JP2017019763A - Prolyl tripeptidyl peptidase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide prolyl tripeptidyl peptidase (PTP) inhibitors excellent in safety, and to provide detection methods which can simply detect PTP inhibitory effects in gingiva.SOLUTION: A PTP inhibitor contains a protein consisting of a specific amino acid sequence derived from Oryza sativa, as an active ingredient. A PTP inhibitor comprises an amino acid sequence in which one or some amino acids are deleted, substituted, inserted, and/or added in the amino acid sequence; and comprises a protein having inhibitory activity to PTP, or an amino acid sequence having 90% or more homologous to the amino acid sequence; and contains as an effective ingredient a protein having inhibitory activity to PTP. A detection method of PTP inhibitory activity comprises contacting a structure containing gelatin with PTP in a medium under the presence of a PTP inhibitor to detect PTP activity.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a prolyl tripeptidyl peptidase inhibitor and a periodontal disease preventive and / or therapeutic agent containing a protein contained in a cereal plant-derived material as an active ingredient. The present invention also relates to a simple method for detecting prolyl tripeptidyl peptidase inhibitory effect.

  Periodontal disease is said to affect approximately 60% of adults. Periodontal disease is known to cause gingival swelling, bleeding, discomfort, regression / decrease of gingiva supporting the teeth, gingival pain, bad breath, etc. when periodontal disease develops. There are a dozen types of aerobic or anaerobic bacteria as the causative agents of periodontal disease bacteria. Some periodontal bacteria have been recognized not only for periodontal disease but also for diseases such as arteriosclerosis, which is a social problem.

  Periodontopathic bacteria have a special metabolic system and cannot use sugar as an energy source. They decompose the gum collagen and ingest amino acids to obtain energy. The anaerobic bacterium Porphyromonas gingivalis (hereinafter simply referred to as “P. gingivalis”) is considered the main causative agent of periodontal disease. It is known that P. gingivalis produces many powerful protease enzymes such as peptidases inside and outside the cells, and these enzymes degrade gingival collagen. Enzymes produced by P. gingivalis include collagenase, gingipain (trypsin-like protease), prolyl tripeptidyl peptidase (hereinafter simply referred to as “PTP”), dipeptidyl peptidase IV, and aminopeptidase N (hereinafter simply referred to as “APN”). For example).

  Collagen contained in the gingiva is a protein having a three-helical structure and containing a lot of proline. A general protease cannot act on collagen having a three-helical structure, but it can act on collagenase. Peptide fragments produced by degradation by collagenase contain a large amount of proline (Pro), and therefore cannot be easily decomposed into amino acids by APN. PTP and dipeptidyl peptidase IV have an action of removing proline from peptide fragments by catalyzing cleavage at X-X-Pro units or X-Pro units. The peptide fragment from which proline has been removed is decomposed into amino acid units by APN. Therefore, PTP cleaved by X-X-Pro units plays an extremely important role in the degradation of collagen with many Gly-X-Pro repeats.

  A gene encoding PTP was isolated by Potempa et al. (Non-patent Document 1). The inventors of the present invention clarified the steric structure of an enzyme by expressing a gene encoding PTP in E. coli and performing crystallization, and performing X-ray crystal diffraction on the obtained crystal (Non-patent Document 2). And 3). PTP is composed of a dimer, and the monomer is composed of a propeller domain and a catalytic domain. The active site is composed of three catalytic residues, Ser, His, and Asp. It has been reported that a hydrophobic pocket exists as a mechanism for recognizing a substrate, and that proline contained in the substrate is recognized by fitting into a PTP pocket. Protease inhibitors are being developed for the treatment and prevention of periodontal disease, and H-Ala-Ile-pyrrolidin-2-yl boronic acid is chemically synthesized as a PTP-specific inhibitor (Non-patent document 5). The structure of the complex of PTP and the inhibitor has been elucidated, and details of the catalytic mechanism of PTP have been clarified. However, since such an inhibitor is covalently bonded to PTP, there are doubts on safety and it is difficult to put it into practical use as a pharmaceutical product.

  In order to develop an active ingredient of a highly safe preventive or therapeutic agent for periodontal disease, a search for a protease inhibitor derived from a natural product such as a plant has been conducted. It has been reported that a protein extracted from polished rice has inhibitory activity against arginine-gingipain (Patent Document 1). Moreover, it has been reported that the protein (Os03g0277300) which is a rice origin component exhibits an antibacterial action with respect to periodontal disease bacteria (patent document 2). On the other hand, it has been reported that the protein (Os03g0277300), which is a rice-derived component, did not show antimicrobial activity (Non-patent Document 4).

  Conventionally, PTP inhibitory activity was detected using alanyl, phenylalanyl, prolyl, and beta naphthylamide (hereinafter referred to as “Ala-Phe-Pro-βNA”) as a substrate. (Non-Patent Document 5). However, the substrate of such a conventional method is a synthetic substrate containing only 3 amino acids, and there is a problem that measurement of PTP inhibitory activity requires a complicated operation.

JP 2009-84161 A JP 2013-60416 A

Banbula, A., Mark, P., Bugno, M., Silberring, J., Dubin, A., Nelson D., Travis, J., and Potempa, J., (1999). J. Biol. Chem. 274, 9246-9252 Nakajima, Y., Ito, K., Xu, Y., Yamada, N., Onohara, Y., Ito T., and Yoshimoto, T., Acta Cryst. F61, 1046-1048 (2005) Ito, K., Nakajima, Y., Ichihara, E., Ogawa, K., Egawa, T., Xu, Y., and Yoshimoto, T., J. Mol. Biol., 362, 228-240 (2006 ) Taniguchi M1, Ikeda A, Nakamichi S, Ishiyama Y, Saitoh E, Kato T, Ochiai A, Tanaka T., Peptides. 2013 Oct; 48: 147-55.doi: 10.1016 / j.peptides.2013.08.011. Epub 2013 Aug 21. Xu, Y., Nakajima, Y., Ito, K., Zheng, H., Oyama, H., Heiser, U., Hoffmann, T., Gaertner, UT., Demuth. HU., And Yoshimoto, T. , J. Mol. Biol., 375 (3), 708-719 (2008)

  An object of the present invention is to provide a prolyl tripeptidyl peptidase inhibitor and a periodontal disease preventive and / or therapeutic agent excellent in safety. Moreover, this invention makes it a subject to provide the detection method which can detect easily the prolyl tripeptidyl peptidase inhibitory effect in a gingiva.

As a result of intensive studies on substances having PTP inhibitory activity, the present inventors focused on the fact that rice bran contains a protein having PTP inhibitory activity, and that the protein having PTP inhibitory activity is a protein having a molecular weight of 25 KDa. Further, the present invention was completed by analyzing the function by purifying and identifying the protein.
The present inventors have also found that PTP inhibitory activity can be detected by using a structure containing gelatin as a periodontal disease model, and completed the present invention.

That is, this invention consists of the following.
1. Prolyl tripeptidyl peptidase (PTP) inhibitor containing any protein selected from the following (a) to (c) as an active ingredient:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;
(B) In the amino acid sequence shown in SEQ ID NO: 1, it contains an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added, and has an inhibitory activity against prolyl tripeptidyl peptidase (PTP) A protein having
(C) a protein comprising an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1 and having an inhibitory activity against prolyl tripeptidyl peptidase (PTP).
2. 2. The prolyl tripeptidyl peptidase (PTP) inhibitor according to item 1 above, wherein the active ingredient is a protein comprising the amino acid sequence shown in SEQ ID NO: 1.
3. 3. The prolyl tripeptidyl peptidase (PTP) inhibitor according to 1 or 2 above, wherein the protein of the active ingredient has a molecular weight of 25 kDa.
4). A protease inhibitor of periodontal disease fungus Porphyromonas gingivalis containing a protein comprising the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient.
5. The periodontal disease bacteria growth inhibitor containing the inhibitor of any one of the preceding clauses 1-4.
6). A preventive and / or therapeutic agent for periodontal disease comprising the inhibitor according to any one of items 1 to 4 or the growth inhibitor of periodontal disease bacteria according to item 5 above.
7). The inhibitor according to any one of items 1 to 4, including the isolation of an active ingredient from a cereal plant-derived material, the periodontal disease bacterial growth inhibitor according to item 5, or the periodont according to item 6 A method for producing a disease prevention and / or treatment agent.
8). A method for detecting PTP inhibitory activity, comprising a step of contacting PTP with a structure containing gelatin in a medium in the presence of a PTP inhibitor to detect PTP activity.

The PTP inhibitor according to the present invention acts as a periodontal disease protease inhibitor. Since the PTP inhibitor of the present invention uses a protein contained in a cereal plant-derived material as an active ingredient, it has an effect that it is excellent not only in effectiveness but also in safety. Furthermore, the PTP inhibitor of the present invention is useful because it exerts an action of suppressing the growth of periodontal disease bacteria. In the PTP inhibitor of the present invention, the active ingredient is a protein, and since the protein is degraded in the stomach, the possibility of side effects is low. The active ingredient of the PTP inhibitor of the present invention can be produced from an edible part (for example, old rice) or a waste product (for example, rice bran) after separating the edible part. Waste can be used effectively and can be manufactured at low cost.
Further, in the conventional method for detecting PTP inhibitory activity, the inhibitory activity against the action of PTP on the synthetic substrate (Ala-Phe-Pro-βNA) was confirmed. However, in the method for detecting PTP inhibitory activity of the present invention, the synthetic substrate By using gelatin closer to gingival collagen as a substrate, it is possible to more accurately predict the growth inhibitory action of periodontal disease bacteria in vivo and the action of suppressing gingival regression in periodontal disease. This is because gelatin is obtained by denaturing and / or degrading natural collagen. Moreover, according to this detection method, it is possible to easily and easily detect the PTP inhibitory activity, and thus the growth inhibitory effect of periodontal disease bacteria and the gingival regression inhibitory action in periodontal disease.

It is a figure which shows the process of isolating and refine | purifying the protein which has PTP inhibitory activity from rice bran. Example 1 It is a figure which shows the fractionation result (FIG. 2a-d) of a chromatography and the result of SDS-electrophoresis (FIG. 2e) in each process of isolation / purification of the protein which has PTP inhibitory activity from rice bran. Example 1 It is a figure which shows the result of having analyzed the amino acid sequence about 25 kDa protein obtained by SDS-electrophoresis using TOF-MS. Example 1 It is a figure which shows the structure (FIG. 4a) of the recombinant gene for expressing the protein which has PTP inhibitory activity of this invention by gene recombination, and the result (FIG. 4b) which carried out SDS-electrophoresis of protein. (Example 2) It is a figure which shows the result of having confirmed PTP inhibitory activity about the protein obtained by gene recombination. (Example 2) It is a figure which shows the result of having confirmed the stability of the protein of this invention refine | purified from the rice bran. (Experimental example 1) It is a photograph figure which shows the result of having confirmed the PTP inhibitory activity by the protein of this invention using a periodontal disease model. (Example 3) It is a photograph figure which shows the result which confirmed the periodontal disease bacteria growth inhibitory effect by the protein of this invention refine | purified from the rice bran. Example 4

  The present invention relates to a PTP inhibitor, and the PTP inhibitor of the present invention acts as a periodontal disease protease inhibitor. As periodontal disease bacteria that cause periodontal disease, dozens of aerobic or anaerobic bacteria are known. Examples of periodontal disease bacteria include Porphyromonas gingivalis (hereinafter simply referred to as “P. gingivalis”), Tannerella forsythensis, Treponema denticola, Prevotella interticola, Media (Prevotella intermedia), Actinobacillus actinomycetemcomitans, etc. are mentioned. In the present invention, the periodontal disease bacteria is preferably P. gingivalis.

  PTP is an enzyme involved in the degradation of gingival collagen. PTP is a serine protease belonging to the prolyl oligopeptidase family (POP family), and exhibits a degrading activity against peptides whose Pro is the third residue from the N-terminus. The PTP inhibitor of the present invention has an action of inhibiting the degradation activity of PTP. PTP is one of periodontal disease proteases. Periodontal disease protease is a general term for enzymes that catalyze the hydrolysis of peptide bonds (-CO-NH-) and includes proteinases and peptidases. Examples of the protease of P. gingivalis in the present invention include gingipain (trypsin-like protease), PTP and the like produced by P. gingivalis.

The protein contained as an active ingredient in the PTP inhibitor of the present invention is any protein selected from (a) to (c).
(A) A protein comprising the amino acid sequence shown in SEQ ID NO: 1.
(B) in the amino acid sequence shown in SEQ ID NO: 1, comprising one or several (preferably 1 to 15) amino acid sequences deleted, substituted, inserted and / or added, and against PTP A protein having inhibitory activity.
(C) a protein comprising an amino acid sequence having homology of 90% or more (preferably 95% or more, more preferably 98% or more) with the amino acid sequence shown in SEQ ID NO: 1 and having an inhibitory activity against PTP.
The protein (b) having a mutation such as deletion, substitution, insertion and / or addition or the protein having a specific homology of (c) may exist in nature, and It may be obtained by introducing a mutation on the basis thereof. Means for introducing mutations are known per se. For example, site-directed mutagenesis, gene homologous recombination, primer extension or polymerase chain reaction (hereinafter abbreviated as PCR) is used alone or in appropriate combination. it can. For example, the method described in the book (edited by Sambrook et al., “Molecular Cloning, Laboratory Manual Second Edition”, 1989, Cold Spring Harbor Laboratory; Muramatsu Masami, “Lab Manual Genetic Engineering”, 1988, Maruzen Co., Ltd.) or by modifying those methods. Ulmer's technology (Ulmer, KM, Science), 1983, Vol. 219, p.666 -671) can also be used. In the case of a protein, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the protein upon introduction of mutation, for example, homologous amino acids (polar amino acids, nonpolar amino acids, Mutual substitution between hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisioned.

The amino acid sequence shown in SEQ ID NO: 1 is shown below:
Met Ala Leu Arg Thr Leu Ala Ser Arg Lys Thr Leu Ala Ala Ala Ala Leu Pro Leu Ala Ala Ala Ala Ala Ala Arg Gly Val Thr Thr Val Ala Leu Pro Asp Leu Pro Tyr Asp Tyr Gly Ala Leu Glu Pro Ala Ile Ser Gly Glu Ile Met Arg Leu His His Gln Lys His His Ala Thr Tyr Val Ala Asn Tyr Asn Lys Ala Leu Glu Gln Leu Asp Ala Ala Val Ala Lys Gly Asp Ala Pro Ala Ile Val His Leu Gln Ser Ala Ile Lys Phe Asn Gly Gly Gly His Val Asn His Ser Ile Phe Trp Asn Asn Leu Lys Pro Ile Ser Glu Gly Gly Gly Asp Pro Pro His Ala Lys Leu Gly Trp Ala Ile Asp Glu Asp Phe Gly Ser Phe Glu Ala Leu Val Lys Lys Met Ser Ala Glu Gly Ala Ala Leu Gln Gly Ser Gly Trp Val Trp Leu Ala Leu Asp Lys Glu Ala Lys Lys Leu Ser Val Glu Thr Thr Ala Asn Gln Asp Pro Leu Val Thr Lys Gly Ala Asn Leu Val Pro Leu Leu Gly Ile Asp Val Trp Glu His Ala Tyr Tyr Leu Gln Tyr Lys Asn Val Arg Pro Asp Tyr Leu Ser Asn Ile Trp Lys Val Met Asn Trp Lys Tyr Ala Gly Glu Val Tyr Glu Asn Ala Thr Ala
The amino acid sequence of SEQ ID NO: 1 is based on the base sequence of the gene registered in Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp/) as Os05g0323900. The amino acid sequence of SEQ ID NO: 1 is encoded by the 1st to 693rd base sequence of the gene of Os05g0323900, the number of amino acid residues is 231 and the estimated molecular weight is 24998.26. The protein having the amino acid sequence shown in SEQ ID NO: 1 has high temporal stability even in a state dissolved in a liquid, and has excellent utility in application. The protein also has an activity of inhibiting the action of PTP on gelatin closer to gingival collagen than a synthetic substrate. In periodontal disease, PTP acts on a peptide fragment decomposed by collagenase, and is considered to be an enzyme that controls the growth and proliferation of periodontal disease bacteria or gingival regression in periodontal disease. The PTP inhibitor of the present invention is expected to be able to effectively suppress the growth and proliferation of periodontal disease bacteria and gingival regression caused by PTP in periodontal disease.

  The protein which is the active ingredient of the present invention may be a part or all of the protein. The protein, which is the active ingredient of the present invention, as a whole has a molecular weight of about 25 kDa when detected using SDS electrophoresis. A part of the protein (partial peptide) that can be used as the inhibitor of the present invention comprises an active site region that can exhibit PTP inhibitory activity. The size of the partial peptide is not particularly limited, but the total number of amino acid residues is about 100 or less, more preferably about 50 residues or less, and still more preferably about 20 residues or less. It is considered that the smaller the size, the easier the handling, the higher the production efficiency, and the reduction of side effects such as antigenicity. Furthermore, the protein which is an active ingredient of the present invention may be a complex with another substance such as another protein or a low molecular compound as long as the protein can exhibit PTP inhibitory activity. The molecular weight of the complex may be anything. Examples of the other protein in the complex include those having a function of solubilizing the protein that is the active ingredient of the present invention.

  The protein that is the active ingredient of the present invention may be a derivative of the protein as long as it can exhibit PTP inhibitory activity. In the protein derivatives, some or all of the functional groups such as amino group, carboxyl group, hydroxyl group and thiol group in the amino acid sequence of the protein are modified with other appropriate substituents (such as phosphate group and acetyl group). When you receive it. Furthermore, the derivative of the protein of the present invention may include a pharmaceutically acceptable salt of the protein. Preferred examples of the salt include alkali metal or alkaline earth metal salt such as sodium salt or calcium salt, hydrohalide salt such as hydrofluoric acid salt, hydrochloride salt, nitrate salt, sulfate salt, phosphate salt and the like. Inorganic acid salts, lower alkyl sulfonates such as ethane sulfonate, organic acid salts such as fumarate, succinate, and acetate. Furthermore, the protein may be a solvate. Examples of such solvates include hydrates, alcohol solvates such as ethanol solvates, and ether solvates.

  The protein which is an active ingredient of the present invention can be extracted and produced from a cereal plant-derived material. Examples of the cereal plant-derived material include cereal plants, preferably gramineous plants, more preferably rice, barley, wheat, rye, cotton, corn, corn, and the like, and most preferably rice. Examples of the cereal plant-derived material of the present invention include waste generated after collecting an edible part from a cereal plant, and examples include rice bran that is generated after milling. The cereal plant-derived material may contain not only waste but also an edible portion. The cereal plant-derived material may use the endosperm portion of rice as a main material, and may use old rice. The protein fraction can be separated from the cereal plant-derived material according to a conventional method. For example, the protein fraction can be obtained by grinding or suspending cereal plant-derived material in a solvent such as water or phosphate buffer, collecting the supernatant by filtration or centrifugation, and diluting or concentrating as necessary. Can be obtained. Then, use methods commonly used for protein separation and purification, such as salting out using ammonium sulfate, precipitation with organic solvents, hydrophobic chromatography, ion exchange chromatography, and gel filtration chromatography. Thus, the target protein fraction can be separated. The protein that is the active ingredient may be used after separation and purification. However, as long as it contains an effective amount of the protein, the extract or the like may be used as it is without separation and purification. The protein of the present invention is obtained by grinding or suspending the cereal plant-derived material in a solvent, collecting the supernatant by filtration or centrifugation, performing salting out after dilution or concentration, and precipitating with an organic solvent, Thereafter, the protein fraction obtained by collecting the active fraction by hydrophobic chromatography may be used.

  Proteins that are active ingredients can also be produced by known genetic engineering techniques. That is, based on the base sequence shown in Os05g0323900, a gene encoding a protein that is an active ingredient is isolated or synthesized, a recombinant expression vector is constructed, and this is introduced into an appropriate host cell as a recombinant protein. It can be produced by expression. Alternatively, it can be produced by an in vitro transcription / translation system. When the protein which is an active ingredient of the present invention is a complex with other proteins, these proteins can be produced by expressing them as fusion proteins by a known technique. When the host cell is Escherichia coli, a recombinant protein can be prepared from DNA consisting of the base sequence shown in SEQ ID NO: 2 described later.

  A protein as an active ingredient can also be produced by a solid phase synthesis method (Fmoc method, Boc method) or a liquid phase synthesis method according to a known general peptide synthesis protocol.

  That the obtained protein has PTP inhibitory activity can be confirmed by a known method for measuring PTP activity. Specifically, it can be performed by a PTP inhibitory activity confirmation experiment using Ala-Phe-Pro-βNA described in Examples described later as a substrate. Alternatively, it can be carried out by the method for detecting PTP inhibitory activity of the present invention using gelatin closer to gingival collagen as a substrate. Furthermore, it can be confirmed by a known method for measuring periodontal disease protease activity that the protein has a periodontal disease protease inhibitory action.

  Furthermore, the PTP inhibitor of the present invention exhibits an action of suppressing the growth of periodontal disease bacteria, and can also be used as a periodontal disease bacteria growth inhibitor. In addition, the PTP inhibitor of the present invention can be used as an agent for preventing and / or treating periodontal disease. The present invention also extends to a method for preventing and / or treating periodontal disease using a PTP inhibitor. In vivo, gelatin is produced as a large peptide fragment from gingival tissue by the action of collagenase. However, periodontal disease bacteria cannot assimilate this in the gelatin state. By decomposing the produced gelatin into even smaller amino acids by the action of PTP, it is taken up by periodontal pathogens for the first time, allowing growth of periodontal pathogens. Since the PTP inhibitor of the present invention inhibits the degradation of gelatin by PTP, it is considered that the growth of periodontal disease bacteria can be inhibited. The PTP inhibitor of the present invention can be advantageously used for the prevention and / or treatment of periodontal disease because it can also suppress the growth of periodontal disease bacteria and suppress the regression of gingiva.

  A composition may be sufficient as the PTP inhibitor of this invention, and the compounding ratio or content ratio of the active ingredient in the said composition will not be specifically limited if it is sufficient quantity for the desired PTP inhibitory effect being acquired. Usually, it is about 0.0001-20 mass% of the whole composition, Preferably it is 0.001-10 mass%, More preferably, it is 0.001-5 mass%. The PTP inhibitor of the present invention may contain components other than the active ingredient as long as the PTP inhibitory action of the active ingredient is not hindered. Ingredients other than the active ingredient are not particularly limited, and can be appropriately selected according to the use described later.

  The PTP inhibitor of the present invention can be used as food and drink, pharmaceuticals, quasi drugs, feed, food additives, feed additives, and the like. Especially, it is preferable to use for food-drinks, a pharmaceutical, and a quasi drug. The PTP inhibitor of the present invention is preferably an oral composition. The periodontal disease protease inhibitor of the present invention is an oral composition administered to mammals including humans, and can also be administered to non-human mammals such as domestic animals and pet animals.

  The food and drink comprising the PTP inhibitor of the present invention is mixed with food hygienically acceptable additives, special purpose food, food for specified health use, nutritional functional food, health food, nutritional supplement, enteral It can be processed into nutritional foods and beverages. The form of the food or drink is not particularly limited, and can be provided as a solid food, a liquid food including a beverage, or other various forms of food or drink. Specific examples of the beverage include fruit juice beverages, soft drinks, and alcoholic beverages. Further, it may be in a form of being diluted with water or the like when ingested. Examples of the solid food include tablets (tablets) and sugar-coated tablets including candy, troches, etc., granule forms, powder forms such as powdered drinks, forms of block confectionery such as biscuits, forms of capsules, jelly, etc. There are various forms of food. Examples of the semi-solid food include a paste form such as jam and a gum form such as chewing gum.

  In addition to the PTP inhibitor of the present invention, food materials and food additives can be blended in the food and drink as long as the intended effects are not impaired. A food material is a material that is generally used as a raw material for food. Except for pharmaceuticals and quasi-drugs specified by the Pharmaceutical Affairs Law and food additives specified by the Food Sanitation Law, they are used for food and drink. Everything is included. A food additive is a substance added for the purpose of processing or storing food. Examples of food additives are those specified by the Ministry of Health, Labor and Welfare's “designated additive list”, “existing additive list item list”, “natural fragrance-based raw material list”, and “food that is generally used for food and drink. Food additives listed in the “List of Items Used as Additives”, etc., those whose safety has been confirmed by international organizations such as JECFA, and food additives that have been approved for use in other countries such as the US and Europe Preservatives, shelf-life improvers, antioxidants, sweeteners, colorants / pigments, emulsifiers, thickening gelling agents, quality improvers, seasonings, acidulants, strengthening agents, fragrances, enzymes, etc. are categorized.

  The pharmaceutical and quasi drug containing the PTP inhibitor of the present invention can be formulated by appropriately blending a pharmaceutically acceptable carrier and additives in addition to the PTP inhibitor. Specifically, oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions and emulsions can be used. Carriers or additives that can be blended are not particularly limited. For example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.

  Additives include binders, excipients such as crystalline cellulose, swelling agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, peppermint, red mono oil Alternatively, a flavoring agent such as cherry is used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc.

  In addition, the composition for oral cavity containing the PTP inhibitor of the present invention comprises known active (medicinal) ingredients, abrasives, wetting agents, thickeners, surfactants, fragrances, sweeteners in addition to the active ingredients. Colorants, preservatives, pH adjusters, and the like can be appropriately selected and blended. Specifically, effective (medicinal) components such as enzymes, anti-plaque agents, antibacterial agents, vitamins, fluorides, metal salts; calcium hydrogen phosphate, calcium carbonate, aluminum hydroxide, aluminum oxide, anhydrous silicic acid, etc. Wetting agents such as sugar alcohols such as lactitol, 1,3-butylene glycol, 1,2-pentanediol, polypropylene glycol, dipropylene glycol, etc .; polyethylene glycol, glycerin, sodium carboxymethylcellulose (CMC / sodium salt), thickeners such as carrageenan and xanthan gum; surfactants such as sodium lauryl sulfate; fragrances such as menthol, mint oil, l-carvone, sweeteners such as saccharin and steviosides, various pigments, etc. Coloring agents and the like are exemplified.

  The active ingredient of the PTP inhibitor of the present invention is an ingredient contained in the cereal plant-derived material, has high safety, has a mild action, and is preferably taken or used for a long time. In addition, when the PTP inhibitor of the present invention is used in combination with other antibacterial agents, an additive or synergistic effect can be expected. The PTP inhibitor of the present invention is also expected to have an inhibitory action on periodontal disease bacterial proteases of mammals such as dogs other than humans.

  The present invention includes a method for detecting PTP inhibitory activity, which comprises the step of contacting PTP with a structure containing gelatin in a medium in the presence of a PTP inhibitor and detecting PTP activity. In the detection method of the present invention, the PTP activity in the absence of a PTP inhibitor or the PTP activity in the presence of another PTP inhibitor is used as a reference, and the PTP activity in the presence of the target PTP inhibitor is used as the reference. By comparing with PTP activity, PTP inhibitory activity can be detected. The reference PTP activity may be detected at the same time as the PTP activity in the presence of the target PTP inhibitor, or may be detected in advance.

Furthermore, this detection method preferably includes the following steps 1) and 2).
1) contacting a structure containing gelatin with PTP in a medium in the presence and absence of a PTP inhibitor;
2) A step of detecting PTP inhibitory activity by a PTP inhibitor by comparing PTP activity in the presence and absence of the PTP inhibitor.
In the method for detecting PTP inhibitory activity of the present invention, the experimental system in which a structure containing gelatin and PTP are brought into contact can be used as a periodontal disease model (gingiva model) because gelatin close to gingival collagen is used as a substrate. It is thought that. In addition, the PTP inhibitor used in the detection method of the present invention may be a candidate substance for a PTP inhibitor or may not have been confirmed to have a PTP inhibitory activity. The detection method of the present invention can also be used as a screening method for PTP inhibitors. In vivo, gelatin is produced as a large peptide fragment from gingival tissue by the action of collagenase, and the produced gelatin is broken down into smaller amino acids by the action of PTP. Only after the amino acid is decomposed, it is taken up by periodontal disease bacteria, and periodontal disease bacteria can grow. The method for detecting the PTP inhibitory activity of the present invention can be used as a method for screening a substance having an action of suppressing gingival regression and / or an effect of inhibiting the growth of periodontal bacteria. It can be used as a method for screening therapeutic agents and / or inhibitors.
Note that a method including a step of bringing a structure containing gelatin into contact with PTP in a medium in the absence of a PTP inhibitor can also be used as a method for detecting PTP activity.

  In step 1), the medium may be any medium that does not affect the action of PTP and / or the action of the PTP inhibitor, such as water or a buffer solution. Preferably, a Tris-HCl buffer solution, a phosphate buffer solution or the like can be used. The conditions (temperature, time, etc.) for bringing the structure containing gelatin into contact with PTP are not particularly limited as long as the action of PTP and the action of the PTP inhibitor can be confirmed. For example, the contact may be performed at room temperature for 1 hour to 7 days.

  There are two methods for detecting the degrading enzyme inhibitory effect of a degrading enzyme inhibitor: a system in which a degrading enzyme is contained in a medium (aqueous liquid) and a system in which a degrading enzyme and a degrading enzyme inhibitor are contained in a medium (aqueous liquid) Inhibitory activity is confirmed by comparing the action of the system on the substrate. In order to confirm the inhibitory activity, it is convenient to use an indicator substance. In the method for detecting a PTP inhibitory activity of the present invention, a structure containing gelatin contacts the medium of the above two systems, and PTP decomposes gelatin. When the gelatin is decomposed, the indicator substance contained in the gelatin layer flows out into the medium (aqueous liquid), or when the gelatin layer decomposes and disappears, the indicator substance in the lower layer of the gelatin layer is It flows into the medium (aqueous liquid) and the indicator substance is contained in the medium. As a result, the index substance contained in the medium can be detected by appearance, concentration measurement, etc., and the effect of the PTP inhibitor substance can be detected or determined. The indicator substance contained in the structure may be a pigment or the like that exhibits color itself. Alternatively, in the method for detecting PTP inhibitory activity of the present invention, the indicator substance may be colored by reacting with gelatin or a degradation substance produced by degrading gelatin with PTP. Such an indicator substance may be contained in a medium (aqueous liquid) and detected by coloration or the like to detect or determine the PTP inhibitory activity. If the indicator substance is a dye, it is preferable because it is easy to detect even with an external appearance or a simple colorimeter.

  The structure containing gelatin is a structure containing at least gelatin, and may be a structure in which part or all of the surface is coated with gelatin, or a single gelatin such as plate gelatin. The structure may be one in which part or all of the surface thereof is covered with a gelatin film including a single or a plurality of gelatin layers. In order to detect PTP inhibitory activity in a short time, the surface area of the structure is preferably large. For example, a sheet-like structure is preferable as the structure having a large surface area. The structure containing gelatin is preferably one containing an indicator substance, preferably a dye. The indicator substance may be contained in the structure in such a manner that the indicator substance is eluted when the gelatin layer on the surface of the structure is decomposed. For example, if there is an indicator substance under a gelatin layer having a certain thickness, it is possible to evaluate the time for which the gelatin having a certain thickness is decomposed. The indicator substance may be included in the structure by separately providing an indicator substance layer below the gelatin layer, or may be included in the structure by being included in the gelatin layer. If a soluble indicator substance is contained in the gelatin layer, the indicator substance is eluted in the medium liquid in accordance with the decomposition of gelatin. By appropriately adjusting the uniformity of the quality of gelatin, the thickness of the gelatin layer, the concentration of the indicator substance, the thickness of the indicator substance layer, etc., it becomes possible to detect the PTP inhibitory activity with high accuracy. When gelatin is decomposed by PTP, the form of the structure changes, or the indicator substance contained in the structure is eluted into the medium, and PTP activity can be detected.

  Gelatin is denatured and / or converted into a single polypeptide chain that is soluble in water by treating collagen molecules having a molecular weight of about 100,000 with heat, acid, alkali, or the like. Alternatively, it is a polymer chain aggregate that has been decomposed and has a molecular weight distribution in the range of tens of thousands to millions. As the gelatin in the present invention, acid-treated gelatin and other various modified gelatins (including polymerized gelatin) used for photographic film and photographic paper can be preferably used.

  As the structure containing gelatin of the present invention, a silver salt color photographic film (preferably a color negative film, a color positive film, or a reversal film) is preferably used. Alternatively, paper-based color photographic printing paper can be preferably used as well. Commercially-produced silver salt color photographic films and color photographic printing papers can be used. This is because they are inexpensive and easily available, and have extremely high uniformity such as gelatin quality, gelatin layer thickness, dye concentration, and dye layer thickness. Silver salt photographic film is produced by applying silver particles and color former (coupler) together with gelatin to a sheet (or film) base material such as polyester or acetate and drying it. It has a structure in which a gelatin layer is provided thereon. Specific examples of the polyester include polyethylene terephthalate, and specific examples of the acetate include cellulose acetate. Silver grains are silver halide grains. A silver salt photographic film having a structure in which a laminated gelatin layer is provided on a base material can be obtained by simultaneously coating and drying silver particles and a coupler together with gelatin. The gelatin layer is preferably water-resistant by crosslinking with an appropriate crosslinking agent, and is preferably prevented from being dissolved during development processing. The silver salt color photographic film is preferably a three-layer emulsion type color film. The three-layer emulsion type color film has a red-sensitive layer, a green-sensitive layer, and a blue-sensitive layer. As a result, each coupler that develops red, green, and blue colors is laminated in gelatin. Is. A silver salt color photographic film that has been exposed and developed can be used. The exposure light may be white light or light of a specific wavelength, such as red, blue, green, etc., and the color may be developed to a hue suitable for absorption analysis after development by adjusting the exposure conditions. The exposed film was subjected to normal development processing. In parallel with the color development of the coupler in the development processing, the reduced silver particles were oxidized to hypo silver in the bleaching step and dissolved and removed in the fixing step. It is preferable to use one. In the developed silver salt color photographic film, the dye molecules formed by the coupler are contained in the gelatin layer.

  “PTP” is not limited as long as it is a PTP enzyme, and may be an extract or a synthetic product. For example, soluble PTP expressed by gene recombination according to the description in Non-Patent Document 3 may be used.

  In step 2), PTP activity is compared in the presence and absence of a PTP inhibitor. PTP activity can be detected by confirming the degree of dissolution due to gelatin degradation. In this detection method, the degree of degradation of gelatin may be confirmed by confirming the morphological change of gelatin itself (degree of dissolution or swelling in water) and the amount of indicator substance (for example, elution of the indicator substance into the medium). By comparing the PTP activity in the presence or absence of the PTP inhibitor, the PTP inhibitory activity by the PTP inhibitor can be detected. In this detection method, detection of PTP activity in the absence of a PTP inhibitor may or may not be performed every time, but is preferably performed every time. The change in the form of gelatin and the elution of the indicator substance can be visually confirmed, and the PTP inhibitory activity by the PTP inhibitor can be measured or detected easily. In particular, the elution of the dye can be quantified by measuring the absorbance at a specific wavelength.

  Hereinafter, representative examples of the present invention will be described with reference to examples, but the technical scope of the present invention is not limited to the examples and experimental examples.

(Example 1) Purification of protein having PTP inhibitory activity from rice bran Reagent According to the method described in Non-Patent Document 3, the PTP gene of periodontal disease bacteria was expressed in Escherichia coli to prepare soluble PTP. Nessler's reagent was purchased from Wako Pure Chemical. The synthetic substrate Ala-Phe-Pro-βNA was purchased from BACHEM. Toyopearl HW-65 and DEAE-Toyopearl gels from Toyo Soda were used for electrophoresis gel (e-PAGEL purchased from Ato). Sephacryl S-200 and Ni-Sepharose Fast Flow were manufactured by GE Healthcare Japan

2. Preparation of sample from rice bran First, a buffer solution (20 mM Tris-HCl pH 7.2) was mixed with rice bran to prepare a raw material solution. The raw material solution was placed in a centrifuge tube and centrifuged at 15000 rpm for 10 minutes at 5 ° C. with a centrifuge. After centrifugation, the supernatant was placed in a glass container for sample storage. Stored at -80 ° C until use.

3. PTP inhibitory activity confirmation experiment
Add buffer (20 mM Tris-Hcl pH7.2) 70 μL, synthetic substrate 2 mM (Ala-Phe-Pro-βNA) 10 μL, protein solution (inhibitor) 10 μL to 96-well plate, and keep warm in a 37 ° C constant temperature bath. did. After 10 μl of enzyme was added, 10 minutes later, 50 μL of Fast Garnet GBC (containing 4% acetate buffer pH 4.0 containing Triton X-100 10%) was added to stop the reaction. After stopping the reaction, the absorbance at OD 550 nm was measured with an absorptiometer.
The enzyme inhibition rate was calculated as follows. First, a value ΔOD obtained by subtracting the blank OD from the measured absorbance (OD) was calculated. Blank is 10 μL of buffer instead of sample. Subsequently, the enzyme inhibition rate was calculated by the following formula.
Enzyme inhibition rate = 100− {ΔOD (with inhibitor) / ΔOD (without inhibitor)} × 100

4). Purification of protein with PTP inhibitory activity About 500 g of rice bran was added with a buffer solution (20 mM Tris-HCl pH 7.2) at 20 g / 50 mL, filtered with medical gauze, and centrifuged at 15,000 rpm for 15 minutes at 5 ° C. separated. To the supernatant separated after centrifugation, ammonium sulfate was added so as to be 40% saturated, left in a refrigerator at 5 ° C. for about 1 day, and centrifuged again at 5 ° C. for 15 minutes at 15000 rpm.
Ammonium sulfate was added to the supernatant separated after centrifugation so as to be 80% saturation, left in a refrigerator at 5 ° C. for about 1 day, and centrifuged again at 5 ° C. for 15 minutes at 15000 rpm. In the purification experiment of the protein having PTP inhibitory activity, all operations including storage, chromatography and centrifugation were performed at 5 ° C. in order to prevent inactivation of the protein.

  After centrifugation, the supernatant was discarded to obtain a precipitate. The precipitate was dissolved in a solution of 20 mM Tris-HCl pH 7.2 containing 40% saturated ammonium sulfate and subjected to hydrophobic chromatography with Toyopearl HW-65C in a cold room (4 ° C.) (column size 5 cm × 15 cm). . For each fraction, the amount of protein was measured by measuring the absorbance at 280 nm, and the PTP inhibitory activity was measured by a PTP inhibitory activity confirmation experiment. The result is shown in FIG. The portion where the peak of PTP inhibitory activity was collected was collected, added to the collected solution so that ammonium sulfate was 80% saturated, left in a refrigerator for about 1 day, and centrifuged at 15,000 rpm for 15 minutes at 5 ° C.

  After centrifugation, the supernatant was discarded to obtain a precipitate. The precipitate was dissolved with 20 mM Tris-HCl pH 7.2. The solution was dialyzed using a Visking tube. Dialysis was performed by changing the external solution until the Nessler reagent did not change color. The dialyzed solution was subjected to ion exchange chromatography using DEAE Toyopearl in a low temperature chamber (4 ° C.) (column size: 5 cm × 15 cm). The result is shown in FIG. For each fraction, the amount of protein was measured by measuring the absorbance at 280 nm, and the PTP inhibitory activity was measured by a PTP inhibitory activity confirmation experiment. The part having the peak PTP inhibitory activity was collected. To the collected solution, ammonium sulfate was added so as to become 80% saturated, and left in a refrigerator for about 1 day, followed by centrifugation at 15,000 rpm for 15 minutes at 5 ° C.

  After centrifugation, the supernatant was discarded to obtain a precipitate. The precipitate was dissolved with Tris-HCl pH 7.2. The solution was dialyzed using a Visking tube. Dialysis was performed by changing the external solution until the Nessler reagent did not change color. The dialyzed solution was subjected to gel filtration chromatography using Sephacryl S-200 in a cold room (4 ° C.) (column size 2.5 cm × 35 cm). The result is shown in FIG. For each fraction, the amount of protein was measured by measuring the absorbance at 280 nm, and the PTP inhibitory activity was measured by a PTP inhibitory activity confirmation experiment. The part having the peak PTP inhibitory activity was collected. To the collected solution, ammonium sulfate was added to 80% saturation, left in a refrigerator for about 1 day, centrifuged at 15000 rpm for 15 minutes, and the supernatant was discarded to obtain a precipitate.

  Since the obtained protein contained a small amount of impurities, gel filtration chromatography (rechromatography) was again performed using Sephacryl S-200 by the same method as described above. The portion where the peak of PTP inhibitory activity was collected was collected, and the sample was subjected to SDS electrophoresis with and without heating according to a conventional method. A dark band having a molecular weight of 25 kDa in common and a minor band that was not heated were observed (FIG. 2e).

5. Identification of a protein having PTP inhibitory activity Among the separated proteins, a protein fraction having a molecular weight of 25 kDa is cut out from an electrophoresis gel by a conventional method, and matrix-assisted laser ionization (MALDI) -time-of-flight (TOF) mass spectrometer MS / MS analysis was performed using AXIMA Performance (manufactured by Shimadzu Corporation) (FIG. 3a). From the obtained MS / MS data, a sequence was searched using a database (Rice Annotation Project DataBase (http://rapdb.dna.affrc.go.jp/index.html)) to identify a protein (FIG. 3b). ).
As a result, it was found that the protein having a molecular weight of 25 kDa obtained by purification corresponds to rice Os05g0323900.

(Example 2) Production of a protein having PTP inhibitory activity by gene recombination After subjecting a cDNA encoding Os05g0323900 protein to restriction enzyme treatment by a conventional method, the restriction enzyme-treated fragment is converted into a polynucleotide encoding Trx (thioredoxin). And fused with His-Tag and introduced into the vector pET-32a (FIG. 4a). The cDNA encoding the Os05g0323900 protein was prepared by chemical synthesis by optimizing the base sequence encoding the amino acid sequence of the protein from the rice database to the E. coli codon for expression in E. coli. The chemically synthesized cDNA is shown below. In the following base sequences, a BamHI site (ggatcc) is added on the 5 ′ side, and an XhoI site (ctcgag) is added on the 3 ′ side for cloning.
CDNA encoding Os05g0323900 protein (SEQ ID NO: 2):
ggatccATGGCCCTTCGGACTCTCGCCTCGCGCAAAACCTTGGCCGCCGCAGCGCTGCCACTCGCTGCGGCAGCAGCTGCACGTGGTGTTACCACAGTGGCATTGCCGGATCTGCCCTATGACTATGGCGCGTTAGAACCGGCCATTAGTGGCGAGATTATGCGCCTGCATCACCAGAAACACCATGCCACGTATGTGGCGAACTACAACAAAGCCCTTGAACAGCTGGATGCTGCAGTAGCGAAAGGGGATGCACCAGCGATTGTCCATCTGCAGAGTGCGATCAAGTTCAATGGTGGAGGCCACGTGAATCACAGCATCTTCTGGAACAACCTCAAACCGATCTCTGAAGGAGGTGGTGATCCGCCTCATGCGAAACTGGGTTGGGCAATTGACGAGGATTTTGGCTCCTTTGAAGCTCTGGTCAAAAAGATGAGCGCGGAAGGTGCGGCGTTGCAAGGCTCAGGGTGGGTTTGGTTAGCCCTGGACAAAGAAGCGAAAAAGCTGTCGGTGGAAACCACTGCGAATCAAGACCCTCTTGTAACGAAAGGCGCTAATCTGGTTCCGTTACTAGGGATAGATGTCTGGGAGCATGCCTATTACCTGCAGTACAAGAACGTTCGTCCGGATTATCTGAGCAACATCTGGAAAGTGATGAACTGGAAATATGCGGGCGAAGTGTACGAGAATGCTACCGCCctcgag

The vector was introduced into E. coli, and a protein in which Trx and Os05g0323900 were fused (Trx-Os05g0323900 fusion protein) was expressed in E. coli. After culturing the E. coli, the E. coli was collected by centrifugation (15000 rpm, 15 min). The recovered E. coli is washed with 20 mM Tris-HCl, pH 7.4 containing 10 mM Na, lysed with a solution containing nuclease, centrifuged, and then the supernatant is added to a 7-fold amount of 20 mM Na phosphate buffer. Dissolved and adsorbed on a Ni-Sepharose column (Ni-Sepharose Fast Flow, manufactured by GE Healthcare Japan Co., Ltd.) and eluted with the same phosphate buffer containing 250 mM Imidazole to isolate and purify the Trx-Os05g0323900 fusion protein (Figure 4b). The molecular weight of Trx was 18786, and the molecular weight of the Trx-Os05g0323900 fusion protein was about 44 kDa.
The purified Trx-Os05g0323900 fusion protein was dissolved in 20 mM Tris buffer so that the amount of protein was 430 μg / mL to prepare a sample. The amount of protein was quantified by absorbance at 280 nm. A PTP inhibitory activity confirmation experiment was performed in the same manner as in Example 1 except that the amount of the protein solution added was changed using the prepared sample. As a control, a protein containing only Trx was expressed in the same manner as the Trx-Os05g0323900 fusion protein, dissolved in 20 mM Tris buffer so that the protein amount was 430 μg / mL, and a control sample was prepared.

  As a result, it was found that the protein containing only the control Trx did not show PTP inhibitory activity, whereas the Trx-Os05g0323900 fusion protein (“Trx-Os05g0323900” in FIG. 5) showed PTP inhibitory activity. (FIG. 5).

(Experimental example 1) Confirmation of stability of protein having PTP inhibitory activity purified from rice bran
As a protein having PTP inhibitory activity, a protein purified by the steps up to hydrophobic chromatography in the same manner as in Example 1 was used. The protein was lyophilized after purification and stored at room temperature for about 2 months. The protein was dissolved in 20 mM Tris-HCl pH 7.2 buffer to prepare a 1 mg / mL PTP inhibitor solution. The prepared PTP inhibitor solution was kept at 37 ° C., and inhibitory activity was confirmed after 0 hours, 0.25 hours, 1 day, 4 days, 6 days, 12 days, and 15 days. Confirmation of the inhibitory activity was carried out in 3 of Example 1. The same experiment was conducted.

  The result is shown in FIG. The PTP inhibitor dissolved in the solution and maintained an inhibitory activity of 90% or more even when kept at 37 ° C. for 2 weeks, indicating that the stability was high.

(Example 3) Confirmation of action of protein having PTP inhibitory activity in periodontal disease model In order to confirm the action of inhibiting the degradation of gingiva by the protein having PTP inhibitory activity of the present invention, it was applied to a gelatin film used for photographic film. The inhibition of PTP degradation was evaluated. As a protein having PTP inhibitory activity, a protein purified by the steps up to hydrophobic chromatography in the same manner as in Example 1 was used. A color photographic film is obtained by multilayer coating of a plastic film surface with a gelatin film containing silver halide grains together with a coupler as a color former. Since the gelatin film is water-resistant using a cross-linking agent, it usually does not dissolve even when immersed in water for a long time at room temperature. In this example, a color photographic negative film was exposed to white light as a photographic film and developed (bleached by Fuji Photo Film, product number: FUJICOLOR SUPERIA X-TRA400) after being developed, bleached and fixed. A photographic film colored in gray as a whole by using three color couplers to form a gelatin film containing three color dyes in each gelatin layer was used in this example. The above photographic film was placed in a 20 mM Tris-HCl pH 7.2 solution containing 1 mg / mL PTP (prepared in Example 1) and 5 mg / mL of a protein having PTP inhibitory activity purified from rice bran as described in Example 1. After immersion, changes were observed after 4 hours and 4 days. This experiment was performed at room temperature.

  The result is shown in FIG. The upper part of FIG. 7 shows the results after 4 hours, and the lower part of FIG. 7 shows the results after 4 days. (A) does not contain a protein having PTP inhibitory activity, and is a control solution containing only PTP, and (B) shows the result of a solution containing PTP and a protein having PTP inhibitory activity. By dissolving PTP in the crosslinked gelatin film on the photographic film, the dye molecules contained in the gelatin film were dissolved and the solution was colored. In (A), coloring of the solution was observed after 4 hours, so that at least partial dissolution of the gelatin film was observed. In (B), no change was observed in appearance, and a change was observed in the gelatin film. There wasn't. Further, in (A), it was confirmed that the gelatin film existing on the surface of the photographic film was completely dissolved after 4 days. On the other hand, in (B), although the gelatin film was peeled off from the film, the peeled gelatin film itself was dissolved. Without leaving the original film shape. Therefore, it was shown that the protein having PTP inhibitory activity of the present invention can also inhibit the action of PTP on gelatin.

(Example 4) Effect of a protein having PTP inhibitory activity on the growth of periodontal disease bacteria (P. gingivalis) About a protein having PTP inhibitory activity purified from rice bran, P. gingivalis (JCM12257 strain: RIKEN) The effect on proliferation of the product obtained from Kenken BioResource Center (ATCC 33277) was confirmed. As the medium, Trypticase Soy blood agar medium was used. The medium contains Trypticase soy agar (Becton Dickinson) 40g, Horse blood (Cosmo Bio) 50ml, and sterilized water 950ml. What was done was used. As a protein having PTP inhibitory activity, a protein purified by hydrophobic chromatography and freeze-dried by the same method as in Example 1 was dissolved in a buffer solution (20 mM Tris-HCl pH 7.2) at 1 mg / ml. The solution was used. The culture was performed at 37 ° C. under anaerobic conditions using a plate using an anaerobic culture kit manufactured by Mitsubishi Gas Chemical Co., Inc.

By the following two methods (A) and (B), addition of a protein having PTP inhibitory activity and inoculation of periodontal disease bacteria were performed. In (A) and (B), a magic line was drawn on the back surface of the plate, and the plate was divided into left and right. In addition, periodontopathic fungi previously grown on a separate plate were used.
(A) Before inoculation, a protein solution (1 mg / ml) having a PTP inhibitory activity was aseptically and evenly added dropwise to the left half of the plate using a pipette, absorbed into the medium, and dried. The right half of the plate did not absorb a protein solution (1 mg / ml) having PTP inhibitory activity. Periodontal disease bacteria (P. gingivalis) that had been grown in advance were applied to the entire surface of the plate while moving the ase left and right according to a normal inoculation method.
(B) Periodontopathic fungi were applied to the entire surface of the plate with ase as in (A). Thereafter, a protein solution having PTP inhibitory activity (1 mg / ml) was carefully absorbed in the left half of the plate in the same manner as in (A). The right half of the plate did not absorb a protein solution (1 mg / ml) having PTP inhibitory activity.

The results are shown in FIG. The upper part of FIG. 8 is a photograph after inoculating by the above methods (A) and (B) and culturing at 37 ° C. for 2 days. The left and right boundary lines shown in the photograph are based on the lines on the back of the plate. The lower part of FIG. 8 is a control and is a photograph after inoculating periodontal pathogens on a plate that has not absorbed a protein solution having PTP inhibitory activity and then culturing at 37 ° C. for 2 days.
In both (A) and (B), in the left half of the plate (the side labeled “(PTPI)” in the figure), the growth of periodontal disease bacteria is inhibited, and the right half of the plate (“(NON)” in the figure). On the side marked as), growth was not inhibited. Therefore, it was confirmed that the protein having PTP inhibitory activity purified from rice bran tends to prevent the growth of periodontal disease bacteria. It was considered that the growth of periodontopathic bacteria was suppressed by inhibiting the degradation of the protein (including proline-binding portion) contained in trypticase soybean agar by periodontal pathogen PTP.

As explained above, a protein having a novel PTP inhibitory activity could be isolated and purified from a cereal plant-derived material. The protein had PTP inhibitory activity even when expressed by genetic recombination. The PTP inhibitor of the present invention containing the protein as an active ingredient has an effect that it is excellent not only in effectiveness but also in safety. Furthermore, since the PTP inhibitor of the present invention exerts an action of suppressing the growth of periodontal disease bacteria, it can be widely used for the treatment and prevention of periodontal disease, and is greatly used for health management centering on oral care. Can contribute. In addition, in the PTP inhibitor of the present invention, the active ingredient is a protein, and the protein is degraded in the stomach, so that the possibility of side effects is low. Furthermore, the active ingredient of the PTP inhibitor of the present invention can be produced from the edible part (for example, old rice) or the waste (for example, rice bran) after separating the edible part. Waste can be used effectively and can be manufactured at low cost.
Further, according to the method for detecting PTP inhibitory activity of the present invention, the inhibitory activity against the action of PTP on gelatin closer to gingival collagen than the synthetic substrate can be confirmed. According to this detection method, the growth inhibitory effect of periodontal disease bacteria and the action of suppressing gingival regression in periodontal disease can be predicted more accurately. Further, the present invention is useful because it can be easily and easily detected.

Claims (8)

Prolyl tripeptidyl peptidase (PTP) inhibitor containing any protein selected from the following (a) to (c) as an active ingredient:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;
(B) In the amino acid sequence shown in SEQ ID NO: 1, it contains an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added, and has an inhibitory activity against prolyl tripeptidyl peptidase (PTP) A protein having
(C) a protein comprising an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1 and having an inhibitory activity against prolyl tripeptidyl peptidase (PTP). The prolyl tripeptidyl peptidase (PTP) inhibitor according to claim 1, wherein the active ingredient is a protein comprising the amino acid sequence shown in SEQ ID NO: 1. The prolyl tripeptidyl peptidase (PTP) inhibitor according to claim 1 or 2, wherein the protein of the active ingredient has a molecular weight of 25 kDa. A protease inhibitor of periodontal disease fungus Porphyromonas gingivalis containing a protein comprising the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient. The periodontal disease bacteria growth inhibitor containing the inhibitor of any one of Claims 1-4. The periodontal disease prevention and / or treatment agent containing the inhibitor of any one of Claims 1-4, or the periodontal disease bacteria growth inhibitor of Claim 5. The inhibitor of any one of Claims 1-4 including isolation of an active ingredient from a cereal plant-derived material, the periodontal disease bacteria growth inhibitor of Claim 5, or Claim 6. Of the preparation of a preventive and / or therapeutic agent for periodontal disease. A method for detecting PTP inhibitory activity, comprising a step of contacting PTP with a structure containing gelatin in a medium in the presence of a PTP inhibitor to detect PTP activity.