxidoreductase was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Using a 10-20% gradient polyacrylamide gel and a CLEARLY Stained Protein Ladder (Takara Bio Inc.) as a marker, the amount of citrulline oxidoreductase contained in each crude enzyme solution was confirmed. The results showed no significant differences in expression levels between citrulline oxidoreductase (WT) and almost all mutants. Furthermore, when measured using the measurement method described below, activity toward citrulline was confirmed for citrulline oxidoreductase (WT) and all mutants.

[0216] [Citrulline oxidase activity measurement] The crude enzyme solution obtained by the above method was subjected to measurement of citrulline oxidase activity. 725 μl of the reagent having the composition shown in Table 1 was incubated at 37°C for 5 minutes, and then 25 μl of the crude enzyme solution was added and mixed. The A per minute at 37°C was measured using a spectrophotometer (U-3900, Hitachi High-Tech Science Corporation). 555 Amount of change (ΔA S ) was measured.

[0217] Next, 25 μl of potassium phosphate buffer solution at pH 7.0 containing 0.1% BSA was added instead of the crude enzyme solution and mixed. 555 The change in activity (ΔA0) was measured. 4-Aminoantipyrine (4-AA) was from Wako Pure Chemical Industries, Ltd., citrulline was from Sigma-Aldrich, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) was from Dojindo Laboratories, and horseradish peroxidase (POD) was from Toyobo.

[0218] [Table 1]

[0219] Citrulline oxidase activity was calculated based on the following formula. Oxidase activity (U / ml) =(ΔA S -ΔA0)×750×df / (39.2×0.5×25) =1.53×(ΔA S -ΔA0)×df 39.2: millimolar extinction coefficient (mM) of 4-AA-TOOS condensed dye for light of wavelength 555 nm -1 cm -1 ) df: Dilution rate of crude enzyme solution

[0220] [Citrulline oxidase activity measurement results] As a measure of improved citrulline reactivity, we also confirmed the reactivity to arginine. Table 2 shows the results of measuring the citrulline oxidase activity of CitOX (WT), mutants E62Q, D74N, D74R, E92Q, E92R, E208N, E208R, E224Q, E224R, D402N, D402Q, D402R, E486M, E486Q, the double mutant D402Q / E486Q, and the double mutant D402R / E486Q. [Table 2]

[0221] The results in Table 2 indicate that the ratio of citrulline reactivity to arginine reactivity increased in all mutants, significantly improving reactivity to citrulline. Furthermore, the arginine and citrulline contents in a sample can be estimated from the reaction ratio of arginine to citrulline. Furthermore, when similar activity measurements were performed using the ultrasonicated supernatant obtained by culturing E. coli BL21(DE3) transformed with only the pET-22b(+) vector, no activity toward citrulline was observed.

[0222] [Evaluation of pH dependence of citrulline oxidase activity] To evaluate the pH dependence of citrulline oxidase activity, the citrulline oxidase activity was measured using the above-described method, except that a pH 6.0 potassium phosphate buffer was used instead of the 150 mM pH 7.0 potassium phosphate buffer. Table 3 shows the results of measuring the citrulline oxidase activity of CitOX(WT), the double mutant D402Q / E486Q, and the double mutant D402R / E486Q treated with a pH 6.0 buffer. [Table 3]

[0223] The results in Table 3 show that treatment with a buffer solution at pH 6.0 improves the reactivity to citrulline compared to treatment with a buffer solution at pH 7.0.

[0224] Furthermore, the citrulline oxidase activity at pH 6.0 of the other citrulline oxidases prepared above was measured in the same manner as above. The measurement results for each mutant are shown in Table 4. [Table 4]

[0225] The results of measuring the citrulline oxidase activity of PjCitOX (WT) and its mutants are shown in Table 5. [Table 5]

[0226] The results of measuring the citrulline oxidase activity of PWCitOX(WT) and its mutants are shown in Table 6. PWCitOX(E354Q), PWCitOX(E383Q), PWCitOX(E354Q), PWCitOX(D403R), PWCitOX(D403N), PWCitOX(E487H), and PWCitOX(E487Q) are mutants of PWCitOX at amino acid residues corresponding to the 353rd, 353rd, 402nd, and 486th amino acid residues of PWCitOX. [Table 6]

[0227] The results of measuring the citrulline oxidase activity of Pn2CitOX(WT) are shown in Table 7. [Table 7]

[0228] [Purification of citrulline oxidase] Anion exchange chromatography was performed using a column of 1.5 ml of Q-Sepharose resin equilibrated with 10 mM potassium phosphate buffer (pH 7.5). The cell-free extract dialyzed against 10 mM potassium phosphate buffer (pH 7.5) was adsorbed. After washing the column with 7.5 mL of 10 mM potassium phosphate buffer, the NaCl concentration was gradually increased stepwise using 10 mM potassium phosphate buffer (pH 7.5) and 10 mM potassium phosphate buffer (pH 7.5) containing 1000 mM NaCl to elute the enzyme. Fractions showing activity were collected.

[0229] Next, gel filtration chromatography was performed using a liquid chromatography system (AKTA avant25, column: HiLoad 26 / 60 superdex200 equilibrated with 10 mM potassium phosphate buffer containing 150 mM NaCl). The fractions obtained above were injected, and the enzyme was fractionated with 10 mM potassium phosphate buffer containing 150 mM NaCl. The activity of each fraction obtained was measured, and fractions showing activity were pooled.

[0230] The degree of purification was confirmed by SDS-PAGE. If contaminating proteins were found, anion exchange chromatography was performed again. Each fraction was confirmed by SDS-PAGE, and fractions that showed a single band and activity were pooled and concentrated by ultrafiltration. The Cit / Arg ratio of the resulting citrulline oxidase preparation was measured, and the results were equivalent to those obtained using the crude enzyme solution.

[0231] [Quantitative determination of citrulline using citrulline oxidase] In the activity measurement method described above, solutions containing various concentrations of citrulline solution (5, 10, 20, or 50 mM) were prepared. Then, 25 μl of a solution containing a citrulline oxidase preparation was added and mixed, and the A was measured using a spectrophotometer (U-3900) at 37°C for 5 minutes. 555 The changes were measured. 555 The correlation between enzyme activity (U / ml) calculated from the change was evaluated using the vertical axis and citrulline concentration on the horizontal axis.

[0232] FIG. 3 shows the correlation between citrulline concentration and enzyme activity (U / ml). FIG. 3(a) shows the measurement results for CitOX (WT) treated with a pH 6.0 buffer solution, and FIG. 3(b) shows the measurement results for CitOX (E486Q) treated with a pH 6.0 buffer solution. For CitOX (WT), the coefficient of determination (R), which is an index of the correlation between citrulline concentration and enzyme activity (U / ml), was 0.01 in the range of 5 mM to 20 mM. 2 ) was 1.00, which indicates a correlation between citrulline concentration (μM) and enzyme activity (U / ml). In addition, in the CitOX mutant E486Q, the coefficient of determination (R ), which is an index of the correlation between citrulline concentration and enzyme activity (U / ml), was 0.00 in the range of 5 mM to 20 mM. 2 ) was 0.93, indicating a correlation between citrulline concentration (μM) and enzyme activity (U / ml). Therefore, it was demonstrated that citrulline can be quantified using CitOX(WT) and CitOX(E486Q).

[0233] [Quantitative determination of citrulline using citrulline dehydrogenase] Citrulline dehydrogenase activity was assessed for CitOX (WT) and the CitOX mutant E486Q treated with pH 6.0 buffer. Citrulline dehydrogenase activity was measured using 2,6-dichlorophenolindophenol (DCIP). Specifically, citrulline dehydrogenase activity was measured according to the following procedure: 2.05 mL of 100 mM phosphate buffer (pH 6.0), 0.6 mL of citrulline solution, and 0.15 mL of 2 mM DCIP solution were mixed and incubated at 37°C for 5 minutes. Next, 0.1 mL of 15 mM PMS solution and 0.1 mL of enzyme sample solution were added to initiate the reaction. The absorbance was measured at the start of the reaction and over time. The decrease in absorbance at 600 nm per minute (ΔA600) associated with the progress of the enzyme reaction was determined using a spectrophotometer (U-3900, Hitachi High-Tech Science). Citrulline dehydrogenase activity was calculated according to the following formula: 1 U of citrulline dehydrogenase activity is defined as the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of 20 mM citrulline at 37°C. Citrulline solutions (5, 10, or 20 mM) or ion-exchanged water were used as substrates. Dehydrogenase activity (U / ml) =(ΔA S -ΔA0)×3.0×df / (14.18×0.1×1.0) =2.12×(ΔA S -ΔA0)×df 14.18: Millimolar extinction coefficient (mM) of DCIP dye for light of 600 nm wavelength -1 cm -1 ) df: dilution rate of enzyme solution

[0234] FIG. 4 shows the correlation between citrulline concentration and enzyme activity (U / ml) in an example of the present invention. FIG. 4(a) shows the measurement results for CitOX (WT) treated with a buffer solution of pH 6.0, and FIG. 4(b) shows the measurement results for CitOX (E486Q) treated with a buffer solution of pH 6.0. For CitOX (WT), in the range of 5 mM to 20 mM, the coefficient of determination (R), which is an index of the correlation between citrulline concentration and enzyme activity (U / ml), was 0.01. 2 ) was 0.99, which indicates that there is a correlation between citrulline concentration (mM) and enzyme activity (U / ml). In addition, for CitOX (E486Q), the coefficient of determination (R 2 ) was 0.98, which indicates a correlation between citrulline concentration (mM) and enzyme activity (U / ml). Therefore, it was demonstrated that citrulline can be quantified using the dehydrogenase reaction.

[0235] [Determination of citrulline by citrulline dehydrogenase in electrochemical measurements] On screen-printed electrodes (DropSens, product number DRP-C110), 15 μL of 100 mM phosphate buffer (pH 6.0) containing 1 M sodium chloride and 200 mM potassium ferricyanide and 4 μL of CitOX (E486Q) solution were applied and mixed. The device was then connected to an ALS 814D electrochemical analyzer using a dedicated connector (DRP-CAC). Cyclic voltammetry measurements were performed at a sweep rate of 20 mV / s over the range of 0 to +600 mV (Ag / AgCl). Next, 1 μL of citrulline solution at various concentrations was added, and the oxidation current at +0.4 V was recorded and plotted for citrulline concentrations of 0 to 26 mM. The results showed that the oxidation current increased with increasing citrulline concentration (see Figure 5). In the range of 0 mM to 26 mM, the coefficient of determination (R 2) was 0.98, which indicates that there is a correlation between the citrulline concentration (mM) and the oxidation current value (μA) at +0.4 V. This demonstrates that citrulline can be quantified by electrochemical measurement.

[0236] [Quantitation of citrulline-containing peptides using citrulline dehydrogenase] We investigated whether citrullinated peptides could be quantified by citrulline dehydrogenase when all of the PAD4 substrate peptides (FFDSHKWHRDFFYSD) were citrullinated by PAD4. Specifically, a final concentration of 6.7 mM of FFDSHKWH(Cit)DFFYSD (Peptide Institute, Inc.) was used as the citrullinated peptide, and a final concentration of 20 μM of CoCl2 was mixed with Pfu Aminopeptidase I (Takara Bio Inc.) as the peptidase and incubated at 75°C for 14 hours. The solution obtained after the peptidase reaction was used as the substrate solution, and citrulline dehydrogenase activity was assessed using CitOX (E62Q) treated with a pH 6.0 buffer solution. Specifically, activity was measured according to the procedure described above for measuring citrulline dehydrogenase activity. However, the wavelength of absorbance was measured at 520 nm, and the millimolar extinction coefficient (mM) of DCIP for light at a wavelength of 520 nm was used. -1 cm -1 ) was calculated as 6.8.

[0237] Fig. 6 shows the relationship between the citrullinated peptide concentration and the change in absorbance per minute in one embodiment of the present invention. Fig. 6 shows the activity measurement results of CitOX (E62Q) treated with a buffer solution of pH 6.0. In the range of 0.01 mM to 0.22 mM, the coefficient of determination (R 2 ) was 1.00, which indicates that there is a correlation between the citrullinated peptide concentration (mM) and the change in absorbance. This demonstrates that the citrullinated peptide can be quantified by using the citrulline dehydrogenase of the present invention.

[0238] [Evaluation of PAD4 activity using uncitrullinated peptides (arginine peptides) and citrulline dehydrogenase] It was also found that the citrullination rate could be calculated by reacting PAD4 in blood with the peptide (FFDSHKWHRDFFYSD) and quantifying the resulting FFDSHKWH(Cit)DFFYSD. Thus, PAD4 activity in blood samples could also be evaluated.

[0239] As described above, the present invention provides a method for quantifying citrulline by adding citrulline oxidase or citrulline dehydrogenase to a sample containing citrulline, a citrulline oxidase or citrulline dehydrogenase for quantifying citrulline added to a sample containing citrulline, a composition for quantifying citrulline containing citrulline oxidase or citrulline dehydrogenase added to a sample containing citrulline, and a kit for quantifying citrulline containing citrulline oxidase or citrulline dehydrogenase added to a sample containing citrulline, which can be used to quantify the concentration of citrulline, a biomarker for diseases associated with abnormal protein citrullination, such as multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, psoriasis, prion disease, liver fibrosis, chronic obstructive pulmonary disease, and cancer. It also provides a method for assessing PAD activity.

Claims

1. Citrulline oxidoreductase is added to the sample, When the citrulline oxidoreductase is citrulline oxidase, the concentration of citrulline is determined by quantifying the hydrogen peroxide produced by adding the citrulline oxidase; or When the citrulline oxidoreductase is citrulline dehydrogenase, the method comprises adding the citrulline dehydrogenase to reduce the mediator, and determining the concentration of citrulline; the citrulline oxidoreductase has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 1 and has citrulline oxidoreductase activity; or the citrulline oxidoreductase has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 31 and has citrulline oxidoreductase activity; or the citrulline oxidoreductase has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 60 and has citrulline oxidoreductase activity; or A method for quantifying citrulline, wherein the citrulline oxidoreductase has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of citrulline oxidoreductase set forth in SEQ ID NO: 81 and has citrulline oxidoreductase activity.

2. The citrulline oxidoreductase has the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted; or The citrulline oxidoreductase has the amino acid sequence of SEQ ID NO: 31 in which one or more amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted; or The citrulline oxidoreductase has the amino acid sequence of SEQ ID NO: 60 in which one or more amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted; or The method for quantifying citrulline according to claim 1, wherein the citrulline oxidoreductase has an amino acid sequence in which one or more amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted in the amino acid sequence of SEQ ID NO:

81.

3. Citrulline oxidoreductase derived from Pseudomonas sp. was added to the sample, When the citrulline oxidoreductase is citrulline oxidase, determining the concentration of citrulline by quantifying the hydrogen peroxide produced by adding the citrulline oxidase; or When the citrulline oxidoreductase is citrulline dehydrogenase, A method for quantifying citrulline, comprising adding the citrulline dehydrogenase to reduce a mediator and determine the concentration of citrulline.

4. The method for quantifying citrulline according to any one of claims 1 to 3, wherein the citrulline oxidoreductase has a reactivity ratio (Cit / Arg) of citrulline to arginine of 1% or more.

5. A citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 31 and having citrulline oxidoreductase activity; or A citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 60 and having citrulline oxidoreductase activity; or A composition for quantifying citrulline, comprising a citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of citrulline oxidoreductase set forth in SEQ ID NO: 81 and having citrulline oxidoreductase activity.

6. A citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 31 and having citrulline oxidoreductase activity; or A citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 60 and having citrulline oxidoreductase activity; or a citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 81 and having citrulline oxidoreductase activity; A kit for quantifying citrulline, comprising either a reagent that reacts with hydrogen peroxide or a mediator that is reduced by adding the citrulline oxidoreductase.

7. A citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 31 and having citrulline oxidoreductase activity; or A citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 60 and having citrulline oxidoreductase activity; or A sensor chip comprising a citrulline oxidoreductase having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of citrulline oxidoreductase set forth in SEQ ID NO: 81 and having citrulline oxidoreductase activity.

8. a citrulline oxidoreductase (excluding the citrulline oxidoreductase set forth in SEQ ID NO: 1) having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 1 and having citrulline oxidoreductase activity; either a reagent that reacts with hydrogen peroxide or a mediator that is reduced by adding the citrulline oxidoreductase; A composition for quantifying citrulline, comprising:

9. a citrulline oxidoreductase (excluding the citrulline oxidoreductase set forth in SEQ ID NO: 1) having a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 1 and having citrulline oxidoreductase activity; A kit for quantifying citrulline, comprising either a reagent that reacts with hydrogen peroxide or a mediator that is reduced by adding the citrulline oxidoreductase.

10. A sensor chip comprising a citrulline oxidoreductase (excluding the citrulline oxidoreductase set forth in SEQ ID NO: 1) that has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 1 and has citrulline oxidoreductase activity.

11. A sensor comprising the sensor chip according to claim 7 or 10.

12. A method for evaluating the activity of peptidylarginine deiminase, using the method for quantifying citrulline according to any one of claims 1 to 4.

13. A peptide that can be a substrate for peptidylarginine deiminase is added to the sample; adding a protease or peptidase to the sample; adding citrulline oxidoreductase to the sample; When the citrulline oxidoreductase is citrulline oxidase, the concentration of citrulline is determined by quantifying the hydrogen peroxide produced by adding the citrulline oxidase; or When the citrulline oxidoreductase is citrulline dehydrogenase, the method comprises adding the citrulline dehydrogenase to reduce the mediator, and determining the concentration of citrulline; the citrulline oxidoreductase has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 1 and has citrulline oxidoreductase activity; or the citrulline oxidoreductase has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 31 and has citrulline oxidoreductase activity; or the citrulline oxidoreductase has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of the citrulline oxidoreductase set forth in SEQ ID NO: 60 and has citrulline oxidoreductase activity; or A method for evaluating the activity of a peptidylarginine deiminase, wherein the citrulline oxidoreductase has a full-length amino acid sequence identity of 90% or more to the amino acid sequence of citrulline oxidoreductase set forth in SEQ ID NO: 81 and has citrulline oxidoreductase activity.

14. The citrulline oxidoreductase has the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted; or The citrulline oxidoreductase has the amino acid sequence of SEQ ID NO: 31 in which one or more amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted; or The citrulline oxidoreductase has the amino acid sequence of SEQ ID NO: 60 in which one or more amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted; or The method for evaluating the activity of peptidylarginine deiminase according to claim 13, wherein the citrulline oxidoreductase has an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 81 have been modified or mutated, or deleted, substituted, added, and / or inserted.

15. A peptide that can be a substrate for peptidylarginine deiminase is added to the sample; adding a protease or peptidase to the sample; adding citrulline oxidoreductase derived from Pseudomonas to the sample; When the citrulline oxidoreductase is citrulline oxidase, the concentration of citrulline is determined by quantifying the hydrogen peroxide produced by adding the citrulline oxidase; or When the citrulline oxidoreductase is citrulline dehydrogenase, the method for evaluating peptidylarginine deiminase activity comprises adding the citrulline dehydrogenase to reduce a mediator and determine the concentration of citrulline.

16. The method for evaluating peptidylarginine deiminase activity according to any one of claims 13 to 15, wherein the citrulline oxidoreductase has a ratio of reactivity with citrulline to reactivity with arginine (Cit / Arg) of 1% or more.

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