JP2979722B2 - Bioactive polypeptide, method for producing the same, and antibacterial agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel physiologically active polypeptide, a method for producing the same, and an antibacterial agent as its use.

[0002]

2. Description of the Related Art In invertebrates such as insects, it is known that an antibacterial active substance is induced to appear in body fluids by vaccination with a vaccine or the like (European Journal.
Of Biochemistry, Volume 106, Page 7 (1
980)). The properties of these substances, the mechanism of antibacterial action, etc., have not yet been sufficiently elucidated.

[0003] In recent years, even in higher mammals, semen (Nature Vol. 249, p. 725 (1979))
And serum (Biochemistry Vol. 20, No. 5973)
Page (1981)) reveals the existence of a protein having a strong antibacterial activity and a broad antibacterial spectrum, and the importance of antibacterial proteins in body fluids of animals or insects is generally emphasized.

[0004] The present inventors have found that the common fly (Sarcopha
It has been clarified that a polypeptide having strong antibacterial activity against Escherichia coli and Bacillus subtilis appears in its body fluid by damaging the body surface of the larva of Ga. peregrina) (Japanese Patent Application Laid-Open No. 59-13730). Gazette, JP-A-61-12
2299). Furthermore, they found that a polypeptide having antibacterial activity (antibacterial polypeptide) was also present in the culture supernatant of cultured cells derived from Aspergillus oryzae, purified it, and clarified its physicochemical properties and structure. JP-A-63-185997).

[0005] The antibacterial polypeptide derived from the common fly is, as is observed in other insects, specifically induced at the time of damage to the body surface of the larva of the common fly, and is therefore a kind of biological agent for preventing bacterial infection. Considered to be a protective substance. Furthermore, since proteins having a plurality of antibacterial activities are found, it is considered that these proteins are interrelated and constitute a biological defense mechanism of Sentinia flies.

[0006] These antimicrobial polypeptides derived from Aspergillus oryzae have a broad antimicrobial spectrum and a strong antimicrobial activity, and are expected to be edible antimicrobial agents because they are proteins.

[0007]

However, there is always a need for new antibacterial agents having a high effect against undesirable pathogens and other microorganisms, and even more novel antibacterial agents have been used in antibacterial polypeptides derived from the common fly. It has been desired to discover a polypeptide and to develop a method for producing the polypeptide.

[0008] The present invention provides a novel antimicrobial polypeptide derived from the common fly, a method for producing the same, and a method for using the same.

[0009]

That is, the present invention provides a physiologically active polypeptide represented by the following amino acid sequence formula, and the following amino acid sequence: A method for producing a physiologically active polypeptide, characterized by obtaining a polypeptide represented by the formula, and an antibacterial agent containing a physiologically active polypeptide represented by the following amino acid sequence as a main component.

H ₂ N-Leu-Thr-Cys-Glu
-Ile-Asp-Arg-Ser-Leu-Cys-
Leu-Leu-His-Cys-Arg-Leu-L
ys-Gly-Tyr-Leu-Arg-Ala-Ty
r-Cys-Ser-Gln-Gln-Lys-Val
-Cys-Arg-Cys-Val-Gln-COO
H.

[0011] The polypeptide provided by the present invention is obtained from the culture supernatant of cells derived from Aspergillus oryzae, and is particularly a basic polypeptide rich in many cysteines and basic amino acids such as arginine, lysine and histidine. It is. That is, the polypeptide of the present invention is represented by the following amino acid sequence formula.

H ₂ N-Leu-Thr-Cys-Glu
-Ile-Asp-Arg-Ser-Leu-Cys-
Leu-Leu-His-Cys-Arg-Leu-L
ys-Gly-Tyr-Leu-Arg-Ala-Ty
r-Cys-Ser-Gln-Gln-Lys-Val
-Cys-Arg-Cys-Val-Gln-COO
H.

[0013] The method for producing the polypeptide of the present invention comprises the steps of: culturing cells derived from Aspergillus oryzae, and purifying and separating the cells from the culture supernatant by a method used for fractionation and purification of general high molecular weight proteins. It is about doing. For example, the polypeptide of the present invention can be obtained with high purity from a culture supernatant of cells of the fly fly embryo by ordinary methods such as ion exchange chromatography, heat treatment, gel filtration, and adsorption chromatography. Since this substance is a protein,
It is effective to use an increase in antibacterial activity per protein as an index.

The polypeptide obtained in the present invention can be used as an antibacterial agent in various compositions depending on the purpose. That is, it can be used as an antibacterial agent alone or in combination with an appropriate liquid, solid, or the like. Also,
It can also be used in combination with other drugs.

For example, it can be used as a preservative and disinfectant for contact lens cleaning solutions, ointments, soaps, shampoos, medicines, foods, feeds, pet foods, toothpastes, and the like.

Further, the polypeptide of the present invention can be used as an agent for treating and preventing bacterial diseases. In this case, any administration method and dosage can be selected depending on the type and symptom of the disease, as long as the antimicrobial polypeptide of the present invention has activity.

When the polypeptide of the present invention is used as a drug for an antibacterial agent, when a therapeutic agent containing an effective amount of the polypeptide of the present invention is clinically administered, it is administered by the oral or parenteral route. The dosage forms include tablets, dragees, pills, capsules, powders, troches, solvents, suppositories, injections, and the like, which are prepared from pharmaceutically acceptable excipients (e.
xcipient). The following can be exemplified as the excipient. Lactose, sucrose, glucose, sorbitol, mannitol, potato starch, amylopectin, other various starches, cellulose derivatives (eg, carboxymethyl cellulose, hydroxyethyl cellulose, etc.), gelatin, magnesium stearate, polyvinyl alcohol, calcium stearate, polyethylene glycol wax Vegetable oils such as gum arabic, talc, titanium dioxide, olive oil, peanut oil, sesame oil, paraffin oil, neutral fat base, ethanol, propylene glycol, physiological saline,
Sterile water, glycerin, colorants, seasonings, thickeners, stabilizers, isotonic agents, buffers, and the like, and other pharmaceutically acceptable excipients.

The antimicrobial agent of the present invention contains 0.001 to 85% by weight, preferably 0.005 to 6% by weight of the polypeptide of the present invention.
0% by weight can be contained.

The dose of the therapeutic agent of the present invention mainly depends on symptoms, but it is 1 to 500 m / day of adult body weight.
g, preferably 1 to 50 mg.

Since the antibacterial agent according to the present invention is a protein, it is considered that it has little toxicity at least when taken orally. Therefore, the antibacterial polypeptide is particularly useful in utilizing as human and animal drugs and food or feed additives.
For example, by adding the present antimicrobial polypeptide to foods such as gums and toothpaste, it is useful for sterilization in the oral cavity and prevention and treatment of caries and periodontal disease. In addition, in fish culture, it is possible to prevent the cultured fish from being infected by adding the polypeptide of the present invention to the feed used.

In other words, in other words, the polypeptide of the present invention can be used as a preservative or pharmaceutical preparation containing the substance as an active ingredient and as a food additive.

[0022]

The present invention will be described in more detail with reference to production examples, antibacterial action and the like.

Example 1 Production Example (a) Cell culture A medium of Mitsuhashi et al. (Sodium chloride 7 g / l, potassium chloride 0.2 g / l, calcium chloride dihydrate 0.2 g)
/ G, magnesium chloride hexahydrate 0.1 g / l, sodium dihydrogen phosphate dihydrate 0.2 g / l, sodium hydrogen carbonate 0.12 g / l, glucose 4 g / l, lactalbumin hydrolysate Contains 6.5 g / l and yeast grate 5 g / l. (pH 6.5) after aseptic filtration, and then sentinel fly-derived cells (NIH shape-
4 cells) were inoculated at 1 × 10 ⁶ cells / ml. 25
And cultured at 7 ° C for 7 to 10 days.
^{When the} number of cells reached ⁷ cells / ml, the cells were passaged, and the culture supernatant was collected by centrifugation.

(B) Separation and Purification of Antimicrobial Polypeptide To 900 ml of the culture supernatant obtained in the above (a), 2,700 ml of a phosphate buffer (pH 6.0) was added to adjust the pH and salt concentration. The obtained solution was passed through a CM cellulose column (3.0 × 9.0 cm), and washed with the same phosphate buffer as described above. Thereafter, the same phosphate buffer containing 520 mM NaCl was passed, and the eluate was collected in 4 ml portions, and the absorbance at 280 nm and the antibacterial activity were measured. Antimicrobial activity is in Journal of Biochemistry No. 21
In accordance with Volume 1 724-734 pages (1983) the method described, Staphylococcus Aure cormorant scan (Stapnyroc
occus aureus) IFO12732. The result is shown in FIG.

The fractions showing the antibacterial activity were collected and 100
The mixture was heated at 10 ° C. for 10 minutes, the generated precipitate was removed by centrifugation, and the obtained supernatant was concentrated by ultrafiltration. The obtained concentrated liquid is applied to a Sephadex G-50 column (1.5
× 120 cm) and eluted with a phosphate buffer containing 130 mM NaCl. The eluate was fractionated by 4 ml, and the absorbance at 280 nm and the antibacterial activity were measured using the same method as described above. The results are shown in FIG.

In FIG. 2, the fractions showing the antibacterial activity were collected and fractionated by HPLC reverse phase chromatography. The result is shown in FIG. The target antimicrobial polypeptide SP-5 was obtained by collecting the peaks shown in FIG. HPLC conditions are as follows.

Column: Synchropack RP-P Eluent: Solution A 0.05% trifluoroacetic acid / water Solution B 0.05% trifluoroacetic acid / acetonitrile Gradient conditions: Solution B in solution A is 15% to 40%
Linear gradient up to 2 ml / min.

Table 1 summarizes the above purification operations.

[0029]

[Table 1]

(C) Amino acid sequence of SP-5 The antimicrobial polypeptide SP-6 fractionated by the above-mentioned HPLC was converted to a gas-phase sequencer (Applied Biosystems 4
The resulting PTH-amino acid was identified and analyzed with a PTH-analyzer (Applied Biosystems, Inc., Model 120A), and the amino acid sequence was determined. The results were as follows.

H ₂ N-Leu-Thr-Cys-Glu
-Ile-Asp-Arg-Ser-Leu-Cys-
Leu-Leu-His-Cys-Arg-Leu-L
ys-Gly-Tyr-Leu-Arg-Ala-Ty
r-Cys-Ser-Gln-Gln-Lys-Val
-Cys-Arg-Cys-Val-Gln-COO
H.

(D) Analysis of the amino acid composition of SP-5 The antibacterial polypeptide SP-5 fractionated by the above PHLC
Was hydrolyzed with 6N hydrochloric acid containing 4% thioglycolic acid in a vacuum sealed tube at 110 ° C. for 22 hours. The amino acid in this hydrolyzate was analyzed by Hitachi Amino Acid Analyzer Model 835, and as a result, the molar ratio of the amino acid composition of SP-5 was as shown in Table 2.

[0033]

[Table 2]

(However, the molar ratio of leucine was 6.0.
As shown. The numbers in parentheses indicate the number of each amino acid residue calculated from the amino acid sequence).

The results of the amino acid composition analysis were not inconsistent with the results obtained by the amino acid sequence analysis.

(D) Measurement of antibacterial activity Journal of Biochemistry, vol. 211, no. 7
Table 2 shows the results of measuring the antibacterial activity of SP-5 according to the method described on pages 27 to 734 (1983).

[0037]

[Table 3]

[0038]

According to the present invention, a novel physiologically active polypeptide can be provided. The bioactive polypeptide of the present invention has antibacterial activity and has almost no toxicity. Therefore, various applications utilizing this property can be considered,
In particular, it can be expected to be used as an antibacterial agent or food additive containing this substance as an active ingredient. In particular, since this substance is a protein and has excellent heat stability, it is promising as an antibacterial food additive for foods requiring heat treatment in the processing step.

In addition, according to the method of the present invention, the polypeptide can be commercially produced on a large scale by expanding the culture scale of cells derived from the fly fly embryo.

[0040]

[Sequence list] Sequence length: 34 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Leu Thr Cys Glu Ile Asp Arg Ser Leu Cy
s Leu Leu His Cys ArgLeu Lys Gly Tyr Leu Arg Ala T
yr Cys Ser Gln Gln Lys Val Cys Arg Cys ValGln

[Brief description of the drawings]

FIG. 1 is a chromatogram showing an elution pattern when a CM cellulose column is used in a purification step of an antibacterial polypeptide according to the present invention.

FIG. 2 is a chromatogram showing an elution pattern when Sephadex G-50 is used.

FIG. 3 is a chromatogram showing an elution pattern by HPLC, which is a final purification step in purification of an antibacterial polypeptide.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification code FI A61K 38/00 A61K 37/02 (C12P 21/02 C12R 1:91) (58) Fields surveyed (Int.Cl. ⁶ , DB Name) C07K 14/435 C12P 21/00-21/02 A23L 3/3526 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A bioactive polypeptide represented by the following amino acid sequence formula: H ₂ N-Leu-Thr-Cys-Glu-Ile-A
sp-Arg-Ser-Leu-Cys-Leu-Le
u-His-Cys-Arg-Leu-Lys-Gly
-Tyr-Leu-Arg-Ala-Tyr-Cys-
Ser-Gln-Gln-Lys-Val-Cys-A
rg-Cys-Val-Gln-COOH 2. Sentinel flies (Sarcophaga peregrin)
The biologically active polypeptide according to claim 1, which is produced by a cultured cell derived from the embryo of a). 3. An antibacterial agent containing a physiologically active polypeptide represented by the following amino acid sequence formula as a main component. H ₂ N-Leu-Thr-Cys-Glu-Ile-A
sp-Arg-Ser-Leu-Cys-Leu-Le
u-His-Cys-Arg-Leu-Lys-Gly
-Tyr-Leu-Arg-Ala-Tyr-Cys-
Ser-Gln-Gln-Lys-Val-Cys-A
rg-Cys-Val-Gln-COOH 4. The red fly (Sarcophaga peregrin)
a) obtaining a polypeptide represented by the following amino acid sequence formula from a culture supernatant of embryo-derived cultured cells: H ₂ N-Leu-Thr-Cys-Glu-Ile-A
sp-Arg-Ser-Leu-Cys-Leu-Le
u-His-Cys-Arg-Leu-Lys-Gly
-Tyr-Leu-Arg-Ala-Tyr-Cys-
Ser-Gln-Gln-Lys-Val-Cys-A
rg-Cys-Val-Gln-COOH