JP7570063B2 - Composition for inhibiting ACE or suppressing hypertension, its method of manufacture, enzyme preparation, polynucleotide, and transformant - Google Patents

Description

The present invention relates to a composition for inhibiting angiotensin I-converting enzyme (hereinafter referred to as "ACE") or suppressing hypertension, which contains a peptide having ACE inhibitory activity, a method for producing the composition, an enzyme preparation containing a synthetic enzyme capable of producing the peptide, a polynucleotide having a gene for the synthetic enzyme, and a transformant into which the gene has been introduced.

The biggest risk factor for cardiovascular disease (such as stroke or myocardial infarction) is high blood pressure. Providing food that can prevent or improve high blood pressure would lead to improved healthy life expectancy and reduced medical costs. For example, Non-Patent Document 2 reports that sesame peptides have ACE inhibitory activity. ACE is an enzyme that hydrolyzes angiotensin I to produce angiotensin II. Angiotensin II promotes the secretion of aldosterone, which increases blood pressure. Therefore, when the amount of angiotensin II produced is reduced by ACE inhibition, the increase in blood pressure is suppressed.

Euglena (genus name: Euglena, Japanese name: Midorimushi) is a unique single-celled organism that combines the animal properties of flagellar movement with the plant properties of photosynthesis. Euglena cells (hereinafter referred to as "Euglena cells") can grow under various nutritional conditions and can contain various nutrients. For this reason, various research and development efforts have been made to utilize Euglena cells as a biological resource such as food. For example, the blood pressure lowering agent described in Patent Document 1 is a cell powder obtained by culturing Euglena cells in a medium containing a large amount of docosahexaenoic acid (hereinafter referred to as "DHA"), centrifuging the culture medium after the culture to collect the precipitated Euglena cells, and drying them. Patent Document 1 reports that when stroke-prone spontaneously hypertensive rats were fed with feed containing this blood pressure lowering agent, the increase in blood pressure in the rats was suppressed.

Japanese Patent Application Publication No. 8-133980

E. S. Kempner, et al., "The Molecular Biology of Euglena gracilis. III. General Carbon Metabolism", Biochemistry, 1965, volume 4, issue 12, pp.2735-2739 E. S. Kempner, et al., "The Molecular Biology of Euglena gracilis IX. Amino Acid pool Composition", The Journal of Protozoology, 1974, volume 21, issue 2, pp.363-367 Daisuke Nakano, and 15 others, "Antihypertensive Effect of Angiotensin I-Converting Enzyme Inhibitory Peptides from a Sesame Protein Hydrolysate in Spontaneously Hypertensive Rats", Bioscience, Biotechnology, and Biochemistry, 2006, volume 70, issue 5, pp.1118-1126 Gietz RD, et al., "Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.", Methods in enzymology, 2002, volume 350, pp.87-96 M. Cramer, and 1 other, Arch. Mikrobiol., 1952, volume 17, pp.384-402 J. A. Schiff, and 2 others, Methods Enzymol., 1971, volume 23, pp.143-162 L. E. Koren, and 1 other, The Journal of Protozoology, 1967, volume 14, supplement, p.17 Yasuhito Shomura, and 9 others, "Structural and enzymatic characterization of BacD, an L-amino acid dipeptide ligase from Bacillus subtilis", Protein Science, 2012, volume 21, issue 5, pp.707-716 Francis C. Neuhaus, "The Enzymatic Synthesis of D-Alanyl-D-alanine I. PURIFICATION AND PROPERTIES OF D-ALANYL-D-ALANINE SYNTHETASE", The Journal of Biological Chemistry, 1962, Volume 237, No. 3, pp. 778-786

Non-Patent Document 1 reports that free amino acids such as L-methionine and L-arginine are produced in Euglena cells. It is also known that taking n-3 polyunsaturated fatty acids (hereinafter referred to as "n-3 PUFAs") such as DHA or eicosapentaenoic acid, or sulfur-containing amino acids such as L-methionine, can prevent hypertensive diseases. It is also known that L-arginine has ACE inhibitory activity. Based on these findings, those skilled in the art have traditionally believed that the blood pressure rise suppression effect reported in Patent Document 1 was due to the n-3 PUFAs and free amino acids contained in Euglena cells as active ingredients.

On the other hand, Non-Patent Document 2 reports that Euglena cells can contain peptides having arginine residues in the molecule. The inventors of the present application (hereinafter referred to as "the inventors") have repeatedly studied whether this peptide can be effectively used for purposes other than as a protein source. Since this peptide is a natural product produced in Euglena cells, it is thought that a wide variety of peptides with different amino acid sequences are mixed. In the past, it was hardly clear what specific amino acid sequences are mixed as peptides having arginine residues in the molecule contained in Euglena cells. Just as sesame peptides were discovered from sesame, the inventors conceived that it would be desirable to discover peptides having ACE inhibitory activity from the wide variety of peptides contained in Euglena cells. In addition, they conceived that it would be even more desirable to discover a gene for a synthetic enzyme capable of producing a peptide having ACE inhibitory activity from the chromosomal DNA of Euglena cells, and to efficiently produce the peptide using this synthetic enzyme, its gene, or a microorganism such as Euglena, and mass-produce a food composition that is easy to prevent or improve high blood pressure.

Therefore, the object of the present invention is to discover a peptide having ACE inhibitory activity from Euglena cells and a gene for a synthetic enzyme capable of producing the peptide, and to provide a composition for inhibiting ACE or suppressing hypertension containing the peptide, a method for producing the composition, an enzyme preparation containing the synthetic enzyme, a polynucleotide having the gene, and a transformant into which the polynucleotide has been introduced.

In order to solve the above-mentioned problems, the composition of the present invention is an enzyme preparation containing a dipeptide synthesizing enzyme, wherein the dipeptide synthesizing enzyme has an amino acid sequence of one type of polypeptide selected from the group consisting of (a), (b), and (c) below: (a) a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1; (b) a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 1; (c) a polypeptide consisting of an amino acid sequence in which one or more and not more than 20 amino acid residues have been substituted, inserted, deleted, and/or added to the amino acid sequence set forth in SEQ ID NO: 1; and the dipeptide synthesizing enzyme is an enzyme preparation having one or more enzyme activities selected from the group consisting of L-arginyl-L-asparagine (Arg-Asn) ligase activity, L-arginyl-L-glutamine (Arg-Gln) ligase activity, L-asparaginyl-L-arginine (Asn-Arg) ligase activity, and L-glutamyl-L-arginine (Gln-Arg) ligase activity.

The method for producing a composition according to the present invention is a method for producing a composition for inhibiting ACE or suppressing hypertension, and includes the steps of preparing an enzyme preparation according to the present invention, and in the presence of one or more compounds selected from the group consisting of L-arginine (Arg) and its salts, and one or more compounds selected from the group consisting of L-asparagine (Asn), L-glutamine (Gln), and their salts, and the enzyme preparation, causing an enzymatic reaction with the dipeptide synthesizing enzyme to produce a dipeptide compound having angiotensin I converting enzyme (ACE) inhibitory activity, which is one or more compounds selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and their salts.

The polypeptide of the present invention is a polynucleotide having a base sequence encoding a dipeptide synthase, wherein the base sequence encoding the dipeptide synthase is a base sequence of one type of polynucleotide selected from the group consisting of the following (d), (e), (f), and (g): (d) a polynucleotide having the base sequence set forth in SEQ ID NO: 2; (e) a polynucleotide having a base sequence having 90% or more identity to the base sequence set forth in SEQ ID NO: 2; (f) a polynucleotide having a base sequence in which one or more and not more than 20 bases are substituted, inserted, deleted, and/or added to the base sequence set forth in SEQ ID NO: 2; (g) a polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence set forth in SEQ ID NO: 2; and the base sequence encoding the dipeptide synthase is a polynucleotide having a base sequence encoding an enzyme having one or more enzymatic activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity.

The recombinant vector of the present invention is a recombinant vector comprising the polynucleotide of the present invention.

The transformant of the present invention is a microorganism transformed with the polynucleotide of the present invention or the recombinant vector of the present invention.

In the transformant of the present invention, the microorganism may be Euglena or yeast.

The kit of the present invention is a kit having one or more selected from the group consisting of the enzyme preparation of the present invention, the polynucleotide of the present invention, the recombinant vector of the present invention, and the transformant of the present invention.

Alternatively, the method for producing a composition according to the present invention is a method for producing a composition for inhibiting ACE or suppressing hypertension, comprising the steps of preparing a medium containing the transformant according to the present invention and a nitrogen source capable of being assimilated by the transformant, and culturing the transformant in the presence of the medium to produce a dipeptide compound having ACE inhibitory activity, the dipeptide compound being one or more compounds selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and salts thereof.

In the method for producing the composition of the present invention, the nitrogen source may include one or more compounds selected from the group consisting of Arg and its salts, and one or more compounds selected from the group consisting of Asn, Gln, and their salts.

The composition of the present invention is a composition for inhibiting ACE or suppressing hypertension, and contains one or more selected from the group consisting of the transformant of the present invention, the transformant cultured in a medium containing a nitrogen source capable of nitrogen assimilation by the transformant, a dried product of the transformant, and a cell lysate of the transformant, and thus contains a dipeptide compound having ACE inhibitory activity, which is one or more compounds selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and salts thereof.

The composition of the present invention may contain the dipeptide synthase or a denatured protein thereof.

In the composition of the present invention, the content of the dipeptide compound may be 0.10 mg/L or more.

Alternatively, the composition of the present invention is a composition consisting of or comprising a fraction of water-soluble components containing a peptide derived from Euglena cells and having a molecular mass of less than 4.0 kDa, the composition not containing lipids derived from the cells, proteins derived from the cells and having a molecular mass of 4.0 kDa or more, and polypeptides derived from the cells and having a molecular mass of 4.0 kDa or more, the peptide derived from the cells and having a molecular mass of less than 4.0 kDa containing a dipeptide compound having ACE inhibitory activity, which is one or more compounds selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and salts thereof, for use in inhibiting ACE or suppressing hypertension.

In each of the composition and the method for producing the composition according to the present invention, the Euglena is preferably one or more protozoa selected from the group consisting of Euglena gracilis, its Z strain, and Euglena gracilis bacillaris variant. The enzymatic activity of the dipeptide synthesizing enzyme in each of the enzyme preparation and the polypeptide according to the present invention is preferably one or more enzymatic activities selected from the group consisting of Arg-Asn ligase activity and Arg-Gln ligase activity. The salt in the dipeptide compound is preferably a sodium salt or a potassium salt. The composition according to the present invention is preferably a food composition. The transformant according to the present invention in the method for producing the composition according to the present invention is preferably immobilized on a support so as to be culturable in order to produce the dipeptide compound. In terms of being more suitable for producing the dipeptide compound, the microorganism in the transformant according to the present invention is preferably Euglena. When the microorganism is Euglena, the nitrogen source in the method for producing the composition according to the present invention is preferably one or more compounds selected from the group consisting of ammonia nitrogen, free amino acids, peptides, and salts thereof. Alternatively, from the viewpoint of suitability for the production of food compositions, the microorganism in the transformant according to the present invention is preferably a yeast, more preferably Saccharomyces cerevisiae. From the same viewpoint, the microorganism is preferably a yeast strain (NITE ABP-03293). In each of the specification, claims, and drawings of the present application, the description of the accession number "NITE ABP-03293" for the yeast strain is preferably corrected to the description of the accession number "NITE BP-03293" or "NITE-BP-3293" when the yeast strain is received after the filing of the present application. From the viewpoint of removing impurities, the composition according to the present invention is preferably a composition that does not contain lipids derived from the transformant according to the present invention, proteins derived from the transformant and having a molecular mass of 4.0 kDa or more, and polypeptides derived from the transformant and having a molecular mass of 4.0 kDa or more.

<Terminology>
In the present specification, with regard to free amino acids or salts thereof, L-arginine is also referred to as "Arg", L-asparagine is also referred to as "Asn", L-aspartic acid is also referred to as "Asp", L-glutamine is also referred to as "Gln", and L-glutamic acid is also referred to as "Glu", and an enzyme capable of synthesizing a dipeptide using a free amino acid or a salt thereof as a substrate is also referred to as a "dipeptide synthesizing enzyme". In the present specification, with regard to free dipeptides or salts thereof, L-arginyl-L-asparagine is also referred to as "Arg-Asn", L-arginyl-L-glutamine is also referred to as "Arg-Gln", L-asparaginyl-L-arginine is also referred to as "Asn-Arg", and L-glutamyl-L-arginine is also referred to as "Gln-Arg", and in the formulas of each of these four types of dipeptides, "-" indicates a peptide bond between the carboxy group and the amino group at the α-position. In the present specification, the enzyme activity as a ligase capable of synthesizing Arg-Asn using Arg and Asn as substrates is also referred to as "Arg-Asn ligase activity", a polypeptide having Arg-Asn ligase activity is also referred to as "Arg-Asn ligase", the enzyme activity as a ligase capable of synthesizing Arg-Gln using Arg and Gln as substrates is also referred to as "Arg-Gln ligase activity", and a polypeptide having Arg-Gln ligase activity is also referred to as "Arg-Gln ligase". the enzyme activity as a ligase capable of synthesizing Asn-Arg using Asn and Arg as substrates is also referred to as "Asn-Arg ligase activity", a polypeptide having Asn-Arg ligase activity is also referred to as "Asn-Arg ligase", the enzyme activity as a ligase capable of synthesizing Gln-Arg using Gln and Arg as substrates is also referred to as "Gln-Arg ligase activity", and a polypeptide having Gln-Arg ligase activity is also referred to as "Gln-Arg ligase".

In this specification, "content" means the total content when two or more kinds of the corresponding compound are contained. In this specification, one or more compounds selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and their salts having ACE inhibitory activity are also referred to as "the dipeptide compound". In this specification, an enzyme having an amino acid sequence of one type of polypeptide selected from the group consisting of (a), (b), and (c) described above with respect to the enzyme preparation of the present invention and having one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity is also referred to as "the enzyme". In this specification, the enzyme preparation of the present invention is also referred to as "the enzyme preparation," the polynucleotide of the present invention is also referred to as "the polynucleotide," the kit of the present invention is also referred to as "the kit," the first embodiment of the composition of the present invention is also referred to as "the composition 1," the first embodiment of the method for producing the composition of the present invention is also referred to as "the method 1," the second embodiment of the composition of the present invention is also referred to as "the composition 2," the second embodiment of the method for producing the composition of the present invention is also referred to as "the method 2," the third embodiment of the composition of the present invention is also referred to as "the composition 3," and the third embodiment of the method for producing the composition of the present invention is also referred to as "the method 3."

In this specification, the term "Euglena" refers to one or more protozoa selected from the group consisting of species belonging to the genus Euglena (green algae) in the zoological classification and their variants. In this specification, the water-soluble component extracted from Euglena cells is also referred to as "Euglena aqueous extract". In this specification, "polynucleotide" refers to DNA or RNA, preferably DNA. In this specification, "stringent conditions" refer to conditions under which specific hybrids are formed and non-specific hybrids are not formed. In this specification, "food composition" refers to one or more compositions selected from the group consisting of processed foods, seasonings, food additives, supplements, and food ingredients blended therewith.

According to the composition of the present invention, the dipeptide compound, which is one or more compounds selected from the group consisting of Asn-Arg, Arg-Asn, Gln-Arg, Arg-Gln, and salts thereof, has stronger ACE inhibitory activity than L-arginine and can be ingested for ACE inhibition or blood pressure elevation suppression. According to the method for producing the composition of the present invention, it is possible to produce the composition of the present invention. According to the enzyme preparation of the present invention, it contains a dipeptide synthesizing enzyme and can be used to generate the dipeptide compound. According to the polynucleotide of the present invention, it has a base sequence that codes for the amino acid sequence of the dipeptide synthesizing enzyme. According to the transformant of the present invention, it can be used when producing the enzyme preparation of the present invention or when producing the composition of the present invention.

1 is a flow chart illustrating a first embodiment of a method for producing a composition according to the present invention. 4 is a flow chart illustrating a second embodiment of a method for producing a composition according to the present invention. 4 is a flow chart illustrating a third embodiment of a method for producing a composition according to the present invention. Total ion chromatograms obtained by LC/MS (liquid chromatography mass spectrometry) from the samples and standard samples (Gln-Arg and Arg-Gln) according to Experimental Examples 1 to 3. In Fig. 4 to Fig. 7, the vertical axis represents detection intensity, and the horizontal axis represents retention time (min). Chromatograms of selected ion monitoring at m/z=70 obtained from the samples according to Experimental Examples 1 to 3 and the standard samples (Gln-Arg and Arg-Gln) by LC/MS/MS (liquid chromatography mass spectrometry using a tandem quadrupole mass spectrometer). This is a chromatogram from Figure 5 with further noise removed in order to measure the area of the peak due to Arg-Gln. The area of the peak due to Arg-Gln is shaded in black. 1 shows total ion chromatograms obtained by LC/MS from the samples according to Experimental Examples 1 to 3 and the standard samples (Asn-Arg and Arg-Asn). Graph showing the ACE inhibition rate by Arg-Gln, Gln-Arg, Arg-Asn, or Asn-Arg. The vertical axis represents the ACE inhibition rate (%), and the horizontal axis represents the dipeptide content (mg/L). n=2. (a) is a graph showing the diastolic and systolic blood pressures of male SHR/NCrlCrlj rats (SHR rats) in the control group that freely ingested saline and SHR rats in the experimental group that freely ingested sample water containing a dipeptide isolated and purified from Euglena cells at 5, 6, and 7 weeks after the start of the experiment (the time when the control group rats were able to freely ingest saline and the experimental group rats were able to freely ingest sample water). In (a), the vertical axis shows blood pressure (mmHg), # shows a significant difference at p<0.07 in one-way analysis of variance, and ab shows a significant difference at p<0.05 in the Tukey-Kramer method. (b) is a graph showing the blood concentration of angiotensin II in each of the control group rats and the experimental group rats at 7 weeks after the start of the experiment, and the vertical axis shows the blood concentration (pg/mL). For both (a) and (b), n=6. * indicates a significant difference at p<0.05 in one-way analysis of variance. A photograph of an electrophoretic gel showing that a purified product of dipeptide ligase derived from a yeast strain (NITE ABP-03293) was subjected to SDS-PAGE electrophoresis, suggesting that the molecular mass of the dipeptide ligase was about 111 kDa. In the figure, "Purified enzyme" indicates the lane in the electrophoretic gel in which the purified product of dipeptide ligase derived from a yeast strain (NITE ABP-03293) was electrophoresed. Graphs showing the results of a test to confirm the amounts of Asn-Arg, Arg-Asn, Gln-Arg, and Arg-Gln produced by enzymatic reaction with dipeptide ligase derived from yeast strain (NITE ABP-03293). In (a), the vertical axis of the graph shows the Asn-Arg content, in (b), the vertical axis of the graph shows the Arg-Asn content, in (c), the vertical axis of the graph shows the Gln-Arg content, and in (d), the vertical axis of the graph shows the Arg-Gln content. (Arg/Asn)+ and (Arg/Gln)+ are reaction solutions to which a purified product of dipeptide ligase derived from yeast strain (NITE ABP-03293) was added, and (Arg/Asn)- and (Arg/Gln)- are reaction solutions to which this purified product was not added. ** indicates a significant difference at p<0.01 in one-way analysis of variance.

<Enzyme preparation>
In the process of completing the present invention, the present inventors analyzed the composition of a water-soluble component extracted from Euglena cells (hereinafter referred to as "Euglena aqueous extract") and discovered that (i) the Euglena aqueous extract contains Arg-Gln and Arg-Asn, and may also contain Gln-Arg and Asn-Arg. The present inventors unexpectedly discovered that (ii) each of the four dipeptides listed in (i) above has stronger ACE inhibitory activity than Arg. The present inventors also discovered that (iii) Euglena cells may contain Arg-Gln and Arg-Asn at high concentrations of up to about 0.5 mmol/L, and that (iv) the chromosomal DNA of Euglena contains a base sequence encoding a dipeptide synthase capable of producing the four dipeptides listed in (i) above. As far as the present inventors know, there have been no reports discussing the findings listed in (i) to (iv) above prior to the priority date of the present application. Therefore, it is considered that none of the findings listed in (i) to (iv) above was known to a person skilled in the art before the priority date of the present application. Hereinafter, in this specification, a compound having one or more ACE inhibitory activities selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and salts thereof is also referred to as "the dipeptide compound of the present invention". The salt of the dipeptide compound of the present invention may be any pharmacologically acceptable salt. Examples of the salt of the dipeptide compound of the present invention include an alkali metal salt or an alkaline earth metal salt. From the viewpoint of being less likely to precipitate and easy to handle, the salt of the dipeptide compound of the present invention is preferably a sodium salt or a potassium salt.

Based on the findings in (i) to (iv) above, the present inventors considered the following. Euglena cells have the property of accumulating amino acids with multiple nitrogen atoms in the molecule in an environment with an abundance of nitrogen sources (one or more nitrogen compounds that Euglena cells can assimilate nitrogen) (see Non-Patent Documents 1 and 2). For example, Euglena cells have the property of taking up ammonium salts into the cells and easily producing Arg, which has four nitrogen atoms in its molecule, and Asn and Gln, which have two nitrogen atoms in their molecules. This property is thought to have been acquired by Euglena cells through natural selection during the course of evolution as a property that ensures the survival of Euglena in a starvation environment by accumulating as many nitrogen atoms as nutrients in the form of these amino acids in the cells as possible. Furthermore, Euglena cells are believed to have the property of converting Arg, Asn, and Gln produced within the cells into the dipeptide forms Arg-Gln and Arg-Asn and storing them within the cells in order to compact the Arg, Asn, and Gln as much as possible, thereby decreasing the intracellular density and reducing the osmotic pressure. In an experiment using a crude enzyme regarding the dipeptide synthase discovered by the present inventors, substrate specificity was observed for three types of amino acids, Arg, Asn, and Gln, and the reaction was promoted by the addition of adenosine triphosphate (ATP). Therefore, the dipeptide synthase discovered by the present inventors is believed to be an Arg-Asn ligase or Arg-Gln ligase having the enzymatic activity shown in the following chemical reaction formula:

例えば上記した化学反応式中、Amino Acid 1はArgを示し、Amino Acid 2はAsn又はGlnを示す。このため、例えばこの式中、Ｒ１はArgの側鎖部分を示し、Ｒ２はAsn又はGlnの側鎖部分を示す。

For example, in the above chemical reaction formula, Amino Acid 1 represents Arg, and Amino Acid 2 represents Asn or Gln. Therefore, for example, in this formula, R1 represents the side chain portion of Arg, and R2 represents the side chain portion of Asn or Gln.

Because Euglena cells can contain a large amount of Arg-Asn and Arg-Gln, they stably express a dipeptide synthase having one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity and Arg-Gln ligase activity. For this reason, if Arg-Asn ligase or Arg-Gln ligase is extracted from Euglena cells, a certain amount of yield can be easily obtained. In addition, Euglena cells can contain small amounts of Asn-Arg and Gln-Arg, so Asn-Arg ligase and Gln-Arg ligase are also expressed. For these reasons, it is possible to extract a dipeptide synthase capable of producing one or more dipeptides selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, and Gln-Arg from Euglena cells. The reaction mechanism of Asn-Arg ligase and Gln-Arg ligase is considered to be the same as the above-mentioned chemical reaction formula, where amide acid 1 and amide acid 2 are interchanged and R1 and R2 are interchanged. Based on the above-mentioned findings and ideas, the present inventors have come up with the invention of the enzyme preparations described below.

The enzyme preparation according to the present invention (hereinafter referred to as "the enzyme preparation") is an enzyme preparation containing one or more dipeptide synthesizing enzymes. The dipeptide synthesizing enzyme contained in the enzyme preparation has an amino acid sequence of one polypeptide selected from the group consisting of the following (a), (b), and (c), and is an enzyme (hereinafter referred to as "the enzyme") having one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity. (a) is a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 1. (b) is a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence described in SEQ ID NO: 1. The identity of the amino acid sequence here may be 95% or more, or 98% or more. (c) is a polypeptide consisting of an amino acid sequence in which one or more and 20 or less amino acid residues are substituted, inserted, deleted, and/or added to the amino acid sequence described in SEQ ID NO: 1. The number of substituted, inserted, deleted, and/or added amino acid residues herein may be 1 or more and 10 or less, or 1 or more and 5 or less. The "substitution, insertion, deletion, and/or addition" described herein may be a naturally occurring mutation or a mutation introduced by a known method. In each of (a), (b), and (c) above, "polypeptide" refers to a polymer of amino acids in which a large number of amino acids are peptide-bonded.

The amino acid sequence described in SEQ ID NO: 1 is the amino acid sequence of a dipeptide synthase discovered by the present inventors through genetic analysis of the chromosomal DNA of Euglena. The identity of the amino acid sequence can be determined, for example, by searching databases such as Swiss-Prot, PIR, or DAD via the Internet using a program such as BLAST or FASTA. The enzyme may be an enzyme consisting of the amino acid sequence of one type of polypeptide selected from the group consisting of (a), (b), and (c) described above. Alternatively, the enzyme may be an enzyme consisting of an additional amino acid sequence bound to the amino acid sequence of one type of polypeptide selected from the group consisting of (a), (b), and (c), so long as the enzyme activity is not substantially impaired. For example, the enzyme may have a tag sequence added thereto, or may be bound to another protein to form a fusion protein. The tag sequence is not particularly limited as long as it does not contradict the purpose of the present invention, but examples thereof include a His tag sequence, that is, an amino acid sequence consisting of about six consecutive histidine residues.

The method for producing the present enzyme or the present enzyme preparation is not particularly limited, but for example, the method includes culturing Euglena or the transformant according to the present invention described later in a medium to increase the cell number, collecting the cultured Euglena or the present transformant from the medium and disrupting the cells, removing lipids from the resulting cell disruption liquid to obtain an aqueous extract, subjecting the extract to a molecular sieve (e.g., gel filtration chromatography, etc.), and recovering and freeze-drying a fraction containing the present enzyme. The freeze-dried product obtained here may itself be used as the present enzyme preparation. Alternatively, from the viewpoint of avoiding moisture absorption and deterioration during long-term storage, it is preferable to mix the freeze-dried product with a pH buffer and adjust the pH to an optimal value to form the present enzyme preparation. From the same viewpoint, it is also preferable to mold the freeze-dried product or the pH-adjusted composition into any dosage form such as a powder, granules, capsules, liquid, or cream by a conventional method to form the present enzyme preparation. As described above, Euglena cells can express not only Arg-Asn ligase and Arg-Gln ligase, but also Asn-Arg ligase and Gln-Arg ligase, so it is considered that an enzyme preparation containing two or more dipeptide synthetases capable of producing the present dipeptide compound can be produced by culturing Euglena or the present transformant obtained by transforming Euglena and recovering a fraction containing the present enzyme in the same manner as described above. Since Arg-Asn and Arg-Gln are more likely to be contained in Euglena cells than Asn-Arg and Gln-Arg, it is considered that the Arg-Asn ligase activity and Arg-Gln activity of the present enzyme are stronger than the Asn-Arg ligase activity and Gln-Arg ligase activity of the present enzyme. From this viewpoint, it is preferable that the enzyme has one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity and Arg-Gln ligase activity.

The enzyme preparation can be used to generate the dipeptide compound, for example, during the production of the first embodiment of the composition according to the present invention, which will be described later. From the viewpoint of efficiently mass-producing the dipeptide compound, the enzyme preparation is preferably in the form of one or more dipeptide synthesizing enzymes including the enzyme preparation. An immobilized enzyme is an enzyme that is bound to a carrier or confined in a certain space in a state in which it is capable of continuous reaction and can be recovered and reused after the reaction. For example, the enzyme preparation may be in a form filled in a space isolated by a semipermeable membrane such as a cellophane membrane so that it can be separated from the dipeptide compound to be produced. From the viewpoint of immobilization while avoiding deactivation, the enzyme preparation in the case of an immobilized enzyme is preferably in a form encapsulated in a polymer gel or bound to an insoluble carrier. Examples of polymer gels include polyacrylamide gel, agar, gelatin, carrageenan, calcium alginate, and the like. Examples of insoluble carriers include carriers such as cellulose, dextrin, resin beads, activated carbon, silica gel, magnetic particles, and polymer membranes. The dipeptide synthase and the carrier can be bound by known techniques, such as an ionic binding method in which the dipeptide synthase is bound to an ion exchange resin or the like through electrostatic interaction, a physical adsorption method in which the dipeptide synthase is adsorbed into the pores of a porous carrier through physical interaction, or an affinity binding method in which the dipeptide synthase is bound to the carrier through an antigen-antibody reaction.

In the case of the present enzyme preparation being an immobilized enzyme, it is more preferable that one or more dipeptide synthases including the present enzyme are bound to an insoluble carrier by affinity binding, from the viewpoint of preventing the dipeptide synthase from being easily detached from the carrier. Since Euglena cells can express Arg-Asn ligase, Arg-Gln ligase, Asn-Arg ligase, and Gln-Arg ligase, for example, by performing the affinity binding method using a homogenate of Euglena cells, it is possible to produce an immobilized enzyme containing these four types of dipeptide synthases bound to an insoluble carrier by an antigen-antibody reaction.

<Polynucleotide>
In this specification, the term "polynucleotide" refers to DNA or RNA, and preferably DNA. The polynucleotide according to the present invention (hereinafter referred to as "the present polynucleotide") is a polynucleotide having a base sequence encoding a dipeptide synthetase, and the base sequence encoding this dipeptide synthetase has a base sequence of one type of polynucleotide selected from the group consisting of the following (d), (e), (f), and (g), and is a polynucleotide having a base sequence encoding a dipeptide synthetase having one or more enzyme activities selected from the group consisting of Asn-Arg ligase activity, Arg-Asn ligase activity, Gln-Arg ligase activity, and Arg-Gln ligase activity. (d) is a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2. (e) is a polynucleotide consisting of a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 2. The identity of the base sequence here may be 95% or more, or 98% or more. (f) is a polynucleotide consisting of a base sequence in which 1 or more and 20 or less bases are substituted, inserted, deleted, and/or added to the base sequence shown in SEQ ID NO: 2. Here, the number of substituted, inserted, deleted, and/or added bases may be 1 or more and 10 or less, or 1 or more and 5 or less. (g) is a polynucleotide that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2.

In this specification, "stringent conditions" are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. In other words, "stringent conditions" are conditions under which DNAs with high base sequence identity, for example DNAs with identity of 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more, hybridize with each other, and DNAs with clearly lower identity than this do not hybridize with each other. Alternatively, "stringent conditions" include, for example, conditions under which washing is performed once, preferably twice or three times, with a solution containing 1xSSC and 0.1% SDS (sodium dodecyl sulfate) at 60°C, which is the washing condition in normal Southern hybridization, preferably a solution containing 0.1xSSC and 0.1% SDS at 60°C, more preferably a solution containing 0.1xSSC and 0.1% SDS at 68°C. Moreover, if the temperature is raised somewhat under the conditions exemplified here, polynucleotides having high identity in the base sequence can be obtained efficiently. Those skilled in the art can achieve similar stringency by appropriately selecting temperature, probe concentration, probe length, ionic strength, time, salt concentration, and the like. Examples of "polynucleotides that hybridize under stringent conditions" include polynucleotides obtained by known hybridization methods such as Southern hybridization or Northern hybridization using all or part of a DNA consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 2 as a probe. The hybridization method can be carried out according to the methods described in, for example, Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Habor Laboratory Press), Current Protocols in Molecular Biology (1994) (Wiley-Interscience), or DNA Cloning 1: A Practical Approach Core Techniques, Second Edition (1995) (Oxford University Press), etc.

The base sequence described in SEQ ID NO: 2 is the base sequence of an open reading frame encoding a dipeptide synthase, which was discovered by the present inventors through genetic analysis of the chromosomal DNA of Euglena. At the time of discovery in 2019, the base sequence described in SEQ ID NO: 2 had about 50% identity to known base sequences encoding ligases possessed by other types of microorganisms. When the base sequence described in SEQ ID NO: 2 is translated, a dipeptide synthase having the amino acid sequence described in SEQ ID NO: 1 is expressed. A person skilled in the art can obtain a polynucleotide having a high identity to the base sequence of this DNA by appropriately introducing substitutions, insertions, deletions, and/or additions to the DNA having the base sequence described in SEQ ID NO: 2 using known methods such as site-directed mutagenesis. By introducing this polynucleotide into a microorganism, it is possible to express a polypeptide having an amino acid sequence identical or similar to the amino acid sequence described in SEQ ID NO: 1, and to collect this polypeptide by extracting it from the microorganism. The thus obtained polypeptide may include a polypeptide having one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity, and functioning as a dipeptide synthesizing enzyme. The identity of the nucleotide sequence may be determined, for example, by searching databases such as DDBJ, EMBL, or GenBank via the Internet using a program such as BLAST or FASTA.

The method for isolating or purifying the polynucleotide is not particularly limited. For example, a primer for PCR (polymerase chain reaction) is designed based on the base sequence of one type of polynucleotide selected from the group consisting of (d), (e), (f), and (g) described above, and a PCR is performed using the chromosomal DNA or cDNA (complementary DNA) library of Euglena as a template to obtain a DNA fragment of the polynucleotide. The DNA fragment obtained by this PCR may be used as a probe to introduce a restriction enzyme digest of Euglena chromosomal DNA into a phage or plasmid, and the polynucleotide may be obtained by colony hybridization or the like using a library or cDNA library obtained by transforming Escherichia coli. Alternatively, the polynucleotide can be obtained by designing a primer for extending outside the known DNA based on the sequence obtained by analyzing the base sequence of the DNA fragment obtained by PCR, digesting the chromosomal DNA of Euglena with an appropriate restriction enzyme, and then performing inverse PCR using the DNA as a template by self-cyclization reaction.

The polynucleotide may be, for example, a genomic DNA or cDNA cloned by the method described below, or may be a synthetic DNA. In hybridization, a DNA or RNA consisting of a complementary sequence to the base sequence described in SEQ ID NO: 2 or a partial sequence thereof is used as a probe to hybridize to the chromosomal DNA of Euglena, and after washing, for example, under the stringent conditions described above, it is confirmed whether the probe significantly hybridizes to the base sequence described in SEQ ID NO: 2 in the chromosomal DNA. The length of the probe may be, for example, 20 or more consecutive bases, or may be 25 or more bases from the viewpoint of easy significant hybridization, preferably 30 or more bases, more preferably 40 or more bases, and even more preferably 80 or more bases. A person skilled in the art can perform hybridization by a known method, for example, using a nylon membrane or a nitrocellulose membrane.

The polynucleotide may have an additional sequence upstream (5' end side) or downstream (3' end side) of the base sequence of one of the polynucleotides selected from the group consisting of (d), (e), (f), and (g). The additional sequence may be, for example, a promoter sequence or an enhancer sequence upstream, and a terminator sequence downstream. The additional sequence and the base sequence of one of the polynucleotides selected from the group consisting of (d), (e), (f), and (g) may be directly linked so that the number of bases intervening between them is zero, or may be indirectly linked via a base sequence of one or more bases and 1,000 bases or less, 500 bases or less, 100 bases or less, 50 bases or less, or 10 bases or less. In the chromosomal DNA of Euglena, taa, which is a triplet corresponding to a stop codon, is directly linked downstream of the base sequence described in SEQ ID NO: 2 so that the number of bases intervening between them is zero. Similarly, in the present polynucleotide, taa may be directly linked downstream of the base sequence of one type of polynucleotide selected from the group consisting of (d), (e), (f), and (g) described above.

From the viewpoint of ease of use in transforming microorganisms, the present polynucleotide is preferably in the form of a recombinant vector containing the base sequence of one type of polynucleotide selected from the group consisting of (d), (e), (f), and (g) described above, and more preferably, this recombinant vector is an expression vector. The present polynucleotide or a recombinant vector containing the same may be commercially available in the form of a DNA solution for gene recombination, for example, by mixing with a pH buffer. The present polynucleotide or a recombinant vector containing the same can be used, for example, to prepare the present transformant, as described below.

<Transformants>
The transformant according to the present invention (hereinafter referred to as "the transformant") is a microorganism transformed with the aforementioned polynucleotide or a recombinant vector containing the polynucleotide. In other words, the transformant can be said to be a microorganism transformed by introducing the polynucleotide or a recombinant vector containing the polynucleotide into the cell. The transformant preferably contains and retains the polynucleotide or a recombinant vector containing the nucleotide in its cell nucleus, and more preferably has the polynucleotide incorporated in its chromosomal DNA. The transformant can be preferably used for producing the dipeptide compound or as a raw material for producing the aforementioned enzyme preparation.

When preparing the present transformant, the type of microorganism (hereinafter referred to as the "host") from which the present polynucleotide or a recombinant vector containing the present polynucleotide is introduced is not particularly limited, but is preferably a microorganism that is edible without sterilization from the viewpoint of safety when taken orally. Examples of the microorganism include koji mold, lactic acid bacteria, natto bacteria, acetic acid bacteria, yeast, green algae, brown algae, red algae, blue-green algae, and Euglena. The present transformant can be used for producing, for example, fermented foods containing the present dipeptide compound depending on the type of the original host. For example, the original host is preferably koji mold when producing soy sauce or miso, lactic acid bacteria when producing cheese, kimchi, or narezushi, etc., preferably natto bacteria when producing natto, acetic acid bacteria when producing vinegar, or yeast when producing bread or alcoholic beverages. The yeast here is preferably a yeast of the genus Saccharomyces, more preferably Saccharomyces cerevisiae, from the viewpoint of ease of producing bread, wine, or beer. Examples of yeasts preferred for wine brewing include Saccharomyces bayanus, etc. When the host is Saccharomyces cerevisiae, the present transformant is not particularly limited as long as it does not contradict the object of the present invention, but an example of the present transformant in this case is a yeast strain received on September 28, 2020 at the Patent Microorganism Depositary Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) with identification mark W303A1/pYES::EgDPL and receipt number NITE ABP-03293 (hereinafter, this yeast strain will be referred to as "yeast strain (NITE ABP-03293)" or, if it is received after the filing of this application, "yeast strain (NITE BP-03293)" or "yeast strain (NITE BP-3293)").

Alternatively, in consideration of mass-producing foods containing the present dipeptide compound and providing them to many people at low cost to prevent or improve high blood pressure in society in general, it is more preferable that the host on which the present transformant is based is Euglena, from the viewpoint of a microorganism capable of growing while containing the present dipeptide compound under various nutritional conditions. In this specification, "Euglena" refers to one or more protozoa selected from the group consisting of species belonging to the genus Euglena (Euglena genus) in the zoological classification and variants thereof. Examples of Euglena include Euglena gracilis, its Z strain (Euglena gracilis Z), Euglena gracilis var. bacillaris, and Euglena viridis. From the viewpoint of ease of culturing, as suitable culture conditions have been thoroughly studied, the "Euglena" in the present specification is preferably one or more protozoa selected from the group consisting of Euglena gracilis, its Z strain, and Euglena gracilis bacilalis variant.

In producing the present transformant from a host, a person skilled in the art can select an appropriate method from known transformation methods according to the type of host. Examples of transformation methods when a true bacterium is used as a host include the electroporation method, in which minute holes are instantaneously opened in the cell membrane by an electric pulse to allow the present polynucleotide or a recombinant vector containing the same to be incorporated into the cell, and a method in which a plasmid or phage DNA or a recombinant vector containing the present polynucleotide is incorporated into the cell of a bacterium that has been made competent in the presence of calcium chloride. Examples of transformation methods when yeast is used as a host include the lithium acetate method (see Non-Patent Document 4). For example, the electroporation method may be used as a transformation method when Euglena is used as a host, but the particle gun method is preferred from the viewpoint of the ease with which the present polynucleotide is incorporated into the cell. The particle gun method is a method in which fine particles of metal such as gold or tungsten coated with the present polynucleotide or recombinant vector are used as bullets and fired at high speed to allow the present polynucleotide or recombinant vector to be incorporated into the cell.

From the viewpoint of easy use for the production of the enzyme preparation or the production of the dipeptide compound, the transformant preferably has an increased transcription amount of the gene encoding the enzyme described above in the description of the enzyme preparation, or an increased expression amount of the enzyme, compared to the original host. The increased transcription amount can be confirmed by comparing the content of mRNA transcribed from a base sequence encoding a dipeptide synthesizing enzyme having one type of polynucleotide base sequence selected from the group consisting of (d), (e), (f), and (g) described above in the description of the polynucleotide, and having one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity, Asn-Arg ligase activity, Arg-Gln ligase activity, and Gln-Arg ligase activity, between the host and the transformant. Examples of methods for evaluating the mRNA content include northern hybridization and RT-PCR. The content of this mRNA may be increased in the present transformant compared to the host, for example, 2.0 times by mass or more, preferably 3.0 times by mass or more, more preferably 5.0 times by mass or more, and even more preferably 10 times by mass or more. Alternatively, the increase in the expression level of the present enzyme can be confirmed by Western blotting using an antibody. The expression level of the present enzyme may be increased in the present transformant compared to the original host, for example, 2.0 times by mass or more, preferably 3.0 times by mass or more, more preferably 5.0 times by mass or more, and even more preferably 10 times by mass or more.

Depending on the type of original host, it is believed that it is possible to produce a transformant having the property of secreting the dipeptide compound outside the cell, unlike Euglena, which continues to contain the dipeptide compound produced intracellularly. In the case where the transformant has such a property, it is preferably immobilized alive on a support so as to be culturable, from the viewpoint of efficiently producing a large amount of the dipeptide compound, and used as a bioreactor for producing the dipeptide compound. The support here is not particularly limited as long as it is insoluble in water, but preferred examples include porous bodies having a large number of pores of a size capable of supporting the transformant, such as glass beads made of phase-separated glass, sintered glass, pumice, or polyurethane foam.

<Kit>
The kit according to the present invention (hereinafter referred to as "the kit") is a kit having one or more selected from the group consisting of the enzyme agent described above, the polynucleotide described above, a recombinant vector containing the polynucleotide, and the transformant described above. The kit can be used as a kit for producing the first embodiment of the composition according to the present invention described below, or a kit for producing the third embodiment of the composition according to the present invention described below. In the case of the kit for producing the third embodiment of the composition according to the present invention, it is preferable that the kit further has a culture solution containing a nitrogen source capable of nitrogen assimilation by the transformant, from the viewpoint of facilitating the production of the third embodiment of the composition according to the present invention. Alternatively, in the case where the kit has one or more selected from the group consisting of the polynucleotide described above, a recombinant vector containing the polynucleotide, and the transformant, it is preferable from the viewpoint of being usable as a kit for producing the enzyme agent.

First embodiment of the composition
The first embodiment of the composition according to the present invention (hereinafter referred to as "the composition 1") is a composition containing the dipeptide compound. For example, the composition 1 can be produced by using the enzyme preparation and an aqueous solution containing Arg and Asn. Alternatively, the composition 1 can be produced by using the enzyme preparation and an aqueous solution containing Arg and Gln. It is known that when a dipeptide is orally ingested, it is absorbed into the body together with hydrogen ions by the H ⁺ -dependent transport carriers of the brush border, and its absorption rate is faster than that of free amino acids. Therefore, it is presumed that when the composition 1 is orally ingested, the dipeptide compound is absorbed into the body and transferred into the blood, thereby exerting an ACE inhibitory effect. When the composition 1 is orally ingested continuously, it exerts an effect of suppressing blood pressure rise, probably due to the ACE inhibitory effect. Therefore, the composition 1 can be used as a composition for inhibiting ACE or suppressing blood pressure rise by oral ingestion.

The content of the dipeptide compound in the composition 1 is preferably 0.10 mg/L or more, more preferably 0.40 mg/L or more, and even more preferably 2.0 mg/L or more, from the viewpoint of easily exerting the ACE inhibitory effect even with a small amount of intake. The content of the dipeptide compound in the composition 1 is preferably 10 mg/L or more, more preferably 30 mg/L or more, and even more preferably 60 mg/L or more, from the viewpoint of easily exerting the ACE inhibitory effect even with a small amount of intake. From the viewpoint of keeping the cost required for purification low, the content of the dipeptide compound in the composition 1 is preferably 500 g/L or less, more preferably 100 g/L or less, and even more preferably 10 g/L or less. In this specification, "content" means the total content when two or more kinds of the corresponding compound are contained. "g" is mass grams.

As a method for measuring the content of the dipeptide compound, a sample purified so as to be substantially free of lipids and proteins is subjected to liquid chromatography mass spectrometry (hereinafter referred to as "LC/MS") or liquid chromatography mass spectrometry using a tandem quadrupole mass spectrometer (hereinafter referred to as "LC/MS/MS"), and the area of the peak detected due to the dipeptide compound contained in the sample is measured. The peak area in the sample is compared with the area of the peak detected by similarly analyzing a sample of the dipeptide compound, and the content of the dipeptide compound in the composition according to the present invention that is the basis of the sample can be calculated taking into account the dilution ratio performed for purification and analysis. Examples of this measurement method are described in Experimental Examples 1 to 3 described later. As a result of such measurement, for example, if the content of the dipeptide compound in the composition according to the present invention that is the basis of the sample is less than 0.10 mg/L, it is preferable to increase the content of the dipeptide compound by concentrating or purifying the composition according to the present invention so that the content is 0.10 mg/L or more, in order to facilitate the exertion of ACE inhibitory action, etc.

The dipeptide compound does not have as strong an ACE inhibitory activity as ACE inhibitors such as perindopril. When an ACE inhibitor is orally taken, blood pressure can drop dramatically in a short time, whereas when the dipeptide compound is orally taken continuously, blood pressure rise can be gradually suppressed or blood pressure can be gradually lowered. For this reason, it is considered that side effects that may occur when an ACE inhibitor is orally taken will hardly occur and will not be a problem when the dipeptide compound is orally taken continuously. For this reason, the composition 1 containing the dipeptide compound is preferably a food composition for ACE inhibition or blood pressure rise suppression. The food composition is, for example, one or more compositions selected from the group consisting of processed foods, seasonings, food additives, supplements, and food materials blended therein. When the composition 1 is a food composition, it may be in a form mixed with one or more known food materials.

When the composition 1 is a processed food, it may take the form of, for example, pickles, dried foods, paste products, powders, canned foods, frozen foods, instant foods, dairy products, confectioneries, luxury items, or beverages, etc., containing the dipeptide compound. When the composition 1 is a beverage, it may take the form of, for example, water, juice, tea, coffee, soft drinks, or alcoholic beverages, etc., containing the dipeptide compound. When the composition 1 is a supplement, it may take the form of, for example, a composition in which the dipeptide compound is mixed with one or more known pharmacologically acceptable additives, and may take the form of a powder, powder, granules, tablets, tablets, pills, capsules, or chewable agents. Examples of known additives include excipients, sweeteners, antioxidants, demulcents, lubricants, diluents, buffers, flavorings, or coloring agents. When the composition 1 is a food additive, it may be, for example, a dry powder used by mixing with one or more foods or their raw materials to obtain a food composition for ACE inhibition or blood pressure elevation suppression. For example, the present composition 1 may be in the form of a dry powder contained in a sachet attached to a food product, and when the consumer opens the sachet and sprinkles the dry powder on the food, the ACE inhibitory effect is exerted in the body of the consumer who eats the food.

From the viewpoint of being able to gently suppress blood pressure rise without side effects by continuous oral intake, the present composition 1 is more preferably a food composition for preventing or improving hypertension. Hypertension is a state in which blood pressure is maintained high above the normal range. A human can be diagnosed with hypertension when his/her systolic blood pressure (hereinafter referred to as "SBP") is 140 mmHg or more and/or his/her diastolic blood pressure (hereinafter referred to as "DBP") is 90 mmHg or more. When the present composition 1 is a food composition for preventing or improving hypertension, it can be distributed on the market as one or more types of health food selected from the group consisting of foods for specified health uses, foods with nutritional functions, foods for the sick, and foods for the elderly. From the viewpoint of easy understanding for consumers, it is preferable that the container or packaging material of this health food is labeled with a label explaining the function that is exerted due to the ACE inhibitory effect by continuous oral intake. The wording of the label is not particularly limited, but examples thereof include "lowering blood pressure", "expecting blood pressure reduction", "suppressing blood pressure rise", "slowing blood pressure rise", "preventing hypertension", or "helping to improve hypertension". From the same viewpoint, the present composition 1 is also preferably a food composition for preventing diseases caused by hypertension. Diseases caused by hypertension are not particularly limited, but examples thereof include cerebral hemorrhage, cerebral infarction, aortic aneurysm, nephrosclerosis, myocardial infarction, cardiac hypertrophy, or ocular fundus hemorrhage. When the present composition 1 is a food composition for preventing diseases caused by hypertension, it may also be distributed on the market as a health food, and in this case, it is preferable that a label explaining the function exhibited due to the ACE inhibitory action, such as "protecting renal function", is attached to the container or packaging material.

From the viewpoint of being less susceptible to contamination with impurities and thus making it easier to keep the purification costs of the dipeptide compound low, it is preferable that composition 1 is a composition produced by the first embodiment of the method for producing a composition according to the present invention, which is described below.

<First embodiment of the method for producing the composition>
In the following description of a first embodiment of the method for producing a composition according to the present invention (hereinafter referred to as "Present Method 1"), there are many commonalities with the above-mentioned present enzyme preparation and present composition 1, and therefore the description of the commonalities will be largely omitted and differences will be mainly described. As shown in Fig. 1, Present Method 1 includes a preparation step S11, an enzyme reaction step S12, and a drying step S14, and is a method capable of producing the above-mentioned present composition 1.

In the preparation step S11, the enzyme preparation described above is prepared. For example, in the preparation step S11, the enzyme preparation is prepared, which has an amino acid sequence of one polypeptide selected from the group consisting of the following (a), (b), and (c): (a) a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence described in SEQ ID NO: 1; (b) a polypeptide consisting of an amino acid sequence in which one or more and 20 or less amino acid residues are substituted, inserted, deleted, and/or added to the amino acid sequence described in SEQ ID NO: 1; and contains a dipeptide synthesizing enzyme having one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity. It is desirable that the enzyme preparation prepared in the preparation step S11 satisfies the preferable items described above for the enzyme preparation.

In the preparation step S11, it is preferable to further prepare a substrate for the dipeptide synthesizing enzyme or to prepare a substrate solution containing the substrate. The substrate for the dipeptide synthesizing enzyme is one or more compounds selected from the group consisting of Arg and its salts (hereinafter also referred to as the "first substrate") and one or more compounds selected from the group consisting of Asn, Gln, and their salts (hereinafter also referred to as the "second substrate"). From the viewpoint of promoting the enzyme reaction of the chemical reaction formula described above in the description of this enzyme, it is preferable to further prepare one or more selected from the group consisting of magnesium salts, ATP, and solutions containing these. From the viewpoint of avoiding the second substrate being deamidated and converted to Asp or Glu before the enzyme reaction, it is also preferable to further prepare a chelating agent or a pH buffer.

In the enzyme reaction step S12, in the presence of the first substrate, the second substrate, and the enzyme agent, the dipeptide synthesizing enzyme contained in the enzyme agent is allowed to undergo an enzymatic reaction to produce the dipeptide compound. In order to carry out the enzymatic reaction, the solution containing the first substrate, the second substrate, and the enzyme agent is kept at a temperature and pH suitable for the enzymatic reaction. In preparing this solution, the order in which the substrate, the enzyme agent, and the solvent are mixed is not particularly limited. For example, water may be added after mixing the first substrate, the second substrate, and the enzyme agent, the enzyme agent may be added to the substrate solution containing the first substrate and the second substrate, or the enzyme agent may be added to the substrate solution containing the first substrate and then the second substrate may be added. In the enzyme reaction step S12, from the viewpoint of promoting the enzymatic reaction of the chemical reaction formula described above in the description of the enzyme agent, it is preferable to further carry out the enzymatic reaction of the dipeptide synthesizing enzyme in the presence of one or more selected from the group consisting of magnesium salt, ATP, pH buffer, and chelating agent. The order in which the magnesium salt, ATP, pH buffer, and/or chelating agent are mixed is not particularly limited.

In the drying step S14, the solution containing the dipeptide compound produced in the previous enzyme reaction step S12 is dried to obtain the present composition 1. As described above in the description of the present composition 1, in the drying step S14, it is preferable to dry the solution so that the content of the present dipeptide compound is, for example, 0.10 mg/L or more. In the drying step S14, the solution containing the present dipeptide compound may be heated and dried, but from the viewpoint of avoiding undesirable heat denaturation, it is preferable to freeze-dry the solution. When producing the present composition 1 in the form of a processed food, it is preferable to further mix the dried product obtained in the drying step S14 with a known food material or the like.

<Second embodiment of the composition>
In the following, in describing a second embodiment of the composition according to the present invention (hereinafter referred to as "Composition 2"), there are many commonalities with Composition 1 described above, and the description of the commonalities will be omitted and differences will be mainly described. Composition 2 is a composition consisting of a fraction of water-soluble components containing a peptide having a molecular mass of less than 4.0 kDa derived from non-genetically modified Euglena cells, or a composition comprising said fraction. From the viewpoint of removing impurities and relatively increasing the content of the dipeptide compound, Composition 2 does not contain lipids derived from Euglena cells, proteins derived from said cells having a molecular mass of 4.0 kDa or more, and polypeptides derived from said cells having a molecular mass of 4.0 kDa or more. Since the dipeptide compound is water-soluble, the peptides derived from said cells having a molecular mass of less than 4.0 kDa in the fraction of water-soluble components derived from said cells contain the dipeptide compound. For this reason, Composition 2 can be used as a composition for inhibiting ACE or suppressing blood pressure elevation by oral ingestion.

The Euglena cells used as the raw material for Composition 2 are preferably cultured and grown in the presence of a nitrogen source that Euglena cells can assimilate, from the viewpoint of easily accumulating a large amount of the dipeptide compound within the cells. Since Euglena cells do not have nitrite reductase, nitrate reductase, or urease, they cannot assimilate nitrite nitrogen (NO ₂ ^- ), nitrate nitrogen (NO ₃ ^- ), or urea (CO(NH ₂ ) ₂ ). On the other hand, examples of nitrogen sources that Euglena cells can assimilate include one or more compounds selected from the group consisting of ammonia nitrogen, free amino acids, peptides, and salts thereof. Ammonia nitrogen is a nitrogen atom of NH ₃ or NH ₄ ⁺ contained in one or more compounds selected from the group consisting of ammonia and ammonium salts. For example, 1.0 mol equivalent of hexaammonium heptamolybdate tetrahydrate ((NH ₄ ) ₆ Mo ₇ O _{24.4H 2} O) corresponds to 6.0 mol equivalent of ammonia nitrogen. Examples of ammonium salts include ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium carbonate, and ammonium chloride. From the viewpoint of facilitating the production of the dipeptide compound by Euglena cells, a preferred nitrogen source is a combination of a compound selected from the group consisting of Arg and salts thereof and a compound selected from the group consisting of Asn and salts thereof. From the same viewpoint, a preferred nitrogen source is a combination of a compound selected from the group consisting of Arg and salts thereof and a compound selected from the group consisting of Gln and salts thereof. From the same viewpoint, a more preferred nitrogen source is a combination of a compound selected from the group consisting of Arg and salts thereof and a compound selected from the group consisting of Asn and salts thereof and a compound selected from the group consisting of Gln and salts thereof.

When Euglena cells grown in a medium are used as the raw material in the present composition 2, the Euglena cells may be dried by heating after growth, but from the viewpoint of avoiding undesirable heat denaturation, it is preferable that the Euglena cells are freeze-dried and dried after growth. The content of the dipeptide compound is relatively increased by the reduction in moisture, which further facilitates the exertion of the ACE inhibitory effect. If the intracellular fluid is reduced or the cell membrane is destroyed by drying, the metabolism in the Euglena cells is suppressed, making the dipeptide compound less likely to be decomposed, and it is considered that the present composition 2 can be easily stored for a long time. Alternatively, from the viewpoint of further avoiding metabolic decomposition and facilitating long-term storage, it is preferable that the Euglena cells grown in a medium in the present composition 2 are cells (cell disruption) that have been dried and crushed after growth. Alternatively, from the viewpoint of further relatively increasing the content of the dipeptide compound, it is preferable that the Euglena cells are degreased, for example, with a non-polar solvent after growth or crushing.

Lipids are substances derived from living organisms and soluble in non-polar solvents. Examples of lipids derived from Euglena cells include n-3 PUFAs. In the present composition 2, lipids derived from Euglena cells are substantially removed as impurities in order to relatively increase the content of the present dipeptide compound. For this reason, the present composition 2 does not contain lipids derived from Euglena cells. In this specification, "does not contain" means that even if a trace amount of a component to be removed remains due to the accuracy of the separation operation, as long as the remaining amount is not to the extent that it does not interfere with the effects of the present invention, it is acceptable because it does not cause a problem in the content or essence of the present invention. In this specification, the mass ratio of the "content (remaining amount) of the component to be removed" to the "content of the present dipeptide compound" (content (remaining amount) of the component to be removed/content of the present dipeptide compound) may be, for example, less than 0.050, preferably less than 0.010, and more preferably less than 0.0010.

For example, Euglena cells may be separated into an oil phase and an aqueous phase, and the aqueous phase fraction may be subjected to a molecular sieve to obtain a fraction of water-soluble components that does not contain lipids derived from Euglena cells, or proteins or polypeptides with a molecular mass of 4.0 kDa or more, and that contains peptides derived from Euglena cells and have a molecular mass of less than 4.0 kDa. This fraction may be used as composition 2. In other words, composition 2 may be a composition essentially consisting of an aqueous Euglena extract that does not contain lipids derived from Euglena cells, or proteins or polypeptides with a molecular mass of 4.0 kDa or more, and a pharmacologically acceptable hydrophilic solvent. An example of the molecular sieve used here is gel filtration chromatography. From the viewpoint of relatively increasing the content of the dipeptide compound, composition 2 is preferably a concentrate obtained by concentrating a fraction of water-soluble components containing a peptide derived from Euglena cells and having a molecular mass of less than 4.0 kDa and substantially removing the hydrophilic solvent, and more preferably a purified product obtained by purifying this concentrate so as to further increase the content of the dipeptide compound. Composition 2 is also preferably a food composition in which the fraction, concentrate, or purified product of water-soluble components containing a peptide derived from Euglena cells and having a molecular mass of less than 4.0 kDa listed above is mixed with one or more known pharmacologically acceptable additives or one or more known food materials.

From the viewpoint of removing impurities and relatively increasing the content of the dipeptide compound, the above-mentioned purified product is preferably a composition that does not contain proteins, polypeptides, or oligopeptides derived from Euglena cells and has a molecular weight of 1,000 or more, for example, by being purified with a molecular sieve such as gel filtration chromatography, and is even more preferably a composition that does not contain proteins, polypeptides, or oligopeptides derived from Euglena cells and has a molecular weight of 500 or more. Furthermore, from the viewpoint of relatively increasing the content of the dipeptide compound having a stronger ACE inhibitory activity than Arg, the present composition 2 is preferably a composition that is purified so as not to contain free amino acids derived from Euglena cells, such as Asn, Asp, Gln, Glu, Arg, L-methionine, L-cysteine, L-homocysteine, and taurine, and salts of these free amino acids, and is even more preferably a composition from which free amino acids and their salts have been substantially removed, including but not limited to these examples of free amino acids.

In addition, the preferred matters regarding the content of the dipeptide compound, the method for measuring it, applications, the form that the food composition can take, or the labeling, etc., of Composition 2 are the same as those described above for Composition 1. From the viewpoint of efficient production, Composition 2 is preferably produced by the second embodiment of the production method of the composition according to the present invention, which will be described next.

<Second embodiment of the method for producing the composition>
In the following, in describing a second embodiment of the method for producing a composition according to the present invention (hereinafter referred to as "Present Production Method 2"), there are many common points with the above-mentioned Present Composition 2, and therefore the description of the common points will be omitted and differences will be mainly described. As shown in Figure 2, Present Production Method 2 includes a preparation step S21, a culture step S22, a collection step S23, a drying step S24, a crushing step S25, a deproteinization step S26, a lipid removal step S27, a concentration step S28, and a purification step S29, and is a method capable of producing Present Composition 2.

In the preparation step S21, live Euglena cells are prepared. For example, wild Euglena can be collected from sunny puddles outdoors, but it is more efficient to obtain experimental Euglena cell lines from a research institute.

In the culture step S22, living Euglena cells are cultured and grown in the presence of a nitrogen source that Euglena cells can assimilate, and the Euglena cells are allowed to produce the dipeptide compound while increasing the cell number. The medium may be a solid medium such as an agar slant medium, which is easy to deal with contamination, but a liquid medium is preferable because it is inexpensive, easy to prepare, easy to stir, and easy to culture Euglena cells at a high density. Examples of liquid media that have been used conventionally for Euglena culture include Kramer-Meyer medium (hereinafter referred to as "CM medium"; see Non-Patent Document 5) shown in Table 1, Huttner medium (see Non-Patent Document 6) shown in Table 2, and Koren-Huttner medium (hereinafter referred to as "KH medium"; see Non-Patent Document 7) shown in Table 3.

The medium used in the culture step S22 may have a composition similar to that shown in Tables 1 to 3. As long as it does not go against the object of the present invention, it is preferable that the medium contains a chelating agent or a pH adjuster. For example, EDTA-Na ₂ (ethylenediaminetetraacetic acid disodium salt) shown in Tables 1 to 3 acts as a chelating agent. Phosphates and citric acid act as pH adjusters. Nitrogen sources that can be assimilated by Euglena cells are, for example, one or more compounds selected from the group consisting of ammonia, ammonium salts, free amino acids, peptides, and salts thereof. For this reason, the medium may contain an ammonium salt, a composition containing a peptide, or ammonia water. Examples of compositions containing peptides include peptone, casamino acid, yeast extract, and corn steep liquor.

In the culture step S22, from the viewpoint of facilitating the growth of Euglena cells so as to contain a large amount of the dipeptide compound, the content of ammonia nitrogen in the medium is preferably 38 mmol/L or more. From the same viewpoint, the content of one or more compounds selected from the group consisting of Glu and its salts (hereinafter referred to as "Glu compounds") in the medium is preferably 3.4 mmol/L or more, and the content of ammonia nitrogen is preferably 13 mmol/L or more. These media can be prepared, for example, by further adding a Glu compound or an ammonium salt to any of the compositions in Tables 1 to 3. From the same viewpoint and from the viewpoint of efficiently increasing the number of cells, the content of the Glu compound in a medium having an ammonia nitrogen content of 14 mmol/L or more is preferably 24 mmol/L or more, more preferably 40 mmol/L or more. From the viewpoint of avoiding an excessive content that would hinder the increase in the number of cells, the content of ammonia nitrogen in the medium is preferably 1.0 mol/L or less, more preferably 100 mmol/L or less. Similarly, from the viewpoint of avoiding inhibition of the increase in cell number, the content of the Glu compound in the medium is preferably 1.0 mol/L or less, more preferably 100 mmol/L or less.

In the culture step S22, Euglena cells can be grown by a culture method that has been used conventionally to increase the number of Euglena cells. For example, the culture vessel may be covered with a thick black cloth, and the culture medium may be kept in the dark to grow Euglena cells. Alternatively, Euglena cells may be grown by providing a light-dark cycle that includes a light period in which the culture medium is irradiated with light and a dark period in which the culture medium is kept in the dark. In the culture step S22, from the viewpoint of growing Euglena cells with nutrients obtained by photosynthesis and efficiently increasing the number of cells, it is preferable to grow Euglena cells by continuously irradiating the culture medium with light. The culture temperature is, for example, 20°C or higher and lower than 34°C, and from the viewpoint of good growth, it is preferable to be 28°C or higher and 30°C or lower. From the same viewpoint, the intensity of light irradiated to the culture medium is preferably 2,000 lux or higher and 8,000 lux or lower. From a similar viewpoint, for example, it is preferable to culture the medium while stirring it with a stirrer, and it is also preferable to culture the medium while shaking it with a shaker at 80 to 120 times per minute. From a similar viewpoint, it is preferable to aerate the medium with air that has passed through a sterilizing filter, or air containing 1% to 5% by mass of carbon dioxide. The initial pH of the medium is, for example, 2.0 to 7.0, and from the viewpoint of efficiently increasing the number of cells, it is preferably 3.0 to 5.0. A small amount of dilute sulfuric acid may be added to the medium to adjust the initial pH. For example, when Euglena cells are grown under these preferable culture conditions by continuously irradiating the KH medium with light, the logarithmic growth phase may be reached in 2 to 3 days from the start of the culture, and the stationary phase may be reached in 4 to 5 days.

Compared with the enzyme reaction step S12 (FIG. 1) described above in the description of the present production method 1, the culture step S22 of the present production method 2 shown in FIG. 2 has the advantage that Arg or its salt is not essential as a medium composition because Euglena cells can metabolize ammonium salts to produce Arg (see Non-Patent Documents 1 and 2). Asn has a problem of being easily deamidated and converted to Asp, and Gln has a similar problem of being easily converted to Glu. On the other hand, Euglena cells can produce Asn from Asp and Gln from Glu, respectively. Therefore, the culture step S22 has the advantage that Asn, Gln, or their salts are not essential as a medium composition. In addition, in the culture step S22, Euglena cells act as a natural concentrator that takes in a nitrogen source and increases the density of nitrogen compounds in the cells, so that the present composition 2 can be produced even if the concentration of the nitrogen source contained in the medium is low. In the culture step S22, even industrial waste generated as a by-product of, for example, food production, can be used as a nitrogen source if it contains one or more nitrogen compounds selected from the group consisting of ammonium salts, amino acids, peptides, and their salts. This is preferable from the viewpoint of facilitating recycling of industrial waste and reducing the production cost of the present composition 2.

In the collection step S23, Euglena cells grown in the medium are collected after the end of the culture period to obtain a collection product with an increased cell density, in order to increase the processing efficiency in the subsequent drying step S24. For example, the medium may be heated and concentrated to increase the cell density in the medium, but from the viewpoint of avoiding undesirable heat denaturation and efficiently collecting the cells in a short time, it is preferable to centrifuge the medium and collect the precipitate (cell pellet) that forms. For example, 300 mL of the medium is dispensed into tubes, and a centrifugal force of about 2,000×G is applied at about 4° C. to collect the pellet that forms at the bottom of the tube. "G" is the standard gravitational acceleration (9.80665 m/s ² ).

In the drying step S24, the cell collection (e.g., concentrated medium or cell pellet) is dried to obtain a dried material containing dried Euglena cells in order to increase the processing efficiency in the subsequent steps (S25 to S27, etc.) and to avoid metabolic decomposition of the dipeptide compound within the Euglena cells. The collection may be dried by heating, but it is preferable to freeze-dry the collection in order to avoid undesirable heat denaturation.

In the crushing step S25, the Euglena cells contained in the dried product are crushed to obtain a crushed product containing crushed Euglena cells, in order to facilitate the removal of lipids and the like in the subsequent steps (S26, S27, etc.). For this purpose, a known cell crushing method for the purpose of extracting proteins from cells can be used. For example, a cell crushing method that combines one or more of the following: osmotic shock method, enzyme digestion method, ultrasonic treatment, French press, crushing with a pestle, crushing with a homogenizer, and crushing with glass beads can be used. From the viewpoint of avoiding undesirable denaturation during crushing, it is preferable to subject the cell suspension obtained by suspending the dried product in a small amount of buffer to the crushing treatment. The osmotic shock method is a method in which the dried product is suspended in a hypotonic solution such as sterile water to rupture the cells. The enzyme digestion method is a method in which various enzymes are added to the cell suspension to digest the cells. However, it is preferable to avoid adding dipeptidase that can decompose the present dipeptide compound. Sonication is a method of disrupting cells by the shear force of ultrasound. French press is a method of forcibly pushing a cell suspension through a small hole under high pressure to disrupt cells by shear force. Pestle grinding is a method of grinding cells in a mortar with a pestle. Homogenizer grinding is a method of homogenizing a cell suspension with a homogenizer, filtering or centrifuging the resulting lysate to remove insoluble matter, and collecting the supernatant. Glass bead grinding is a method of adding glass beads to a cell suspension, repeatedly stirring with a vortex mixer while cooling, filtering or centrifuging the resulting lysate to remove insoluble matter, and collecting the supernatant.

In the deproteinization step S26, the crushed product is subjected to a deproteinization treatment to remove impurities and relatively increase the content of the dipeptide compound to obtain a deproteinized product. For this purpose, a known deproteinization method that has been conventionally used for the purpose of removing proteins from the crushed product can be used. For example, one or a combination of two or more methods selected from the group consisting of protein denaturation precipitation, a method of separating and removing proteins by liquid chromatography, and ultrafiltration can be used. Polypeptides and proteins with larger molecular weights are more easily removed, and the dipeptide compound with smaller molecular weights remains in the deproteinized product.

When performing the protein denaturation precipitation method in the deproteinization step S26, the crushed product is mixed with a precipitant, the protein is precipitated by centrifugation, and the aqueous layer is collected so as not to be contaminated with the precipitate (protein) and treated as the deproteinized product. For example, it is preferable to mix 1.0 part by mass of the crushed product with 0.2 parts by mass or more and 4 parts by mass or less of a precipitant, stir the mixture, leave it in a cool place for 15 minutes or more to precipitate the protein, apply a centrifugal force of about 20,000×G for about 15 minutes to precipitate the protein, and collect the aqueous layer (deproteinized product) so as not to be contaminated with the precipitate (protein). Examples of precipitants include ethanol, methanol, acetone, acetonitrile, trichloroacetic acid, perchloric acid, or a mixture thereof. When performing ultrafiltration in the deproteinization step S26, the crushed product is passed through an ultrafiltration membrane with an ultramolecular weight of 30,000 or less, and the filtrate that has passed through the membrane is collected and treated as the deproteinized product. An example of an ultrafiltration membrane is an Amicon Ultra centrifugal ultrafiltration filter (manufactured by Merck, Amicon is a registered trademark). Proteins and polypeptides with large molecular weights cannot pass through the membrane and are removed. When separating proteins by liquid chromatography (e.g., gel filtration chromatography, ion exchange chromatography, etc.) in the deproteinization step S26, for example, as a preliminary experiment, standard samples of amino acids, dipeptides, tripeptides, polypeptides, and proteins are passed through a column, and the retention time of each standard is recorded by a chromatogram. Then, the column is washed, the crushed product is passed through the washed column, and the fractions with retention times containing amino acids, dipeptides, and tripeptides are collected and treated as deproteinized products. If fractions containing polypeptides and proteins are not collected, the polypeptides and proteins are removed.

In the lipid removal step S27, lipids are removed from the deproteinized product to remove impurities and relatively increase the content of the dipeptide compound, thereby obtaining an aqueous Euglena extract. For example, the deproteinized product may be centrifuged or left to stand in a cool, dark place to separate the components according to their specific gravity, and the lipid layer that forms may be removed to obtain an aqueous Euglena extract. Alternatively, the deproteinized product may be passed through a separation column for liquid chromatography (e.g., reverse phase chromatography), and the fraction containing the water-soluble components that have passed through the column may be collected and treated as an aqueous Euglena extract.

In the lipid removal step S27, it is preferable to mix the deproteinized product with a polar solvent from the viewpoint of easily and quickly removing lipids. The polar solvent may be, for example, methanol, but from the viewpoint of efficiently extracting water-soluble components while avoiding the inclusion of lipids, a solvent having a dielectric constant of 35 or more at 20°C is preferable, for example, acetonitrile, formic acid, water, or a mixture thereof. When the deproteinized product is mixed with a polar solvent, lipids remain in the extractive layer (oil layer) and precipitate, and water-soluble components are extracted into the polar solvent (for example, water), so that the layer of the polar solvent (for example, the water layer) formed after mixing can be collected and treated as an aqueous Euglena extract. Alternatively, from a similar viewpoint, it is also preferable to mix the deproteinized product with a nonpolar solvent. The nonpolar solvent may be, for example, ethyl acetate, chloroform, or a mixture thereof. From a similar viewpoint, the nonpolar solvent is preferably a solvent having a dielectric constant of 4.5 or less at 20°C, for example, hexane, benzene, toluene, diethyl ether, or a mixture thereof. When the deproteinized product is mixed with a nonpolar solvent, lipids are extracted into the nonpolar solvent, and water-soluble components remain in the raffinate layer (aqueous layer) and the precipitate, so that the raffinate layer (aqueous layer) formed after mixing can be collected and treated as an aqueous Euglena extract. From a similar viewpoint, it is also preferable to mix the deproteinized product with a polar solvent and a nonpolar solvent and collect the polar solvent layer (e.g., aqueous layer) formed. From a similar viewpoint, when a polar solvent or a nonpolar solvent is used in the lipid removal step S27, it is preferable to mix the deproteinized product with the solvent after drying, and more preferably to mix the deproteinized product with the solvent after freeze-drying. When collecting the polar solvent layer (e.g., aqueous layer), it is preferable to avoid the interface with the nonpolar solvent layer (oil layer) and the area close to the precipitate, from the viewpoint of being able to remove amphipathic components (e.g., phospholipids, glycolipids) at the interface and insoluble components in the precipitate.

In the concentration step S28, the Euglena aqueous extract is concentrated to obtain a concentrate in order to remove the solvent (e.g., water) and relatively increase the content of the dipeptide compound. For this purpose, the Euglena aqueous extract may be dried by heating, but in order to avoid undesirable heat denaturation, it is preferable to obtain the concentrate by freeze-drying, and it is also preferable to obtain the concentrate by centrifugal concentration using a centrifugal concentrator (centrifugal evaporator).

In the purification step S29, the concentrate is purified to remove impurities and relatively increase the content of the dipeptide compound to obtain a purified product. For this purpose, it is preferable to remove lipids derived from Euglena cells and proteins and polypeptides with a molecular mass of 4.0 kDa or more by one or a combination of two or more selected from the group consisting of molecular sieving (e.g., ultrafiltration or gel filtration chromatography), ion exchange chromatography, and adsorption chromatography. When free amino acids, oligopeptides, polypeptides, proteins, and salts thereof are removed from the concentrate, it is preferable to pass the concentrate through a gel filtration chromatography column to fractionate at certain retention times, and collect fractions with retention times containing dipeptides or tripeptides and treat them as a purified product.

In the present production method 2 described above, the combination of the preparation step S21, the culture step S22, the collection step S23, the drying step S24, and the crushing step S25 functions as a step of preparing a crushed product in which Euglena cells grown in a culture medium and dried are crushed. The combination of the deproteinization step S26, the lipid removal step S27, and the purification step S29 functions as a step of removing lipids derived from Euglena cells and proteins and polypeptides with a molecular mass of 4.0 kDa or more from the crushed product to obtain a fraction of water-soluble components derived from Euglena cells and with a molecular mass of less than 4.0 kDa. The purified product obtained in the purification step S29 can be treated as the present composition 2.

<Third embodiment of the composition>
In the following, in describing the third embodiment of the composition according to the present invention (hereinafter referred to as "Composition 3"), there are many common points between the above-mentioned transformant, Composition 1, Production Method 1, Composition 2, and Production Method 2, and the common points will be omitted and the differences will be mainly described. Composition 3 is a composition containing the dipeptide compound by containing one or more selected from the group consisting of the transformant, the transformant cultured in a medium containing a nitrogen source that the transformant can assimilate, a dried product of the transformant, and a cell lysate of the transformant. Composition 3 contains the dipeptide compound produced by the transformant, and can be used as a composition for inhibiting ACE or suppressing hypertension by oral ingestion. Examples of the present transformant include a present polynucleotide having a base sequence encoding a dipeptide synthetase, the base sequence encoding the dipeptide synthetase being a group consisting of the following (d), (e), (f), and (g): (d) a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 2; (e) a polynucleotide consisting of a base sequence having 90% or more identity to the base sequence set forth in SEQ ID NO: 2; (f) a polynucleotide consisting of a base sequence in which 1 or more and 20 or less bases have been substituted, inserted, deleted, and/or added to the base sequence set forth in SEQ ID NO: 2; (g) a polynucleotide consisting of the sequence a polynucleotide which hybridizes under stringent conditions with a DNA having a complementary base sequence to the base sequence shown in No. 2, and the base sequence encoding the dipeptide synthetase is a base sequence encoding an enzyme having one or more enzymatic activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity, or a recombinant vector containing the polynucleotide. The preferred features of the transformant are as described above.

From the viewpoint of a high content of the dipeptide compound, the transformant used as a raw material in the composition 3 is preferably cultured in the presence of a nitrogen source capable of nitrogen assimilation by the transformant. The nitrogen source capable of nitrogen assimilation varies depending on the type of host from which the transformant originates, and is prepared appropriately according to the type of the original host. For example, if the transformant is derived from a microorganism having nitrite reductase, nitrate reductase, or urease, unlike Euglena, nitrite nitrogen, nitrate nitrogen, or urea can be used as a nitrogen source. From the viewpoint of keeping raw material costs low, the nitrogen source is preferably inorganic nitrogen capable of nitrogen assimilation by the transformant, but from the viewpoint of being capable of nitrogen assimilation regardless of the type of host from which the transformant originates, it is preferable that the nitrogen source is one or more compounds selected from the group consisting of free amino acids, polypeptides, and salts thereof. In addition to the same viewpoint, from the viewpoint of facilitating the production of the dipeptide compound, it is more preferable that the nitrogen source contains one or more compounds selected from the group consisting of Arg and its salts, and one or more compounds selected from the group consisting of Asn, Gln, and their salts.

The method for drying the transformant in the present composition 3 may be, for example, heat drying, but freeze drying is preferred from the viewpoint of avoiding damage to various components useful for maintaining health, such as the present dipeptide compound. In the present composition 3, the treated product in which the present transformant has been subjected to at least a treatment including drying may also be a treated product that has been subjected to a treatment other than drying, so long as the dipeptide compound is not substantially denatured. Examples of treatment other than drying include one or more treatments selected from the group consisting of heating, freezing, pressurization, decompression, cell disruption, and radiation exposure. The treated product may contain the present enzyme or its denatured protein derived from the present transformant. For example, the treated product may be a treated product in which the present transformant or its dried product has not been subjected to deproteinization or purification.

In the present composition 3, the preferred matters regarding the content of the present dipeptide compound, the method for measuring the content, the use, the form that it can take as a food composition, the labeling, the concentration, the purification, and Euglena, etc. are the same as those described above for the present composition 1 and the present composition 2. From the viewpoint of efficient production, the present composition 3 is preferably produced by the third embodiment of the production method of the composition according to the present invention, which will be described next.

<Third embodiment of the method for producing the composition>
In explaining the third embodiment of the method for producing a composition according to the present invention (hereinafter referred to as "Present Method 3"), there are many common points among the above-mentioned Present Method 1, Present Method 2, and Present Composition 3, and explanations of the common points will be omitted and differences will be mainly explained. As shown in FIG. 3, Present Method 3 includes a preparation step S31 and a culture step S32, and is a method capable of producing Present Composition 3 using the Present Transformant. As the Present Transformant, for example, there is a Present Polynucleotide having a base sequence encoding a dipeptide synthetase, the base sequence encoding the dipeptide synthetase being a group consisting of the following (d), (e), (f), and (g): (d) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2; (e) a polynucleotide consisting of a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 2; (f) a polynucleotide consisting of a base sequence in which 1 or more and 20 or less bases are substituted, inserted, deleted, and/or added to the base sequence shown in SEQ ID NO: 2; (g) a polynucleotide consisting of a base sequence having the sequence shown in SEQ ID NO: 2; a polynucleotide which hybridizes under stringent conditions with a DNA having a complementary base sequence to the base sequence shown in No. 2, and the base sequence encoding the dipeptide synthetase is a base sequence encoding an enzyme having one or more enzymatic activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity, or a recombinant vector containing the polynucleotide. The preferred features of the transformant are as described above.

In the preparation step S31, the present transformant and a medium containing a nitrogen source capable of nitrogen assimilation by the present transformant are prepared. Since the present transformant can be cultured under the same conditions as the original host, it is preferable to prepare a medium suitable for culturing the present transformant depending on the type of the original host. For example, when preparing the present transformant in which Euglena is transformed, it is preferable to prepare the CM medium (Table 1), Huttner medium (Table 2), KH medium (Table 3) described above in the explanation of the present method 2, or a medium with a partially modified composition of these media. For example, when preparing the present transformant in which yeast is transformed, a medium generally suitable for culturing yeast may be prepared, such as YNB (Yeast Nitrogen Base) medium, DOB medium in which a carbon source and ammonium sulfate are added to YNB medium, or SD medium in which various amino acids, adenine, etc. are added to DOB medium. Generally, the composition of SD medium contains Arg. From the viewpoint of facilitating the production of the present dipeptide compound, it is preferable that the nitrogen source in the medium contains one or more compounds selected from the group consisting of Arg and its salts, and one or more compounds selected from the group consisting of Asn, Gln, and their salts.

When the present composition 3 is produced in the form of soy sauce or miso containing the present dipeptide by the present production method 3, it is preferable to prepare the present transformant obtained by transforming koji mold and swollen soybeans and rice and/or wheat as a medium in the preparation step S31. When the present composition 3 is produced in the form of cheese, kimchi, or narezushi containing the present dipeptide compound, it is preferable to prepare the present transformant obtained by transforming lactic acid bacteria and milk, Chinese cabbage, or fish meat as a medium. When the present composition 3 is produced in the form of natto containing the present dipeptide compound, it is preferable to prepare the present transformant obtained by transforming natto bacteria and swollen soybeans as a medium. When the present composition 3 is produced in the form of vinegar containing the present dipeptide compound, it is preferable to prepare the present transformant obtained by transforming acetic acid bacteria and saccharified mash as a medium. When the present composition 3 is produced in the form of beer containing the present dipeptide compound, it is preferable to prepare the present transformant obtained by transforming brewer's yeast (e.g., Saccharomyces cerevisiae) and wort cooled to about 5°C as a medium. When producing the present composition 3 in the form of sake containing the present dipeptide compound, it is preferable to prepare the present transformant obtained by transforming Saccharomyces cerevisiae and mash as a medium.When producing the present composition 3 in the form of wine or cider containing the present dipeptide compound, it is preferable to prepare the present transformant obtained by transforming Saccharomyces cerevisiae and grape juice or apple juice as a medium.When producing the present composition 3 in the form of bread containing the present dipeptide compound, it is preferable to prepare the present transformant obtained by transforming Saccharomyces cerevisiae and sponge soybean paste as a medium.

In the culture step S32, the present transformant is cultured in the presence of a medium, and the present dipeptide compound is produced in the present transformant during the process of growing the present transformant and increasing the cell number. When the present transformant is prepared by transforming Euglena, it is preferable to carry out the culture step S32 shown in FIG. 3 in the same manner as described for the culture step S22 (FIG. 2) of the present production method 2 described above. When the present transformant is prepared by transforming a microorganism other than Euglena, the present transformant is cultured under culture conditions suitable for growing the original host and increasing the cell number, while avoiding the proliferation of miscellaneous bacteria. For example, when the present transformant is prepared by transforming Aspergillus oryzae, it is preferable to culture the present transformant in the presence of a medium (soybean, wheat, rice, etc.) at a temperature of 25° C. or higher and 35° C. or lower for several days while fermenting the medium. For example, when the present transformant is prepared by transforming Bacillus subtilis natto, it is preferable to culture the present transformant in the presence of a medium (swollen soybean) at a temperature of 38° C. or higher and 42° C. or lower for about one day while fermenting the medium. For example, when the present transformant is prepared by transforming beer yeast (Saccharomyces cerevisiae), it is preferable to culture the present transformant in the presence of a medium (wort) at a liquid temperature of about 0° C. or higher and about 6° C. or lower for 7 days or longer and not longer than several tens of days while fermenting the medium. For example, when the present composition 3 is produced in the form of wine containing the present dipeptide compound, it is preferable to culture the present transformant obtained by transforming Saccharomyces cerevisiae in the presence of a medium (grape juice) at a temperature of about 10° C. or higher and about 30° C. or lower.

In the present production method 3, the medium that has completed the culture period in the culture step S32 (hereinafter referred to as the "cultured medium") contains the present transformant that has grown and increased in cell number. This cultured medium itself may be treated as the present composition 3. Many of the present transformants contained in the cultured medium metabolize the nitrogen source during the culture period to generate the present dipeptide compound, which is contained within the cells or secreted outside the cells. For this reason, if the cultured medium is orally ingested, blood pressure rise is likely to be suppressed in the body due to the ACE inhibitory effect.

However, the above-mentioned cultured medium itself often has problems in terms of flavor and long-term storage stability for consumption. In order to solve this problem, although not shown, it is preferable to further process the cultured medium so that it is suitable for consumption, depending on the form of the food composition (composition 3) to be produced, as long as the dipeptide compound is not damaged. For example, when the composition 3 is produced as miso, it is preferable to mix salt water with the cultured medium (soybean koji) and pack it in a barrel for long-term aging, from the viewpoint of good flavor and storage stability as miso. For example, when the composition 3 is produced as natto, it is preferable to refrigerate the cultured medium (fermented soybeans) and ripen it, from the viewpoint of good flavor and storage stability as natto. For example, when the composition 3 is produced as vinegar or wine, it is preferable to ripen the cultured medium (fermented mash or grape juice) from the viewpoint of good flavor and storage stability as vinegar or wine. For example, when producing the present composition 3 as sake, it is preferable to squeeze the cultured medium (fermented mash) to separate the sake lees and to heat sterilize it to prevent spoilage, from the viewpoint of improving the flavor and preservation of the sake.

In addition, the cultured medium obtained in the culture step S32 has a high content of components derived from the medium, so that the content of the dipeptide compound produced is relatively low. In addition, when the transformant obtained by transforming Euglena is cultured as in the culture step S22 (FIG. 2) in the present production method 2 described above, the components derived from the medium are not suitable for consumption in terms of flavor, etc. In such a case, it is preferable to remove the components derived from the medium as impurities from the viewpoint of producing the present composition 3 which relatively increases the content of the dipeptide compound and is more likely to exert the ACE inhibitory effect. From this viewpoint, it is more preferable to subject the cultured medium to processing corresponding to one or more steps selected from the group consisting of the collection step S23, drying step S5, crushing step S25, deproteinization step S26, lipid removal step S27, concentration step S28, and purification step S29 shown in FIG. 2 described in the present production method 2 described above, and the more the number of processing steps listed here, the more preferable it is. From the same viewpoint, and also from the viewpoint of reducing purification costs, it is also preferable to concentrate or purify the culture medium so that the content of the present dipeptide compound is, for example, 0.10 mg/L or more and 500 g/L or less, and in this case, the more preferable content is the same as that described above in the explanation of the present composition 1.

On the other hand, depending on the type of the original host, the transformant may contain various functional components useful for maintaining health other than the dipeptide compound, such as paramylon contained in Euglena cells and resistant protein contained in yeast. In order to avoid the loss of such functional components, when further processing is performed on the cultured medium obtained in the culture step 32, it is preferable to avoid performing processing corresponding to one or more steps selected from the group consisting of the deproteinization step S26, the lipid removal step S27, and the purification step S29 shown in Figure 2. For example, it is also preferable to prepare the composition 3 so as to contain one or more dipeptide synthases including the enzyme described above or denatured proteins thereof by performing further processing on the cultured medium obtained in the culture step 32 while avoiding the deproteinization step S26.

<Other embodiments of the method for producing the composition>
The method for producing a composition according to the present invention may be an embodiment obtained by modifying Production Method 2 shown in FIG. 2 or Production Method 3 shown in FIG. 3 as exemplified below, so long as the method does not contradict the object of the present invention. When a large amount of Euglena cells or transformants of the present invention that already contain a large amount of the dipeptide compound can be prepared in the preparation step (S21 or S31), the culture step (S22 or S32) can be omitted from the viewpoint of simplifying the process. In this case, Euglena cells or transformants of the present invention that have already been dried, or Euglena cells or transformants of the present invention that have already been defatted, may be prepared, and the cells may be dead. When a large amount of a dried material or suspension containing Euglena cells or transformants of the present invention that already contain a large amount of the dipeptide compound in a high density can be prepared in the preparation step (S21 or S31), not only the culture step (S22 or S32) but also the collection step S23 and the drying step S24 can be omitted.

In the culture step (S22 or S32), if the water evaporates during culture and a medium with a high cell density is obtained, the collection step S23 may be omitted. In the present method 2, if there is no problem with the efficiency of the processing being deteriorated in each of the steps of the crushing step S25, the deproteinization step S26, the lipid removal step S27, and the concentration step S28, the collection step S23 and the drying step S24 may be omitted from the viewpoint of simplifying the process. If the amount of medium used in the culture step (S22 or S32) is small (e.g., 1.0 L or less), the collection step S23 and the drying step S24 can be performed together. The cultured medium may be dried by heating, but from the viewpoint of avoiding undesirable heat denaturation, it is preferable to freeze-dry the cultured medium, and it is also preferable to centrifugally concentrate the cultured medium.

In the crushing step S25, in order to increase the efficiency of the next deproteinization step S26, it is preferable to concentrate the crushed product before subjecting it to the deproteinization step S26. For this purpose, the crushed product is centrifuged to collect the lower layer formed, or the crushed product is freeze-dried, or the crushed product is centrifuged to concentrate it. From a similar perspective, in the deproteinization step S21, it is preferable to concentrate the deproteinized product and subject it to the next lipid removal step S27. Alternatively, in order to simplify the steps by combining the crushing step S25 and the lipid removal step S27, it is preferable to mix the dried material or cell suspension containing Euglena cells with a non-polar solvent and collect the resulting raffinate layer (aqueous layer). Euglena is a tiny single-celled organism, and when the cells are dried, some of the cell membrane is destroyed, so if the dried Euglena cells are mixed with a non-polar solvent, it is possible to remove lipids from the cells over time. In this case, it is preferable to subject the collected raffinate layer (aqueous layer) to a deproteinization treatment. Alternatively, from the viewpoint of simplifying the process by combining the deproteinization step S26 and the lipid removal step S27, it is also preferable to mix a disruption-treated product containing disrupted Euglena cells with a polar solvent that functions as a protein precipitant, and collect the resulting extraction layer (polar solvent layer or aqueous layer). Alternatively, from the viewpoint of further simplifying the process by combining the disruption step S25, the deproteinization step S26, and the lipid removal step S27, it is also preferable to mix a dried product or cell suspension containing Euglena cells with a polar solvent that functions as a protein precipitant, and collect the resulting extraction layer (polar solvent layer or aqueous layer). An example of a polar solvent that functions as a protein precipitant is an aqueous acetonitrile solution. Alternatively, lipids may be removed from a dried product containing dried Euglena cells, and the resulting aqueous solution may be subjected to a deproteinization treatment, and the resulting deproteinized product may be treated as an aqueous Euglena extract (the present composition 2).

When deproteinization is performed in the purification step S29, the deproteinization step S26 may be omitted. When the content of the dipeptide compound in the composition 2 is not a problem even if it is low, or when the content of a large amount of impurities other than the dipeptide compound is not a problem, one or more steps selected from the group consisting of the deproteinization step S26, the concentration step S27, and the purification step S29 shown in FIG. 2 may be omitted. In this case, the aqueous solution collected in the lipid removal step S27 can be treated as the Euglena aqueous extract (the composition 2). In the present production method 2, from the viewpoint of removing impurities and relatively increasing the content of the dipeptide compound, the collected Euglena cells may be mixed with a nonpolar solvent to obtain cells that have been somewhat defatted, and the cells may be subjected to the drying step S24. From the same viewpoint, a dried product containing dried Euglena cells may be mixed with a nonpolar solvent to obtain a composition containing cells that have been somewhat defatted.

When the present composition 1, the present composition 2, or the present composition 3 is produced as a processed food, it is preferable to further mix a known pharmacologically acceptable additive or a known food material in the present production method 1 shown in FIG. 1, the present production method 2 shown in FIG. 2, or the present production method 3 shown in FIG. 3.

The present invention may be implemented in a form or embodiment in which various improvements, modifications, or variations have been made based on the knowledge of a person skilled in the art without departing from the spirit of the present invention, and may be implemented in a form or embodiment in which any of the invention-specific features are replaced with other technologies within the scope of the same operational effect. The present invention will be specifically explained below using examples, but the present invention is not limited to the following examples.

<Analysis of dipeptides contained in Euglena cells>
The present inventors received a Euglena gracilis cell line for experimental use managed by the Laboratory of Food Metabolism and Nutrition at Osaka Prefecture University. To culture and grow the Euglena cells, 150 mL of the above-mentioned KH medium (Table 3) was prepared in a flask as the medium for Experimental Example 1. In addition, 150 mL of the medium for Experimental Example 2 and the medium for Experimental Example 3, _which have the same composition as the medium for Experimental Example 1 (KH medium) except that they contained more free Glu, ammonium sulfate (( _NH4 ) _2SO4 ), and ammonium phosphate (( _NH4 ) _2HPO4 ) as shown in Table ₄ below, were prepared in separate flasks.

For each of the media according to Experimental Examples 1 to 3, the mouth of the flask was filled with a cotton plug and autoclaved at 2 atm and 121° C. for 15 minutes. The sterilized media was placed in a clean bench to cool, after which a small amount of cell suspension was added so that each medium contained 1.0×10 ⁴ or more and 3.0×10 ⁴ or less Euglena cells, in order to prevent contamination with bacteria. In a culture room maintained at 28° C. or more and 30° C. or less, the media to which the cells had been added was shaken at about 80 rpm with a shaker while continuously irradiating the medium with light of about 5,000 lux to allow the cells to be cultured and grown for 7 days.

The culture medium according to each of Experimental Examples 1 to 3 after the culture was freeze-dried using a freeze dryer to obtain a freeze-dried product. 200 mg of the freeze-dried product was suspended in 5.0 mL of 80% by weight acetonitrile aqueous solution in a tube to prepare a cell suspension. While cooling the cell suspension in the tube with ice, ultrasonic vibration was applied to the cell suspension for 10 seconds using an ultrasonic crusher (manufactured by Tommy Seiko Co., Ltd., model name: UR-21P) three times to obtain a cell disruption solution. A centrifugal force of 6,000 x G was applied to the disruption solution at 4°C for 10 minutes using a centrifuge, and about 5 mL of the supernatant formed by centrifugation was collected. The supernatant was centrifuged using a centrifugal concentrator (manufactured by Tommy Seiko Co., Ltd., model name: CC-105) until it became 200 μL or less, and a concentrate was obtained.

In order to purify the above-mentioned concentrate so that the content of the dipeptide compound is increased, 100 mmol/L hydrochloric acid was prepared as a column equilibration buffer. This buffer was injected into a solid-phase extraction column (Oasis (registered trademark) MCX Column, manufactured by Waters Corporation) to equilibrate it. The concentrates according to each of Experimental Examples 1 to 3 were mixed with 800 μL of 100 mmol/L hydrochloric acid, and the resulting mixture was injected into the equilibrated column. Furthermore, after injecting methanol to wash the inside of the column, methanol containing 500 mmol/L of ammonia was injected to collect the aqueous solution eluted from the column. The collected aqueous solution was dried by centrifugal concentration to obtain a purified product.

To perform LC/MS (liquid chromatography mass spectrometry) and LC/MS/MS (liquid chromatography mass spectrometry using a tandem quadrupole mass spectrometer), an LC-MS/MS (Waters Corporation, LC model number: Alliance e 2965, MS/MS model number: Xevo TQD) was prepared. The purified products according to each of Experimental Examples 1 to 3 were dissolved in a 2.0% by mass aqueous monofluoroacetic acid solution to prepare samples for LC/MS. In addition, as standard samples, 2.0% by mass aqueous monofluoroacetic acid solutions containing 1.0 μmol/L each of Gln-Arg and Arg-Gln, or 1.0 μmol/L each of Asn-Arg and Arg-Asn were prepared. As mobile phase A, an acetonitrile solution containing 0.1% by mass of formic acid was prepared. As mobile phase B, an aqueous solution containing 100 mmol/L of ammonium formate was prepared. The column temperature of an amino acid analysis column (Intrada Amino Acid, manufactured by Intact Corporation, inner diameter 2.0 mm×column length 50 mm) was kept at 40° C., and 20 μL of either a sample for LC/MS or a standard sample was injected into this column. Furthermore, mobile phase A and mobile phase B were controlled in concentration gradient as shown in Table 5 below, and fed to this column at a flow rate of 0.3 mL/min to separate each component, thereby performing LC/MS or LC/MS/MS.

In the total ion chromatogram obtained from the standard samples (Gln-Arg and Arg-Gln) by LC/MS, a peak derived from Gln-Arg was observed at a retention time of 6.49 minutes, and a peak derived from Arg-Gln was observed at a retention time of 6.88 minutes, as shown in FIG. 4. Furthermore, when Gln-Arg or Arg-Gln is subjected to LC/MS/MS, H ₂ NC(:NH)NHCH ₂ ^- ions are generated due to the arginine residues contained in the molecule. The mass-to-charge ratio of this product ion is about 70. Therefore, in the chromatogram of the selected ion monitoring at m/z=70 shown in FIG. 5, a peak derived from Gln-Arg and a peak derived from Arg-Gln were observed at the same retention time.

As shown in FIG. 5, a large peak was observed at a retention time of about 6.88 minutes in each of the samples from Experimental Example 1 to Experimental Example 3, suggesting that all samples contained a large amount of Arg-Gln. At a retention time of about 6.49 minutes in FIG. 5, no peak was observed in Experimental Example 1 and Experimental Example 2, but a small peak was observed in Experimental Example 3. This indicates that the sample from Experimental Example 3 contained a detectable amount of Gln-Arg compared to Experimental Examples 1 and 2. As shown in FIG. 5, many small peaks derived from peptides other than Gln-Arg and Arg-Gln were observed in Experimental Example 1, whereas similar peaks were hardly observed in Experimental Examples 2 and 3. Considering the results of Tables 3 and 4, the medium of Experimental Example 3 contains more Glu compounds and ammonia nitrogen than the medium of Experimental Example 1 (KH medium). This suggests that Arg is more likely to be contained in the form of Arg-Gln or Gln-Arg in Euglena cells grown in the medium.

Using MassLynx mass spectrometry (MS) software (Waters Corporation, version 4.1), noise was removed from the chromatogram shown in Figure 5 to obtain the chromatogram shown in Figure 6. Based on the fact that the area of the Arg-Gln-derived peak in the standard sample in Figure 6 corresponds to 1.0 μmol/L, the content of Arg-Gln in each sample according to Experimental Example 1 to Experimental Example 3 was calculated from the area of the Arg-Gln-derived peak.

As shown in Table 6, the samples of Experimental Examples 2 and 3 contained more than eight times as much Arg-Gln as the sample of Experimental Example 1. This suggests that Euglena cells grown and dried in the medium of Experimental Example 2 or the medium of Experimental Example 3 contained more Arg-Gln than Euglena cells grown and dried in the medium of Experimental Example 1 (KH medium). This is thought to be due to the Euglena cells being grown in a medium with a higher content of Glu compounds and ammonia nitrogen than KH medium, and a larger amount of Arg-Gln being biosynthesized from Glu compounds and ammonia nitrogen within the cells.

In the total ion chromatograms obtained by LC/MS from the standard samples (Asn-Arg and Arg-Asn), as shown in FIG. 7, a peak due to Asn-Arg was observed at a retention time of 6.15 minutes, and a peak due to Arg-Asn was observed at a retention time of 6.79 minutes. This peak due to Asn-Arg was observed in the sample of Experimental Example 1, but not in the samples of Experimental Examples 2 and 3. Furthermore, this peak due to Arg-Asn was observed in the sample of Experimental Example 2, but not in the samples of Experimental Examples 1 and 3. For this reason, it is considered that Asn-Arg and Arg-Asn may or may not be easily contained depending on the composition of the medium in which Euglena cells are grown.

<Evaluation of ACE inhibitory activity of each amino acid and dipeptide>
As free amino acids having ACE inhibitory activity, Asn, Asp, Gln, Glu, and Arg (all manufactured by Kyowa Hakko Kirin Co., Ltd.) were prepared. Separately, a mixed composition was prepared by mixing these five kinds of free amino acids in equal amounts. ACE Kit-WST manufactured by Dojindo Laboratories Co., Ltd. was prepared as a set of substrate solutions and enzyme solutions for measuring ACE inhibitory activity.

A solution for comparative testing was prepared by dissolving any one of the above free amino acids or the mixed composition in 18 mΩ Milli-Q water. According to the measurement operation method described in the ACE Kit-WST instruction manual, 20 μL of the solution for comparative testing, 20 μL of the substrate solution, and 20 μL of the enzyme solution were mixed to prepare 60 μL of a mixed solution. In this mixed solution, the content of the free amino acid was adjusted to, for example, 500 mg/L or 1,000 mg/L. As a control group not containing an ACE inhibitor, a mixed solution was prepared by mixing Milli-Q water instead of the solution for comparative testing. As a control group not containing ACE, a mixed solution was prepared by mixing Milli-Q water instead of the enzyme solution. Each of these mixed solutions was kept at 37° C. for 10 minutes, and then the optical density (hereinafter referred to as "OD ₄₅₀ ") for light with a wavelength of 450 μm was measured. The OD ₄₅₀ measurement value of the control group not containing an ACE inhibitor was considered to indicate an ACE inhibition rate of 0%, and the OD ₄₅₀ measurement value of the control group not containing an ACE inhibitor was considered to indicate an ACE inhibition rate of 100%, and the ACE inhibition rate of each mixture was calculated based on the OD ₄₅₀ measurement results. The average values of the calculation results are shown in Table 7 below.

In addition, Arg-Gln, Gln-Arg, Arg-Asn, and Asn-Arg were prepared by a peptide synthesis service provided by GenScript. These dipeptides were prepared by peptide synthesis using free Arg and Asp, or free Arg and Gln as raw materials. In measuring the ACE inhibition rate of each of these dipeptides, the mixture was made to contain one of the dipeptides Arg-Gln, Gln-Arg, Arg-Asn, and Asn-Arg at a concentration of 0.12 mg/L, 0.62 mg/L, 3.1 mg/L, 15 mg/L, or 77 mg/L, and the ACE inhibition rate was calculated by measuring in the same manner as the measurement of the ACE inhibition rate of the free amino acid described above. The calculated average values are shown in FIG. 8 and Table 8 below.

As shown in FIG. 8 and Table 8, in the mixture containing any one of the four dipeptides, the ACE inhibition rate increased with increasing dipeptide content. Comparing Tables 7 and 8, the ACE inhibition rate of dipeptides is higher than that of free amino acids even in small amounts. From these experimental results, the inventors discovered that each of Arg-Gln, Gln-Arg, Arg-Asn, and Asn-Arg is a dipeptide with stronger ACE inhibitory activity than free amino acids such as Arg. As shown in Table 8, the ACE inhibition rate of the mixture containing 3.1 mg/L to 77 mg/L of dipeptides is high, so if the portion marked "N.D." is re-experimented, experimental data showing ACE inhibitory activity, albeit at a small value, can be obtained.

<Preparation of a composition containing an aqueous Euglena component>
As in the above-mentioned Experimental Example 1, a cell line of Euglena gracilis was provided. In order to culture and grow Euglena, 150 mL of a medium having the composition shown in Table 9 below was prepared in a flask as a medium for Experimental Example 4 by partially changing the composition of the above-mentioned CM medium (Table 1). In addition, 150 mL of each of the media for Experimental Examples 5 to 10, which have the same composition as the medium for Experimental Example 4 (Table 9) except that the content of Glu or ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ) is higher as shown in Table 10, was prepared in a separate flask.

As in the above-mentioned Experimental Example 1, the media for each of Experimental Examples 4 to 10 were autoclaved and cooled, and then a suspension of Euglena cells was added. The cells were grown by culturing for 7 days while shaking and irradiating light. At the end of the culturing period, each medium was stirred, a small amount was taken, and dropped onto a hemocytometer, and a cover glass was attached to the droplet. The number of cells present in a 1.0 mm x 1.0 mm section on the hemocytometer was counted under a microscope, and the number of Euglena cells per 1.0 mL of medium was calculated using the following formula 1.

Ｎ_Ｃ：培地１．０ｍＬあたりの細胞数
Ｎ_Ｎ：１．０ｍｍ^２あたりに存する細胞数の平均値
１０^４：１．０ｍｍ^２に対する容量の変換値

N _C : Number of cells per 1.0 mL of medium N _N : Average number of cells per 1.0 ^mm2 10 ⁴ : Conversion value of volume to 1.0 ^mm2

After the culture, the medium according to each of Experimental Examples 4 to 10 was dispensed into tubes and centrifuged at 4°C with a centrifuge at 2,000×G. The pellet of cells formed by the centrifugation was collected and suspended in a small amount of Tris-HCl buffer to obtain a cell suspension containing Euglena cells at a high density. The cell suspension and the ultrasonic disintegrator's oscillator were placed in a beaker, and the beaker was cooled on ice and repeatedly subjected to vibrations of 20 kHz to obtain a disrupted solution of Euglena cells. The disrupted solution was transferred to a new tube and subjected to a centrifugal force of 6,000×G at 4°C to collect the formed supernatant. The supernatant was mixed with trichloroacetic acid and centrifuged at 6,000×G at 4°C to precipitate proteins, obtaining a deproteinized supernatant. The supernatant was mixed with diethyl ether to extract lipids contained in the supernatant into diethyl ether. The formed diethyl ether layer was removed, and the formed aqueous layer was collected. This aqueous layer was dried using a freeze-dryer to obtain freeze-dried products (compositions comprising an aqueous component of Euglena) according to each of Experimental Examples 4 to 10. The total mass of each of these freeze-dried products was measured.

<Evaluation of ACE inhibitory activity in prototype compositions>
For each of Experimental Examples 4 to 10, 200 μg of the above-mentioned lyophilized product was collected, dissolved in 20 μL of acetate buffer solution at pH 5.0, and further mixed with 40 μL of 0.5% by mass ninhydrin test solution. The mixture of the lyophilized product, acetate buffer solution, and ninhydrin test solution was heated in a boiling water bath for 15 minutes, and then left at room temperature (about 20° C.) for 30 minutes to cool. The mixture that had been left for 30 minutes was mixed with 260 μL of 50% by volume ethanol aqueous solution, and the optical density (OD ₅₉₅ ) of the obtained solution at a wavelength of 595 μm was measured using a spectrophotometer. Based on the calibration curve of a free Arg standard (hereinafter referred to as "Arg equivalent"), the absolute amount of amino acids (the total amount of free amino acids and peptide-constituting amino acids) in the lyophilized product according to each Experimental Example was calculated.

Separately, a part of the lyophilized product according to each of Experimental Examples 4 to 10 was taken and mixed with Milli-Q water to prepare a sample solution for measuring ACE inhibitory activity. Using the above-mentioned ACE Kit-WST, 20 μL of sample solution, 20 μL of substrate solution, and 20 μL of enzyme solution were mixed according to the measurement operation method described in the instruction manual. By this mixing, 60 μL of a mixture containing any of the lyophilized products according to each of Experimental Examples 4 to 10 was prepared so that the absolute amount of amino acid in terms of Arg was 45 mg/L. This mixture was kept at 37° C. for 10 minutes, and then OD ₄₅₀ was measured, and the ACE inhibition rate was calculated as described above. The average value of the calculation results is shown in Table 11 below.

As shown in Table 11, the value of "ACE inhibition rate in a mixed solution with an absolute amount of amino acids of 45 mg/L" was 38% or more in all of Experimental Examples 4 to 10. Considering the values of the ACE inhibition rate shown in Tables 7 and 8, it is presumed that each of the freeze-dried products according to Experimental Examples 4 to 10 contains a large amount of Arg in the form of this dipeptide compound rather than as a free amino acid. In addition, a comparison of Experimental Examples 4 to 7 shown in Table 11 showed that the "number of Euglena cells present per 1.0 mL of medium cultured for 7 days" decreased as the "content of ammonia nitrogen in the medium" increased. On the other hand, compared with Experimental Examples 4 and 5, in Experimental Examples 6 and 7, the value of "ACE inhibition rate in a mixed solution with an absolute amount of amino acids of 45 mg/L" increased as the "content of ammonia nitrogen in the medium" increased. For this reason, when the ammonia nitrogen content in the medium is 38 mmol/L or more, the higher the ammonia nitrogen content, the more difficult it becomes to increase the number of cells, but it is considered that each cell is more likely to accumulate a large amount of the present dipeptide compound.

Comparison of Experimental Example 4 and Experimental Examples 8 to 10 shown in Table 11 showed that when the ammonia nitrogen content in the medium was 15.1 mmol/L, the "number of Euglena cells present per 1.0 mL of medium cultured for 7 days" increased and the "ACE inhibition rate in a mixed solution with an absolute amount of amino acids of 45 mg/L" increased as the Glu content in the medium increased. In addition, compared to Experimental Examples 4 to 7, the "number of Euglena cells used as raw material per 1,000 mg of lyophilized product" was higher in Experimental Examples 8 and 10. Therefore, it is presumed that when the ammonia nitrogen content in the medium was 15.1 mmol/L, the cell number was more likely to increase as the Glu content in the medium increased, and thus a large amount of the dipeptide compound was collected in the lyophilized product.

<Animal testing>
An animal experiment was carried out to verify the ACE inhibitory effect and the like exerted by the oral ingestion of the composition according to the present invention. In order to prepare a prototype of the composition (lyophilized product according to Experimental Example 11) to be used in the animal experiment, Sephadex G-10 manufactured by GE Healthcare Japan Co., Ltd. was prepared as a carrier for gel filtration chromatography and packed into a column with a diameter of 1.5 cm and a length of 15 cm. The exclusion limit of Sephadex G-10 is 700 Da. The exclusion limit is the upper limit of the molecular weight at which molecules passing through the column can be captured and fractionated by the stationary phase (carrier). For this reason, Sephadex G-10 is a carrier suitable for fractionating peptides with relatively small molecular weights such as the present dipeptide compound. As a preliminary experiment, a solution containing Gln-Arg, Glu, and Arg was passed through a Sephadex G-10 packed column at room temperature of about 20° C., and the retention time at which a fraction containing Gln-Arg but substantially not containing Glu or Arg flowed out of the column was examined. In addition, a Euglena gracilis cell line was provided in the same manner as in the above-described Experimental Example 1. A part of the composition of the above-described KH medium (Table 3) was changed, and 150 mL of a medium according to Experimental Example 11 having the composition shown in Table 12 below was prepared in a flask.

Euglena cells were cultured and grown in the same manner as in Experimental Example 1 described above, except that the medium according to Experimental Example 11 was used, the culture medium was freeze-dried, a cell suspension was prepared and subjected to ultrasonic disruption to obtain a disruption treatment solution. This disruption treatment solution was injected into a Sephadex G-10 packed column at a room temperature of about 20°C and fractionated, and a fraction was obtained that flowed out of the column at the same retention time as the dipeptide compound flowed out in the above-mentioned preliminary experiment. The fraction obtained here is a solution containing an aqueous Euglena extract that is substantially free of lipids, proteins, free amino acids and their salts, and polypeptides and oligopeptides with relatively large molecular weights derived from Euglena cells, and that has been purified to increase the content of the dipeptide compound. This fraction was freeze-dried to obtain a freeze-dried product according to Experimental Example 11. The absolute amount of amino acids (the total amount of free amino acids and peptide-constituting amino acids) in the freeze-dried product according to Experimental Example 11 was measured in the same manner as in Experimental Examples 4 to 10 described above. Then, the freeze-dried product of Experimental Example 11 was mixed with physiological saline so that the absolute amount of amino acids was 2.0 mg/L to prepare sample water.

Twelve 12-week-old male SHR/NCrlCrlj rats (hereinafter referred to as "SHR rats") that had been made SPF (specific pathogen free) were purchased from Charles River Japan Co., Ltd. SHR rats are a strain of experimental animals that naturally develop hypertension with age. The animal experiments were conducted in accordance with the "Guide for the Care and Use of Experimental Animals" of the National Institutes of Health. In a breeding room maintained at 20°C or higher and 26°C or lower and a relative humidity of 50% RH or higher and 70% RH or lower, the 12 SHR rats were divided into six control rats and six experimental rats, and were allowed to freely consume feed (hereinafter referred to as "breeding feed") with the composition shown in Table 13 below, and were pre-bred for six days, after which they were fasted for 12 hours.

A non-invasive rat blood pressure monitor (Softlon Corporation, model number: BP-98A-L) was prepared. The experiment started when the rats had completed the 12-hour fasting period. At this point, the rats' systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured at the base of the tail using a rat blood pressure monitor in the atmosphere of a breeding room. After blood pressure was measured at the start of the experiment, the control rats were allowed to freely consume saline and breeding feed, and the experimental rats were allowed to freely consume the above-mentioned sample water and breeding feed, and they were raised in the breeding room for 7 weeks. No abnormalities were observed in the appearance or weight gain of any of the rats during the preliminary or subsequent breeding periods. There were some individual differences between the rats in the control group regarding the amount of saline intake and the amount of sample water intake in the experimental group, but the average values for the six control rats and the six experimental rats were similar. Similarly, although there were some individual differences in the intake of the feed, the average value of the six control rats was similar to that of the six experimental rats. At 5, 6, and 7 weeks after the start of the experiment, SBP and DBP were measured in the same manner as at the start of the experiment. After measuring SBP and DBP at 7 weeks, blood was drawn from the tail vein and centrifuged to obtain serum. The serum and angiotensin II quantification kit for rats (Cloud-Clone Corp. WUHAN, model number: KSA005Ra11) were used to measure the blood concentration of angiotensin II according to the measurement method in accordance with the kit's instructions. The measurement results are shown in Figure 9 (a), Figure 9 (b), and Table 14 below.

As shown in FIG. 9(a) and Table 14, there was no substantial difference between the SBP and DBP at the start of the experiment between the control rats and the experimental rats. On the other hand, the experimental rats showed lower values for both SBP and DBP at 5, 6, and 7 weeks after the start of the experiment than the control rats. At 6 weeks, the experimental rats showed significantly lower SBP and DBP values than the control rats. As shown in FIG. 9(b) and Table 14, the experimental rats showed significantly lower blood pressure levels at 7 weeks after the start of the experiment than the control rats. These results suggest that the experimental rats that were continuously orally administered the lyophilized product of Experimental Example 11 had a gradual suppression of the increase in blood pressure associated with aging from the start of the experiment. This effect is believed to be due to the fact that the dipeptide compound contained in the lyophilized product of Experimental Example 11 exerted an ACE inhibitory effect in the experimental rats, thereby suppressing the production of angiotensin II to a low level. Considering this mechanism of action, the freeze-dried product of Experimental Group 11 is considered to be suitable as a food composition for preventing or improving hypertension not only in SHR rats but also in mammals including humans.

<Preparation of transformants and purification of dipeptide ligase>
In the following, detailed descriptions of the prototype transformants and purification of dipeptide ligase performed by the present inventors will be omitted because those skilled in the art can carry out each of the following methods by appropriately combining known methods as necessary: isolating a polynucleotide encoding dipeptide ligase from Euglena cells, preparing a recombinant vector containing the isolated polynucleotide, introducing the recombinant vector into a microorganism to prepare a transformant, culturing and growing the prepared transformant, and extracting and purifying the enzyme from the grown transformant. Examples of known methods include those described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols in Molecular Biology, John Wiley & Sons Press (1989), etc.

The present inventors conducted a homology search for base sequences using a program such as BLAST (Basic Local Alignment Search Tool) and found the base sequence shown in SEQ ID NO: 2, which has about 50% homology to known base sequences encoding ligases from other microorganisms, from the base sequence of the chromosomal DNA of Euglena gracilis Z strain. Based on the results of the homology search, as described below, they performed cloning using Euglena gracilis Z strain to isolate a polynucleotide (2,970 bp) having a base sequence including the base sequence shown in SEQ ID NO: 2 (2,955 bp) and a promoter sequence upstream of the polynucleotide and a terminator sequence downstream of the polynucleotide, and introduced the isolated polynucleotide into Escherichia coli or yeast to produce a transformant expressing a dipeptide ligase derived from Euglena cells.

The Euglena gracilis Z strain was cultured in KH medium (Table 3) for 6 days with shaking while maintaining the temperature of the KH medium (Table 3) at 26°C in a culture room. The culture medium was centrifuged (1,500 rpm, 6 minutes) to collect the cultured Euglena cells as a precipitate. Total RNA containing mRNA was prepared from the collected Euglena cells by the CTAB (hexadecyltrimethylammonium bromide) method (Nucleic Acids Research, volume 8, Issue 19, pp.4321-4325 (1980)). A cDNA library derived from Euglena cells was created from the prepared total RNA using PrimeScript ^TM II ^1st strand cDNA Synthesis Kit manufactured by Takara Bio Inc. Based on the information on the base sequence described in SEQ ID NO:2 and its upstream and downstream base sequences, a sense primer consisting of the base sequence described in SEQ ID NO:3 and an antisense primer consisting of the base sequence described in SEQ ID NO:4 were designed and prepared. Using the sense and antisense primers, PCR was performed to obtain a PCR amplified product by amplifying a cDNA fragment from a cDNA library derived from Euglena cells. A portion of the PCR amplified product was sampled and subjected to agarose gel electrophoresis to confirm that the PCR amplified product was substantially composed of a cDNA fragment of about 2,970 bp. After confirmation, the PCR amplified product was purified by ethanol precipitation to obtain a purified cDNA fragment.

We prepared BL21(DE)pLysS Competent Cells manufactured by Promega, pCold ^TM II DNA, an expression vector manufactured by Takara Bio Inc. (a 4,392 bp circular plasmid, the URL of the instructions at the time of filing this application is https://catalog.takara-bio.co.jp/PDFS/3362_DS_j.pdf), and pCold ^TM TF DNA, an expression vector manufactured by Takara Bio Inc. (a 5,769 bp circular plasmid, the URL of the instructions at the time of filing this application is https://catalog.takara-bio.co.jp/PDFS/3365_DS_j.pdf). In each of pCold ^TM II DNA and pCold ^TM TF DNA, a purified cDNA fragment was ligated between the Nde I site and the EcoR I site contained in the multicloning site of the circular plasmid to prepare two types of recombinant vectors containing a polynucleotide consisting of a base sequence including the base sequence described in SEQ ID NO: 2, a base sequence including a promoter sequence located upstream, and a base sequence including a terminator sequence located downstream. Each of the two types of recombinant vectors was introduced into the cells of BL21(DE)pLysS Competent Cells, which is an Escherichia coli (E. coli) strain. The E. coli strains into which the two types of recombinant vectors were introduced were cultured on an LB agar medium plate containing ampicillin and chloramphenicol, and the white colonies formed on the medium plate were collected to selectively collect the transformed E. coli strains. Circular plasmid DNA was prepared by the alkaline-SDS method using the E. coli contained in the transformed E. coli strain. The prepared circular plasmid DNA was digested at the Nde I site and EcoR I site by restriction enzyme treatment, and the excised insert DNA was confirmed to be approximately 2,070 bp by agarose gel electrophoresis. Using an ABI PRISM (registered trademark) 3100 Genetic Analyzer manufactured by Thermo Fisher Scientific, it was confirmed that the insert DNA had the base sequence described in SEQ ID NO: 2. This suggests that the prototype E. coli was a transformant that had been transformed by introducing a recombinant vector containing a polynucleotide consisting of the base sequence described in SEQ ID NO: 2 into the cells.

A recombinant vector in which a polynucleotide comprising a base sequence including the base sequence described in SEQ ID NO:2, a base sequence including an upstream promoter sequence, and a base sequence including a downstream terminator sequence was incorporated into pCold ^TM II DNA was used as a template to amplify a cDNA fragment having a base sequence (3,081 bp) encoding a polypeptide comprising the amino acid sequence described in SEQ ID NO:1 and a His tag sequence by PCR. In this PCR, a PCR amplification product was obtained by amplifying this cDNA fragment using PrimeSTAR (registered trademark) Max DNA Polymerase manufactured by Takara Bio Inc., a sense primer comprising the base sequence described in SEQ ID NO:5, and an antisense primer comprising the base sequence described in SEQ ID NO:6. A part of this PCR amplification product was collected and subjected to agarose gel electrophoresis to confirm that the PCR amplification product was substantially composed of a cDNA fragment of about 3,081 bp. After confirmation, the PCR amplification product was purified by ethanol precipitation to obtain a purified product of a cDNA fragment having a base sequence encoding a polypeptide comprising the amino acid sequence described in SEQ ID NO:1 and a His tag sequence.

A fragment of pYES2 Yeast Expression Vector (a circular plasmid of 5,856 bp, URL of the instruction manual at the time of filing of this application https://assets.fishersci.com/TFS-Assets/LSG/manuals/pyes2_man.pdf?_ga=2.240319687.539912641.1601619011-1502215192.1601619011) manufactured by Thermo Fisher Scientific was amplified by PCR. In this PCR, a PCR amplification product was obtained by amplifying a fragment of pYES2 Yeast Expression Vector using the above-mentioned PrimeSTAR (registered trademark) Max DNA Polymerase, a sense primer consisting of the base sequence described in SEQ ID NO: 7, and an antisense primer consisting of the base sequence described in SEQ ID NO: 8. A portion of this PCR amplification product was collected and subjected to agarose gel electrophoresis to confirm that the PCR amplification product was substantially composed of a DNA fragment of about 5,896 bp. After confirmation, the PCR amplification product was purified by ethanol precipitation to obtain a purified amplified pYES2 Yeast Expression Vector fragment.

Using Takara Bio In-Fusion (registered trademark) HD Cloning Kit, a purified product of a cDNA fragment having a base sequence encoding a polypeptide consisting of the amino acid sequence described in SEQ ID NO:1 and a His tag sequence, and a purified product of an amplified pYES2 Yeast Expression Vector fragment, a recombinant vector in which the cDNA fragment was incorporated into the pYES2 Yeast Expression Vector fragment was prepared. This recombinant vector was introduced into the cells of Competent high DH5α, an E. coli strain (E. coli) manufactured by SciTrove. The E. coli strain into which this recombinant vector was introduced was cultured on an LB agar medium plate containing ampicillin, and the white colonies formed on the medium plate were collected to selectively collect the transformed E. coli strain. Plasmid DNA was prepared by the alkaline-SDS method using E. coli contained in the transformed E. coli strain. It was confirmed that the prepared plasmid DNA contained the base sequence described in SEQ ID NO:2 and the base sequence encoding the His tag using the ABI PRISM (registered trademark) 3100 Genetic Analyzer described above. After this confirmation, a recombinant vector in which a cDNA fragment having a base sequence encoding a polypeptide including the amino acid sequence described in SEQ ID NO: 1 and a His tag sequence was incorporated into a fragment of pYES2 Yeast Expression Vector was introduced into the cells of yeast (Saccharomyces cerevisiae) W303-1A strain. The yeast strain into which this recombinant vector was introduced was cultured on an SD (-Ura) medium plate prepared by removing uracil from a solid SD medium containing 2% glucose by mass, and the white colonies formed on the medium plate were collected to selectively collect the transformed yeast strain. The transformed yeast strain collected here is the yeast strain (NITE ABP-03293) described above in the explanation of this transformant.

A part of yeast was collected from a yeast strain (NITE ABP-03293) to induce the expression of a dipeptide ligase containing a His tag. For this purpose, first, an SD (-Ura) medium was prepared by removing uracil from the composition of an SD medium generally used for yeast culture. Then, 3 mL of SD (-Ura) liquid medium was prepared so that the glucose content as a carbon source for yeast was 2% by mass, and 100 mL of SD (-Ura) was prepared so that the glucose content was 0.1% by mass. While the liquid temperature of the 3 mL of SD (-Ura) medium was kept at 30° C., a part of yeast collected from the yeast strain (NITE ABP-03293) was cultured in this medium overnight with shaking. The next morning, the yeast strain was subcultured in 100 mL of SD(-Ura) liquid medium so that the optical density (OD ₆₀₀ ) measured at a wavelength of 600 μm was OD ₆₀₀ =0.12. The temperature of the subcultured SD(-Ura) liquid medium (about 100 mL) was kept at 30° C., and the medium was cultured with shaking until OD ₆₀₀ reached about 0.6. Galactose was added to about ₁₀₀ mL of SD(-Ura) liquid medium at a final concentration of 2% by mass, and the medium was cultured with shaking for 20 hours while keeping the temperature at 30° C., to induce expression of a dipeptide ligase having a His tag in the yeast.

Thereafter, about 100 mL of SD(-Ura) liquid medium was centrifuged (8,000 rpm, 4°C, 10 minutes) using a centrifuge, and yeast was collected as the precipitate formed. The collected yeast was disrupted using a Multi-Beads Shocker (registered trademark), a cell disrupter manufactured by Yasui Kikai Co., Ltd., and then centrifuged (14,000 rpm, 4°C, 10 minutes) to collect the formed supernatant. In addition, a column was prepared that was filled with TALON (registered trademark) Metal Affinity Resin (manufactured by Takara Bio Inc.), which is an immobilized metal affinity chromatography resin using Co ²⁺ that has high specificity for His tags. The collected supernatant was passed through this column, and affinity purification was performed on the dipeptide ligase bound to the His tag contained in the supernatant. A portion of the affinity purified dipeptide ligase was subjected to SDS-PAGE (SDS Poly-Acrylamide Gel Electrophoresis), and the electrophoretic gel shown in FIG. 10 was obtained. From a calibration curve prepared based on the bands of the molecular mass markers shown in this electrophoretic gel, the molecular mass of the purified enzyme (i.e., affinity purified dipeptide ligase) band shown in FIG. 10 was calculated to be about 111 kDa. On the other hand, the molecular mass of the dipeptide ligase bound to the His tag was calculated to be 111,222 Da based on the amino acid sequence described in SEQ ID NO: 1, which is consistent with the molecular mass of about 111 kDa calculated based on FIG. 10. Therefore, it was confirmed that a purified product of a polypeptide containing the amino acid sequence described in SEQ ID NO: 1, that is, a purified product of a dipeptide ligase derived from a yeast strain (NITE ABP-03293) was obtained.

<Preparation of a composition containing a dipeptide using dipeptide ligase>
In order to confirm whether a dipeptide-containing composition can be produced using a purified product of dipeptide ligase derived from the above-mentioned yeast strain (NITE ABP-03293), the present inventors considered carrying out an experiment by applying the ATP/NADH binding system of pyruvate kinase and lactate dehydrogenase described in Non-Patent Document 9, with reference to Non-Patent Document 8. To carry out this experiment, four types of reaction solutions shown in Table 15 below, that is, (Arg/Asn)+, (Arg/Asn)-, (Arg/Gln)+, and (Arg/Gln)-, were prepared.

To prepare each of the four reaction solutions shown in Table 15, first, 50 mmol/L of Tris-HCl buffer solution with a pH of 7.2, 10 mmol/L of potassium chloride, 10 mmol/L of magnesium chloride, 5 mmol/L of ATP (manufactured by Fujifilm Wako Pure Chemical Industries, product name: Adenosine 5'-Triphosphate Disodium Salt Trihydrate, Crystallized, model number: 018-16911), 2.5 mmol/L of phosphoenolpyruvic acid (manufactured by Sigma-Aldrich, product name: Phospho(enol)pyruvic acid monopotassium salt, model number: 860077-250MG), 0.8 mmol/L of NADH (manufactured by Oriental Yeast Co., Ltd., product name: β-Nicotinamide-adenine dinucleotide reduced phosphate), and 10 mmol/L of β-Nicotinamide-adenine dinucleotide reduced phosphate) were mixed with 10 mmol/L of ... A solution containing 30 units/mL of L-lactate dehydrogenase (recombinant, rabbit muscle), product number: 46776003, manufactured by Oriental Yeast Co., Ltd., product name: Pyruvate kinase (from rabbit muscle), product number: 46665002, and 50 units/mL of LDH (recombinant, rabbit muscle), product number: 46776003, manufactured by Oriental Yeast Co., Ltd., product name: L-lactate dehydrogenase (recombinant, rabbit muscle), product number: 46776003) was prepared. When preparing (Arg/Asn)+ and (Arg/Gln)- using this solution, 5.0 mmol/L of Arg (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd., product name: L(+)-Arginine, model number: 019-04611) and Asn (manufactured by the same company, product name: L(+)-Glutamine, model number: 019-04812) were added, respectively, and 5.0 mmol/L of Arg (manufactured by the same company, the above-mentioned L(+)-Arginine) and Gln (manufactured by the same company, product name: L(+)-Glutamine, model number: 076-00521) were added, respectively, when preparing (Arg/Gln)+ and (Arg/Gln)-. The same amount of purified dipeptide ligase derived from a yeast strain (NITE ABP-03293) was added when preparing (Arg/Asn)+ and (Arg/Gln)+, but was not added when preparing (Arg/Asn)- and (Arg/Gln)-.

Using a spectrophotometer, the reaction solutions of (Arg/Asn)+, (Arg/Asn)-, (Arg/Gln)+, and (Arg/Gln)- were kept at a liquid temperature of 26°C and the optical density (OD ₃₄₀ ) was measured for 5 minutes at a wavelength of 340 nm. Compared to before the enzyme reaction, no change was observed in the OD ₃₄₀ measurements for (Arg/Asn)- and (Arg/Gln)- after the enzyme reaction, whereas the OD ₃₄₀ measurements for (Arg/Asn)+ and (Arg/Gln)+ after the enzyme reaction were greatly reduced. This suggests that dipeptides were produced by dipeptide ligase in (Arg/Asn)+ and (Arg/Gln)+ after the enzyme reaction. The enzyme activities of the dipeptide ligase derived from the yeast strain (NITE ABP-03293) were calculated based on the measured _OD340 value and the millimolar extinction coefficient of NADH (ε = 6.22 mmol/L/cm), and the Arg-Asn ligase activity was found to be 18.7 μmol/min/mg protein, and the Arg-Gln ligase activity was found to be 9.2 μmol/min/mg protein.

The dipeptide content of each of the (Arg/Asn)+, (Arg/Asn)-, (Arg/Gln)+, and (Arg/Gln)- after the enzymatic reaction was measured by LC/MS/MS using LC-MS/MS (Alliance e 2965 and Xevo TQD as mentioned above). For this purpose, 200 μL of the solution collected from either (Arg/Asn)+, (Arg/Asn)-, (Arg/Gln)+, or (Arg/Gln)- after the enzymatic reaction was diluted 10-fold with 100 mmol/L hydrochloric acid and centrifuged (15,000 rpm, 4°C, 5 minutes), and the formed supernatant was collected. In addition, a solid-phase extraction column (Oasis® MCX Column as mentioned above) was equilibrated with 100 mmol/L hydrochloric acid. 30 μL of the collected supernatant was injected into the equilibrated solid-phase extraction column. After injecting 1 mL of methanol to wash the inside of the column, 1 mol/L of aqueous ammonia was injected into the column and the aqueous solution eluted from the column was collected. The aqueous solution was concentrated to dryness using a centrifugal concentrator (CC-105 as described above) and then dissolved in a 2% by mass aqueous solution of formic acid to prepare a sample for LC/MS/MS.

The aforementioned Intrada Amino Acid (inner diameter 2.0 mm x column length 50 mm) was prepared as an amino acid analysis column for LC/MS and LC/MS/MS. As mobile phase A, an acetonitrile solution containing 0.1% by mass of formic acid was prepared. As mobile phase B, an aqueous solution containing 100 mmol/L of ammonium formate was prepared. The column temperature of the amino acid analysis column was kept at 40°C, a 2 mass% aqueous formic acid solution prepared so that the content of any one of Arg-Asn, Asn-Arg, Arg-Gln, or Gln-Arg was 1.0 µmol/L was injected into the column, and the concentration gradient was controlled with mobile phase A and mobile phase B as described above in Table 5, and the solution was delivered to the column at a flow rate of 0.3 mL/min to separate each component and purify the dipeptide. The recovery rates of the purified dipeptides were 11 mass% for Asn-Arg, 25 mass% for Arg-Asn, 19 mass% for Gln-Arg, and 31 mass% for Arg-Gln. In addition, as standard samples for LC/MS and LC/MS/MS, 2 mass% aqueous solutions of formic acid were prepared so that the content of any one of Arg-Asn, Asn-Arg, Arg-Gln, and Gln-Arg was 250 μmol/L, 62.5 μmol/L, 15.6 μmol/L, and 3.91 μmol/L, respectively.

The column temperature of the amino acid analysis column (Intrada Amino Acid) was kept at 40°C, and 20 μL of either the LC/MS/MS sample or the standard sample was injected into the column, and the concentration gradient was controlled with mobile phase A and mobile phase B as described above in Table 5, and the liquid was sent to the column at a flow rate of 0.3 mL/min to separate each component, thereby performing LC/MS and LC/MS/MS. When the standard sample was injected into the amino acid analysis column, the retention time at which a peak originating from Arg-Asn, Asn-Arg, Arg-Gln, or Gln-Arg appeared in LC/MS was recorded, and a selected ion chromatogram at m/z = 70 was obtained at this retention time in LC/MS/MS. From this selected ion chromatogram, noise was removed using the above-mentioned MassLynx mass spectrometry (MS) software, and the area of the peak formed due to the arginine residue in the molecule for each dipeptide was measured, and a calibration curve of the concentration based on the peak area was created for each dipeptide. When an LC/MS/MS sample prepared from either (Arg/Asn)+, (Arg/Asn)-, (Arg/Gln)+, or (Arg/Gln)- after the enzyme reaction was injected into an amino acid analysis column, the retention time and calibration curve obtained with the standard sample were used to remove noise from the selected ion chromatogram at m/z=70, and the dipeptide content was measured based on the area measurement value of the peak due to the arginine residue contained in the molecule for each dipeptide, in the same manner as with the standard sample. The dipeptide content was measured five times for each LC/MS/MS sample. The average values after correction (Asn-Arg content: 100/11 times by mass, Arg-Asn: 100/25 times by mass, Gln-Arg content: 100/19 times by mass, and Arg-Gln content: 100/31 times by mass) for each of the five measurements, taking into account the above-mentioned numerical value of the dipeptide recovery rate, are shown in Table 16 and FIG. 11.

As shown in Table 16 and FIG. 11, the Asn-Arg content and Arg-Asn content were significantly higher in (Arg/Asn)+ to which a purified product of dipeptide ligase derived from yeast strain (NITE ABP-03293) was added than in (Arg/Asn)- to which the purified product was not added (p<0.001). Similarly, the Gln-Arg content and Arg-Gln content were significantly higher in (Arg/Gln)+ to which the purified product was added than in (Arg/Gln)- to which the purified product was not added (p<0.001). This suggests that the dipeptide ligase derived from yeast strain (NITE ABP-03293) has one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity. Although no purified dipeptide ligase derived from a yeast strain (NITE ABP-03293) was added, small amounts of Asn-Arg and Arg-Asn were detected in (Arg/Asn)-, and small amounts of Gln-Arg and Arg-Gln were detected in (Arg/Gln)-. This suggests that there may have been some factor in the reaction solutions of (Arg/Asn)- and (Arg/Gln)- that could induce peptide bonds between free amino acids without the intervention of dipeptide ligase.

<Production of a composition for inhibiting ACE or suppressing hypertension using a transformant>
SD (-Ura, +Asn) medium was prepared by removing uracil from the SD medium formulation generally used for yeast culture and adding Asn so that the Asn content was 5.0 mmol/L. 3 mL of SD (-Ura, +Asn) liquid medium prepared so that the glucose content was 2% by mass and 100 mL of SD (-Ura, +Asn) prepared so that the glucose content was 0.1% by mass were prepared. While keeping the liquid temperature of 3 mL of SD (-Ura, +Asn) medium at 30°C, a part of yeast collected from a yeast strain (NITE ABP-03293) was shake-cultured in this medium overnight. The next morning, the transformed yeast was subcultured in 100 mL of SD (-Ura, +Asn) liquid medium so that the measured OD ₆₀₀ value was 0.12. The cultured SD (-Ura, +Asn) liquid medium (about 100 mL) was cultured with shaking until the measured OD ₆₀₀ reached about _0.6 while keeping the liquid temperature at 30°C. Galactose was added to the SD (-Ura, +Asn) liquid medium (about 100 mL) at a final concentration of 2% by mass, and the culture was cultured with shaking for 20 hours while keeping the liquid temperature at 30°C, thereby inducing the expression of dipeptide ligase in yeast and promoting the production of Asn-Arg and Arg-Asn. Then, about 100 mL of the SD (-Ura, +Asn) liquid medium was centrifuged (8,000 rpm, 4°C, 10 minutes) and the precipitate formed was collected. The collected precipitate was subjected to cell disruption using a disrupter (Multi-Beads Shocker (registered trademark) described above), and the resulting cell disruption was centrifuged (14,000 rpm, 4° C., 10 minutes), and the formed supernatant was collected. The collected supernatant had a low OD ₃₄₀ value, suggesting that it contained a large amount of dipeptides.

200 μL of the supernatant collected as described above was diluted 10 times with 100 mmol/L hydrochloric acid, centrifuged (15,000 rpm, 4 ° C, 5 minutes), and 30 μL of the formed supernatant was collected and injected into a solid phase extraction column (Oasis (registered trademark) MCX Column described above) equilibrated with 100 mmol/L hydrochloric acid. Furthermore, 1 mL of methanol was injected to wash the inside of the column, and then 1 mol/L ammonia water was injected into the column, and the aqueous solution eluted from the column was collected. The collected aqueous solution was centrifuged and concentrated to dryness using a centrifugal concentrator (CC-105 described above), and then dissolved in a 2% by mass aqueous formic acid solution to prepare a sample for LC/MS/MS. LC/MS and LC/MS/MS were performed on this LC/MS/MS sample in the same manner as when the Asn-Arg content and Arg-Asn content were measured using (Arg/Asn) + and (Arg/Asn) - shown in Table 16 described above. As a result, in 30 μL of the supernatant injected into the solid-phase extraction column, the Asn-Arg content was calculated to be 110.9 nmol/L, and the Arg-Asn content was calculated to be 72.3 nmol/L. This suggests that a composition for ACE inhibition or blood pressure elevation suppression containing Asn-Arg and Arg-Asn having ACE inhibitory activity can be produced by culturing a part of yeast collected from a yeast strain (NITE ABP-03293) in the presence of an SD (-Ura, +Asn) liquid medium containing Asn and Arg.

SD (-Ura, +Gln) medium was prepared by removing uracil from the SD medium and adding Asn so that the Gln content was 5.0 mmol/L. As described above, compared to the case where yeast was cultured using SD (-Ura, +Asn) medium and the contents of Asn-Arg and Arg-Asn were measured, a part of yeast collected from a yeast strain (NITE ABP-03293) was cultured under the same conditions, except that the medium was changed to SD (-Ura, +Gln), and 30 μL of the supernatant was prepared to be injected into a solid-phase extraction column, and LC/MS and LC/MS/MS were performed. As a result, the Gln-Arg content was calculated to be 17.3 nmol/L and the Arg-Gln content was calculated to be 246.0 nmol/L in the 30 μL of the supernatant injected into the solid-phase extraction column. Therefore, it was suggested that a composition for ACE inhibition or blood pressure elevation suppression containing Gln-Arg and Arg-Gln having ACE inhibitory activity can be produced by culturing a part of yeast collected from a yeast strain (NITE ABP-03293) in the presence of an SD (-Ura, +Gln) liquid medium containing Gln and Arg. For example, it is expected that a composition containing a higher content of the present dipeptide compound can be produced by investigating the optimal amounts of Arg, Asn, and Gln in the medium in the future.

Claims (11)

An enzyme preparation containing a dipeptide synthetase,
The dipeptide synthetase is selected from the group consisting of the following (a), (b), and (c):
(a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1;
(b) a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO:1;
(c) a polypeptide consisting of an amino acid sequence in which one or more and not more than 20 amino acid residues are substituted, inserted, deleted, and/or added to the amino acid sequence set forth in SEQ ID NO:1;
and having an amino acid sequence of one polypeptide selected from the group consisting of
The enzyme preparation is characterized in that the dipeptide synthesizing enzyme has one or more enzyme activities selected from the group consisting of L-arginyl-L-asparagine (Arg-Asn) ligase activity, L-arginyl-L-glutamine (Arg-Gln) ligase activity, L-asparaginyl-L-arginine (Asn-Arg) ligase activity, and L-glutamyl-L-arginine (Gln-Arg) ligase activity. A method for producing a composition for inhibiting ACE or suppressing hypertension, comprising the steps of:
A step of preparing the enzyme agent according to claim 1;
a step of enzymatically reacting the dipeptide synthesizing enzyme with one or more compounds selected from the group consisting of L-arginine (Arg) and salts thereof, one or more compounds selected from the group consisting of L-asparagine (Asn), L-glutamine (Gln), and salts thereof, in the presence of the enzyme agent, to produce a dipeptide compound having angiotensin I converting enzyme (ACE) inhibitory activity, the dipeptide compound being one or more compounds selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and salts thereof;
A method for producing a composition for inhibiting ACE or suppressing hypertension, comprising: A polynucleotide having a base sequence encoding a dipeptide synthetase,
The base sequence encoding the dipeptide synthetase is a group consisting of the following (d), (e), (f), and (g):
(d) a polynucleotide consisting of the base sequence set forth in SEQ ID NO:2;
(e) a polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO:2;
(f) a polynucleotide consisting of a nucleotide sequence in which one or more and 20 or less nucleotides have been substituted, inserted, deleted, and/or added to the nucleotide sequence set forth in SEQ ID NO: 2;
(g) a polynucleotide that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO:2;
having a base sequence of one type of polynucleotide selected from
The polynucleotide, wherein the base sequence encoding the dipeptide synthesizing enzyme is a base sequence encoding an enzyme having one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity. A recombinant vector comprising the polynucleotide described in claim 3. A transformant of a microorganism, which expresses an enzyme encoded by a base sequence encoding the dipeptide synthesizing enzyme described in claim 3, and which has one or more enzyme activities selected from the group consisting of Arg-Asn ligase activity, Arg-Gln ligase activity, Asn-Arg ligase activity, and Gln-Arg ligase activity . The transformant according to claim 5, wherein the microorganism is Euglena or yeast. A kit comprising one or more selected from the group consisting of the enzyme preparation described in claim 1, the polynucleotide described in claim 3, the recombinant vector described in claim 4, the transformant described in claim 5, and the transformant described in claim 6. A method for producing a composition for inhibiting ACE or suppressing hypertension, comprising the steps of:
A step of preparing a medium containing the transformant according to claim 5 or 6 and a nitrogen source capable of nitrogen assimilation by the transformant;
Cultivating the transformant in the presence of the medium to produce a dipeptide compound having ACE inhibitory activity, the dipeptide compound being one or more compounds selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and salts thereof;
A method for producing a composition for inhibiting ACE or suppressing hypertension, comprising: The method for producing a composition for inhibiting ACE or suppressing hypertension according to claim 8, wherein the nitrogen source comprises one or more compounds selected from the group consisting of Arg and its salts, and one or more compounds selected from the group consisting of Asn, Gln, and their salts. A composition for inhibiting ACE or suppressing hypertension, comprising:
The present invention relates to a transformant according to claim 5 or 6, a dried product of the transformant, and a cell lysate of the transformant.
The present invention comprises a dipeptide compound having an ACE inhibitory activity, which is one or more compounds selected from the group consisting of Arg-Asn, Arg-Gln, Asn-Arg, Gln-Arg, and salts thereof,
A composition for inhibiting ACE or suppressing hypertension, characterized in that the content of the dipeptide compound is 0.10 mg/L or more . A composition for inhibiting ACE or suppressing hypertension according to claim 10, comprising the dipeptide synthetase or a denatured protein thereof.

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