JP2000319300A - Antimicrobial peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel compound that has not only excellent antimicrobial activity but also high safety and is useful as a naturally occurring antimicrobial agent applicable over a wide variety of uses, for example, medicines and the like. SOLUTION: This novel compound is a peptide having the amino acid sequence represented by the formula: Phe Ser Gln Ser Cys Ala Pro Gly Ala Asp Pro Lys Ser Arg Leu Cys Ala Leu Cys Ala Gly Asp Asp Gln Gly Leu Asp Lys Cys Val Pro Asn Ser Lys Glu Lys Tyr Tyr Gly Tyr Thr Gly Ala or its salts. This compound is prepared, for example, by fractionating the 3-10 kD fraction from the pepsin hydrolyzate of bovine lactoferrin with the cationic ion-exchanging column, then with the anionic ion-exchanging column, and finally purifying the resultant fraction with the reversed phase chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel antimicrobial peptide. More specifically, the present invention relates to a peptide having a novel amino acid sequence confirmed to have antibacterial activity among peptides fractionated from a hydrolyzate of bovine lactoferrin and determined for its amino acid sequence, and an antibacterial agent containing the peptide. .

[0002]

2. Description of the Related Art Lactoferrin is a glycoprotein having a molecular weight of about 80,000 in which a single polypeptide chain is linked to a sugar chain composed of galactose or mannose. Lactoferrin 1
The molecule has two metal ion binding sites and usually binds iron ions reversibly. Lactoferrin is widely distributed in exocrine fluids such as milk, tears and saliva, and neutrophils, which are a type of white blood cell. Its content is high in colostrum, especially the highest concentration in human colostrum, being present at 5 to 10 mg / ml. The fact that lactoferrin is involved in the body's defense functions is presumed from the fact that it is abundant in colostrum, and it has already been shown to protect against infection, suppress inflammation, regulate the immune system, promote cell proliferation, antioxidant, and promote iron absorption. Biological function has been reported.

On the other hand, in addition to the function of the lactoferrin molecule itself, the effects of various peptides fractionated from the lactoferrin hydrolyzate have also been studied. No. 271068 contains 5 residues consisting of 11 amino acids isolated from lactoferrin hydrolyzate.
Peptides exhibiting various antibacterial properties have been described. Japanese Patent Application Laid-Open No. 05-92994 discloses an antimicrobial peptide consisting of 20 amino acids similarly obtained from a lactoferrin hydrolyzate, and Japanese Patent Application Laid-Open No. 05-238948 discloses an antimicrobial peptide consisting of 25 amino acids. I have. These are all antibacterial effects confirmed in vitro,
Japanese Patent Application Laid-Open No. 08-291198 describes a peptide mixture obtained from a lactoferrin hydrolyzate which has no antibacterial activity in vitro but has an antibacterial activity only in vivo. Thus, there are many unclear points about the antibacterial properties of lactoferrin degradation products, and there is also a mechanism to prevent the establishment of infection by inhibiting the attachment of bacteria to the site of infection in addition to actively killing bacteria. JP 0
7-300425.

[0004]

With the increasing safety orientation in the industry and consumers, there is a demand for a new type of highly safe antibacterial agent in place of a synthetic antibacterial agent. Furthermore, there is a need for the development of a new type of antibacterial agent that can be widely used for various uses such as pharmaceuticals, foods and drinks, and industrial chemicals, and that can be administered not only externally but also orally.

[0005]

SUMMARY OF THE INVENTION The present invention has been made for the purpose of responding to the above-mentioned demands and is not only excellent in antibacterial activity but also high in safety and widely used in various applications. The purpose of this study is to develop a new type of natural-derived antibacterial agent that can be used.

[0006] The present inventors have focused on lactoferrin as a result of various studies to achieve the above object. The present inventors have found that lactoferrin is a safe substance contained in milk, and that peptides obtained from hydrolysates of such substances are also present in degradants produced in normal digestion. It is considered that the safety has already been guaranteed, and it is expected that the chemical stability is expected to be excellent, and a large amount of peptides will be secured by clarifying the amino acid sequence, and more clear antibacterial properties can also be expected And from a viewpoint.

In view of the above, the present inventors have been searching for peptides having antibacterial properties from degradation products of lactoferrin by various proteases from the viewpoint of inhibiting infection by inhibiting adhesion. The antibacterial activity, which was not clear at the time of crude purification, can be confirmed as it goes up. Finally, it was confirmed that the novel peptide having the amino acid composition shown in SEQ ID NO: 1 has strong antibacterial activity. As a result of the discovery of a sex peptide and further studies based on this new finding, the present invention was successfully completed.

That is, the present invention relates to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and an antibacterial agent containing the peptide as an active ingredient.
The peptide includes a wide variety of commonly used salts. Hereinafter, the present invention will be described specifically.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The antimicrobial peptide of the present invention comprises 3 to 10 bovine lactoferrin obtained from pepsin degradation product.
The kDa fraction can be fractionated on a cation exchange column, then fractionated on an anion exchange column, and finally purified by reverse phase chromatography. The antimicrobial peptide of the present invention can also be obtained by chemically synthesizing it using an automatic peptide synthesizer. In addition, by utilizing a gene encoding the amino acid sequence of the peptide of the present invention, it is possible to produce a large amount using a microorganism cell, plant cell, or animal cell into which the gene has been incorporated.

The antimicrobial peptides of the present invention obtained as described above include various Escherichia coli (toxinogenic Escherichia coli ATCC31705, enterohemorrhagic Escherichia coli O157; H7 RIMD0509939 and RIMD0509861),
In vitro against Salmonella enteritidis 1891, Listeria monocytogenes and Yersinia enterocollitica
It was confirmed that o had strong antibacterial activity. In addition, since the present peptide did not inhibit the adhesion of the toxinogenic Escherichia coli ATCC 31705 to immobilized laminin, it was considered that the antibacterial main body acts not on the adhesion but on bactericidal bacteria.

The antimicrobial peptide of the present invention and its salt are in a form which has already undergone a decomposition and denaturation treatment by hydrolysis from the lactoferrin molecule itself, and has become more easily handled, including an improvement in heat resistance. Therefore, it can be used as an antibacterial agent in various forms depending on the purpose. For example, eye drops, mouthwashes, liquids such as emulsions, creams, ointments, pastes such as toothpastes, solids such as powders, solids such as granular soaps, and forms such as application to sterilized paper or diapers However, it can be used. In addition, because of its high safety, it should be used in foods and drinks, foods and drinks for specified health use, health drinks, health foods, nutritional foods, and various other types of foods and drinks (in the present invention, foods include drinks). In addition, this peptide can be added to or mixed with the feed beforehand as a feed for animals, and then fed directly to the feed or mixed with drinking water. Can also.

The peptide of the present invention can be used for preventing the growth of general microorganisms and can also be used as a medicament for treating or preventing bacterial diseases. The peptide of the present invention has no toxicity or very low toxicity. In an acute toxicity test on rats, no acute toxicity is observed even when 500 mg per day is orally administered, showing excellent safety. Therefore, the antimicrobial peptide of the present invention can be used in any dosage and administration method depending on the type and symptom of the disease.

For example, tablets, capsules, granules, powders,
In addition to oral administration with a syrup or the like, enteral or tube administration can be selected. These various preparations are excipients, binders, disintegrants, lubricants, flavors,
It can be formulated using known auxiliaries usually used in the technical field of pharmaceutical formulation, such as a solubilizing agent, a suspending agent and a coating agent. The dosage varies depending on symptoms, age, body weight, administration method, dosage form, etc., but about 0.1 mg to 1500 mg can be administered to an adult at a time. In addition, the peptide of the present invention does not show any particular acute toxicity at least when it is orally administered, even if it is taken in large amounts.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0015]

Example 1 An antimicrobial peptide was purified from a pepsin hydrolyzate of bovine lactoferrin as follows.

Bovine lactoferrin (Wako) was added to 0.1
M citrate buffer (pH 2.2), dialyzed and de-ironed before pepsin (Wako, 169-05742, p.
(H 2.0, 37 ° C., 4 hours), followed by heating at 80 ° C. for 30 minutes to inactivate the enzyme. Using the ultrafiltration membrane (Centriplus, Amicon), the pepsin degradation product was collected from a molecular weight cut-off of 10,000 or less to 3,000 or more.

This fraction is used as a cation exchange column (Waters).
Protein-Pak SP) using 20 mM sodium phosphate buffer pH 7.0 as eluent, 0 M (10 min) → 1.
Fractionation was performed by the linear concentration gradient method of 0 M sodium chloride (30 minutes) (flow rate: 0.5 ml / min) (FIG. 1). Fraction I in FIG. 1 was applied to an anion exchange column (Waters Protein-Pak Q).
Using 0 mM Tris-HCl buffer as eluent, 0 M (1
(0 min) → fractionation by a linear concentration gradient method of 1.0 M sodium chloride (30 min) (flow rate: 0.5 ml / min) (FIG. 2). Further, fraction I in FIG. 2 was purified by reverse phase chromatography using a C4 column to obtain a peak fraction. In this case, 10% acetonitrile / 0.1% trifluoroacetic acid (TFA) 0
Min → 60% acetonitrile / 0.1% TFA for 30 minutes with a linear concentration gradient method (flow rate 1 ml / min). This fraction showed a single peak by analysis with C4 and C18 columns (example of C18 column: FIG. 3).

From the N-terminal amino acid sequence analysis of this peptide and the molecular weight analysis by liquid chromatography-mass spectrometry (LC-MS), the peptide obtained this time was found to be a peptide between bovine lactoferrin between 487 and 529 amino acids , BLf487-529). This peptide (BLf48
7-529) is a novel peptide which has been unknown so far, and has been confirmed to have excellent antibacterial activity as apparent from the following.

[0019]

Example 2 The peptide obtained above (BLf487-52)
The antibacterial activity of 9) was confirmed as follows.

Escherichia coli ATCC
31705) for CFA agar
Was used. Salmonella Enteritidis 189
1), Listeria monocytogenes IID 57
7), enterohemorrhagic E. coli O157 (Escherichia coli RI)
MD 0509939 and RIMD 0509861) and cultivation of Yersinia enterocollitica YE 98013) and measurement of the viable cell count, use BHI broth and BHI ag.
ar was used. ATCC strain is American Type Culture
Collection (Rockville, USA) and the IID strain were obtained from the Osaka University Microbial Disease Research Institute Strain Storage Facility, respectively.

A 0.036% aqueous solution of BLf487-529 and about 4 × 10 ² / ml of each bacterial suspension were cultured at 37 ° C. for 24 hours, and the viable cell count was determined by a plate culture method. Table 1 shows the obtained results. As is clear from the results, when the peptide of the present invention was not added, the number of bacteria of each bacterium was about 10 ⁹ / m
However, when the BLf487-529 peptide was added, the growth of each bacterium was completely suppressed. The toxinogenic E. coli ATCC 31705 acted bacteriostatically and bactericidally against other pathogenic bacteria.

The following bacteria were used as pathogenic bacteria. (A) Escherichia coli ATCC 31705 Toxigenic E. coli (B) Escherichia coli RIMD 0509939 O157; H7 (C) Escherichia coli RIMD 0509861 O157; H7 (D) Salmonella Enteritidis 1891 (E) Listeria monocytogenes IID 577 (F) Yersinia enterocollitica YE 98013

(Table 1) Number of pathogen inoculated bacteria Number of bacteria after 24 hours of culture (actual value) BLf 487-529 without addition (A) 5.8 × 10 ² / ml 2.6 × 10 ⁹ / ml 5.8 × 10 ² / ml ml (B) 7.8 × 10 ² / ml 6.6 × 10 ⁸ / ml <3.3 × 10 / ml (C) 6.3 × 10 ² / ml 1.6 × 10 ⁹ / ml <3.3 × 10 / ml (D) 4.3 × 10 ² / ml 7.6 × 10 ⁸ / ml <3.3 × 10 / ml (E) 4.1 × 10 ² / ml 3.0 × 10 ⁸ / ml <3.3 × 10 / ml (F) 1.0 × 10 ³ / ml 2.0 × 10 ⁸ / ml <3.3 × 10 / ml

[0024]

Example 3 (1) BLf487-529 peptide (lyophilized product) 50 g (2) Lactose 90 g (3) Corn starch 29 g (4) Magnesium stearate 1 g

(1), (2) The total amount and (3) 17 g were mixed, and (3) granulated together with the paste prepared from 7 g. To the obtained granules, 5 g of the remaining amount of (3) and the whole amount of (4) were added and mixed well, and this mixture was compressed with a compression tablet machine to give a tablet 1 containing 50 mg of the peptide BLf487-529 of the present invention per tablet. 2,000 were produced. The dose varies depending on the patient's symptoms and age,
1,500 mg / Kg / Day, preferably 1 to 50 m
g / Kg / Day.

[0026]

As described in detail above, the novel antimicrobial peptide of the present invention shows strong antimicrobial activity against various pathogenic microorganisms, and is excellent in safety as a drug because it is derived from natural products. In addition, it is promising as a novel antibacterial agent that can be applied to various uses, for example, because it is prepared after enzymatic decomposition, heat treatment and the like, and has excellent processing stability and storage stability.

[0027]

[Sequence List] SEQUENCE LISTING <110> Meiji Milk Products Co., Ltd. <120> Antifungal Peptide <130> 6166 <141> 1999-5-14 <160> 1 <210> 1 <211> 43 <212> PRT <212> Artificial Sequence <400> 1 Phe Ser Gln Ser Cys Ala Pro Gly Ala Asp Pro Lys Ser Arg Leu 5 10 15 Cys Ala Leu Cys Ala Gly Asp Asp Gln Gly Leu Asp Lys Cys Val 20 25 30 Pro Asn Ser Lys Glu Lys Tyr Tyr Gly Tyr Thr Gly Ala 35 40

[Brief description of the drawings]

FIG. 1. Bovine lactoferrin pepsin degradation product (molecular weight 3
2 shows the fractionation pattern of a cation exchange column (K / 10K fraction). In the figure, fraction I contains the peptide of the present invention.

FIG. 2 shows the fractionation pattern of the fraction I of FIG. 1 using an anion exchange column. In the figure, fraction I contains the peptide of the present invention.

FIG. 3 shows the results obtained by purifying fraction I of FIG. 2 by reversed-phase chromatography using a C4 column and then confirming the degree of purification with a C18 column. It shows almost a single peak.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/00 ZNA A61K 37/14 14/47 37/18 (72) Inventor Shingo Nakamura Kikyo, Hirosaki City, Aomori Prefecture No. 2-chome 20-1 Kikyono House 2 303 F term (reference) 4C084 AA02 AA07 BA01 BA19 CA59 DA41 MA23 MA35 MA37 MA41 MA43 MA52 NA06 NA07 ZB352 4H011 AA02 BB19 DG05 DH11 4H045 AA10 AA30 BA19 CA43 EA01 GA10 EA01 GA10

Claims (2)

[Claims] 1. A peptide represented by the amino acid sequence of SEQ ID NO: 1 or a salt thereof. 2. An antibacterial agent comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1 or a salt thereof as an active ingredient.