KR20200071421A - An anti-microbial peptide, Teleogryllusine 3 isolated from Teleogryllus emma and its synthetic composition - Google Patents

Abstract

The present invention relates to a novel teleogryllusine 3 peptide isolated from Teleogryllus emma and a composition thereof. The teleogryllusine 3 peptide exhibits antibacterial functions against gram-positive bacteria, gram-negative bacteria and fungi. Since the peptide has little cytotoxicity and strong antibacterial activities, the peptide can be used as antibiotics, food antiseptics, cosmetic preservatives, and pharmaceutical preservatives containing an antibacterial agent or an antifungal agent.

Description

An anti-microbial peptide, Teleogryllusine 3 isolated from Teleogryllus emma and its synthetic composition}

The present invention relates to a novel antibacterial peptide tereogrilysin 3 and its composition derived from a king cricket, and more specifically, a new tereogrilucin 3 peptide showing antibacterial and antifungal activity with minimal cytotoxicity derived from a king cricket and the same It relates to an antibacterial agent or an antibiotic.

Infection with pathogenic microorganisms is one of the most common and fatal causes of human disease, but unfortunately, the antibiotic resistance of pathogenic microorganisms has been caused by the abuse of antibiotics. Indeed, the rate at which pathogenic microorganisms are resistant to new antibiotics is much faster than the rate at which analogs of new antibiotics are developed. For example, pathogenic microbial species such as Enterococcus faecalis , Mycobacterium tuberculosis , and Pseudomonas aeruginosa , which are potentially life-threatening, are known to date. It has developed resistance to all antibiotics.

Tolerance of antibiotics is a phenomenon distinct from resistance to antibiotics, which was first discovered in Pneumococcus sp. in the 1970s and provided important clues to the mechanism of action of penicillin. Resistant species stop growing in the presence of conventional concentrations of antibiotics, but do not die as a result. This resistance occurs because the activity of bacterial autolytic enzymes, such as autolysin, does not occur when antibiotics inhibit cell wall synthase, and this is because penicillin uses endogenous hydrolytic enzymes. By activating it, it kills the bacteria and the bacteria also inhibit their activity, resulting in survival even when treated with antibiotics.

It is clinically very important that pathogenic microorganisms are resistant to antibiotics, because when it becomes impossible to eradicate resistant microorganisms, the effectiveness of antibiotic treatment in clinical infections decreases. In addition, resistance is considered to be a prerequisite for resistance to antibiotics, since it produces strains that survive antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow even in the presence of antibiotics.

Since it is known that the pathogenic microorganisms showing practically all resistances are also resistant, the development of new antibiotics capable of killing the pathogenic microorganisms having such antibiotic resistance is urgent.

Therefore, in order to prevent damages caused by pathogenic microorganisms that are resistant to antibiotics, new antibiotics are needed, and new antibiotics that act independently of autolysine activity are also required. In addition, there is a need to provide such new antibiotics with pharmaceutical compositions for effectively treating infections of pathogenic microorganisms.

On the other hand, some of the substances that play an important role in the process for maintaining homeostasis of organisms are bioactive substances derived from various organisms. Numerous studies have been conducted on a number of bioactive substances, and among them, antibacterial peptides isolated from various organisms are known to act in various ways, including bacteria, fungi, and viruses. In addition, antimicrobial peptides are known to play an important role in host defense and innate immune systems.

Korean Patent Registration No. 10-0625875 (a new peptide separated from Aspergillus nidulans and a pharmaceutical composition containing it as an active ingredient), Korean Patent Registration No. 10-0964136 (separated from Uldo sky larvae) Antibacterial and antifungal peptide genes and synthetic peptides having antibacterial and antifungal activity), Korean Patent Publication No. 10-2011-0013074 (New use and manufacturing method of insect glycosaminoglycan), Korean Registered Patent No. 10-1660381 (Insect N-glycan and uses thereof) are known, but so far, no research has been conducted on new peptides isolated from king crickets and their new uses.

Korean Registered Patent No. 10-0625875 (New peptide isolated from Aspergillus nidulans and pharmaceutical composition containing it as an active ingredient) Korean Registered Patent No. 10-0964136 (Antibacterial and antifungal peptide genes isolated from Uldo sky larvae and synthetic peptides having antibacterial and antifungal activity) Korean Patent Publication No. 10-2011-0013074 (New use and manufacturing method of insect glycosaminoglycan) Korean Registered Patent No. 10-1660381 (Insect N-glycan and its use)

An object of the present invention is to provide an amino acid sequence of the antimicrobial peptide tereogrlysine 3 and its composition isolated from a king cricket that can be usefully used for the prevention and treatment of Gram-negative, benign and human fungal diseases.

The present invention relates to an antibacterial or antifungal peptide (SEQ ID NO: 1) derived from a king cricket ( Teleogryllus emma ) consisting of the amino acid sequence of SEQ ID NO: 1 below.

SEQ ID NO: 1 MFWKNKLFFIVKK-NH ₂

The peptide is characterized by having antibacterial activity against any one or more of Staphylococcus aureus and Escherichia coli 0-157.

The peptide is characterized by having antifungal activity against Candida albicans .

The peptide is characterized by minimal toxicity to the red blood cells of the rat.

The present invention is to provide a pharmaceutical composition, an antibiotic, a food preservative, a cosmetic preservative or a pharmaceutical preservative for an antibacterial or antifungal agent containing the above peptide as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an antibacterial or antifungal peptide derived from a king cricket ( Teleogryllus emma ) consisting of the amino acid sequence of SEQ ID NO: 1.

To explain, in the present invention, in order to identify the peptide gene derived from the king cricket ( Teleogryllus emma ), first, the king cricket is rapidly frozen with liquid nitrogen to separate the entire RNA, and this is used to determine the genetic information of the king cricket transcripts using RNA seq analysis. Based on the sequence of amino acids translated from the respective transcripts, unigene, which is presumed to be a peptide exhibiting antibacterial and antifungal activity, is selected using the properties of the existing antibacterial peptide.

The amino acid sequence of this peptide is MFWKNKLFFIVKK-NH ₂ , composed of 13 amino acids, which is called Teleogryllusine 3.

In addition, the present invention provides a pharmaceutical composition for antimicrobial and antifungal preparations containing it as an active ingredient, an antibiotic or a food preservative, or a cosmetic preservative or a pharmaceutical preservative.

In detail, Staphylococcus aureus , Escherichia coli 0-157, and antifungal as antibacterial activity target bacteria to measure the antibacterial and antifungal activity of the tereogrilusin 3 peptide As an active target bacterium, RDA assay (radial diffusion assay) was performed on Candida albicans , which has antibacterial and antifungal activity (see FIG. 1 ).

In addition, as a result of confirming the ability to destroy erythrocytes, the novel tereogrlysine 3 peptide of the present invention was found to have no cytotoxicity, whereas the bee venom melittin used as a positive control showed very high cytotoxicity (see Table 2). ).

Therefore, the pharmaceutical composition containing the novel tereoglycerine 3 peptide of the present invention as an active ingredient, as well as exhibiting antifungal and antibacterial effects, can be usefully used as a safe antibacterial agent and antifungal agent because of its minimal cytotoxicity.

The pharmaceutical composition may be administered parenterally during clinical administration and may be used in the form of a general pharmaceutical preparation.

That is, the novel peptides of the present invention can be administered in various parenteral formulations, and when formulated, are usually prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

In addition, the novel peptides of the present invention can be used in combination with various carriers allowed as medicaments such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose, or dextran to increase stability or absorption, Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as medicaments.

In the pharmaceutical composition of the present invention, the total effective amount of a novel peptide of the present invention and a peptide having 95% or more homology thereto is a single dose by bolus form or infusion for a relatively short period of time. It can be administered to a patient, and multiple doses can be administered by a fractionated treatment protocol that is administered for a long time. The concentration is the effective dosage of the patient is determined in consideration of various factors such as the patient's age and health status, as well as the route of administration and the number of treatments of the drug. It will be possible to determine an appropriate effective dosage for a particular use as a pharmaceutical composition of a novel peptide.

In addition, the present invention provides an antibiotic, food preservative, cosmetic preservative or pharmaceutical preservative containing novel peptides as active ingredients.

At this time, food preservatives, cosmetic preservatives and pharmaceutical preservatives are additives used to prevent deterioration, decay, discoloration and chemical changes in food or pharmaceuticals, and may include bactericides, antioxidants, and microorganisms such as bacteria, fungi, and yeast. Functional antibiotics such as inhibiting growth and inhibiting the growth or sterilization of decaying microorganisms in food and medicine are also included. The ideal conditions for preservatives, cosmetics and pharmaceutical preservatives in these foods should be non-toxic and should be effective even in trace amounts.

The novel tereoglycerine 3 peptide of the present invention has antibacterial and antifungal activity and has little toxicity as shown in FIGS. 1 and 2, and thus can be usefully used as a preservative, cosmetic, and pharmaceutical preservative in food.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

According to the present invention, as the antimicrobial peptide tereogrlysine 3 and its composition derived from a king cricket are provided, they can be provided as antibiotics, food preservatives, cosmetic preservatives, medicines, etc., because they have minimal cytotoxicity and exhibit antibacterial and antifungal activity. have.

1 is a view showing the antibacterial and antifungal activity of the novel tereoglycerine 3 peptide of the present invention derived from a king cricket.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

Example 1: Selection of antibacterial peptide gene derived from king cricket

In order to identify the peptide gene showing antibacterial or antifungal activity from the king cricket, first, the king cricket was rapidly frozen with liquid nitrogen to separate the entire RNA, and the RNA seq analysis method was used to confirm genetic information about the king cricket transcripts, and each of them was identified. Based on the sequence of amino acids translated from the carcasses, the whole unigene was filtered based on the properties of the existing antibacterial peptides, and as a result, genes supposed to be new antibacterial peptides were selected. The selected amino acid sequence was MFWKNKLFFIVKK-NH ₂ , composed of 13 amino acids, and was named Tereogryllusine 3 (see Table 1).

Peptide Amino acid sequence Teleogryllusine 3 MFWKNKLFFIVKK-NH ₂

Example 2: Synthesis and purification of tereogrlysine 3

The tereogrlysine 3 peptide derived from the king cricket identified in Example 1 was synthesized and separated and purified.

First, in order to synthesize the tereogrlysine 3 peptide, the present inventors used the liquid phase solid-phase method of Merrifield (Merrifield, RB., J. Am. Chem. Soc., 85, 2149, using a Fmoc amino group protecting container) 1963) to prepare a peptide.

The peptide synthesis method was synthesized by a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group of an N _α -amino group of amino acids.

Specifically, a peptide having a carboxyl terminus -NH ₂ type was used as Rink Amide MBHA-Resin as a starting material, and a peptide having a carboxyl terminus -OH type was using Fmoc-amino acid-Wang Resin as a starting material.

Elongation of the peptide chain by coupling of Fmoc-amino acids was performed by DCC ( N -hydroxybenzo-triazole (HOBt)-dicyclo-hexylcar-bodiimide) method.

After coupling the Fmoc-amino acid at the amino terminal of each peptide, the Fmoc group is removed with 20% piperidine/N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM), and then nitrogen Gas dried.

Trifluoroacetic acid (TFA)-phenol-thioanisole-H ₂ O-triisop-ropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) was added to the solution and reacted for 3 hours to remove the protecting group and from the resin. After the peptide was separated, the peptide was precipitated with diethyl ether. The crude peptide obtained in this way was purified with an acetonitrile gradient containing 0.1% TFA to form a purified reverse phase-HPLC column (Delta Pak, C ₁₈ 300 15, 15μm, 19.0 mm× 30, Waters).

After the synthetic peptide was hydrolyzed with 6 N-HCl at 110° C. for 24 hours, the obtained residue was concentrated under reduced pressure, and dissolved in 0.02 N-HCl to measure the amino acid composition with an amino acid analyzer (Hitachi 8500 A).

In addition, the molecular weight was calculated based on the sequence of the synthesized peptide, and the correct molecular weight was measured using a MALDI mass spectrometry (matrix-assisted laser desorption ionization mass spectrometer).

As a result, since the measured molecular weight and the calculated molecular weight matched, it was confirmed that an antibacterial peptide having an accurate amino acid sequence was synthesized.

Example 3: Investigation of the antimicrobial activity of the terogrillusin 3 antimicrobial peptide against pathogenic microorganisms

RDA (radial diffusion assay) was performed to investigate the antibacterial activity against the tereogrlysine 3 peptide. In this case, melittin, a strong antibacterial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antimicrobial peptide was stored at -20°C and dissolved in sterilized tertiary distilled water.

[Measurement of antibacterial activity]

In order to measure the antimicrobial activity of the antimicrobial peptide, first, 3% (w/v) TSB (Tryptic Soy Broth) of the pathogenic bacteria Staphylococcus aureus and Escherichia coli 0-157 ) Incubated at 37° C. and 200 rpm for 18 hours, inoculated in a new medium, and grown for 2 hours to become log phase.

Afterwards, a sterilized under consisting of Citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH7.4) and 1% (w/v) type I (low electroendosmosis) agarose, 0.03% TSB. Put the cultured bacteria (4×10 ⁶ colony forming units/ml) on the underlay gel, mix it, pour it into a square plate, and when the underlay gel hardens, make a hole with a diameter of 3 mm to make peptides by concentration. 5 µl each was placed in the hole.

After incubating at 37°C for 3 hours to spread the peptide, 10 ml of an overlay gel (6% TSB, 1% agarose) was poured and incubated again at 37°C to confirm the formation of a clear zone. The results are shown in FIG. 1.

[Measurement of antifungal activity]

Measurement of antifungal activity of the antimicrobial peptide is then grown overnight with pathogenic fungi of Candida albicans (Candida albicans) a YPD medium (1% yeast extract, 2% peptone, 2% dextrose) 30 ℃, 200 rpm conditions, this new medium And incubated for 2 hours to prepare a log phase.

Then, bacteria cultured on a sterile underlay gel composed of Citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH7.4) and 1% (w/v) type I (low electroendosmosis) agarose, 0.03% TSB ( 4×10 ⁶ colony forming units/ml) were added, mixed, poured into a square plate, and when the underlay gel was hardened, a hole with a diameter of 3 mm was made, and peptides of each concentration were added to each hole of 5 µl.

After incubating at 37°C for 3 hours to spread the peptide, 10 ml of Overlay gel (6% TSB, 1% agarose) was poured and incubated again at 37°C to confirm the formation of a clear zone. It is shown in 1.

[Results of antibacterial activity]

As shown in FIG. 1, it was confirmed that the tereogrilysin 3 peptide has activity against Gram-negative and positive bacteria, which was found to increase in a concentration-dependent manner. This shows that it can be used as a treatment for existing harmful strains.

[Antifungal activity results]

As shown in FIG. 1, as a result of measuring the antifungal activity of the tereogrlysine 3 peptide, a clear zone was formed, and it was confirmed that the activity was increased in a concentration-dependent manner as in the gram-negative bacterium positive bacteria.

Example 4: Cytotoxicity measurement of rat erythrocytes of tereogrlysine 3 peptide

Cytotoxicity was measured on rat red blood cells as follows.

Red blood cells (rat erythrocytes) were washed three times with phosphate-sodium chloride buffer, pH 7.4 (PBS), and then diluted with phosphate-sodium chloride buffer to prepare a 5.0% red blood cell solution.

Then, 100 µl of the 5.0% red blood cell solution was dispensed into a 96-well plate, followed by mixing the stepwise diluted solution of the antibacterial peptide.

Subsequently, phosphate-sodium chloride buffer, pH 7.4 (PBS) was added so that the total amount of the total solution was 200 μl in all tubes, and the cells were incubated for 30 minutes in a 37° C. incubator.

After the incubation, the supernatant was transferred to a 96-well plate by centrifugation for 10 minutes at a rotation speed of 1000 rpm in a centrifuge.

Destructive ability for red blood cells was analyzed by measuring the absorbance under a wavelength of 550 nm with an ELISA reader.

In addition, the value when treated with 0.1% Triton X-100 as a control was calculated as 100% destructive ability, and the case where only red blood cells were added was calculated as 0% destructive ability.

At this time, the breaking ability of the peptide was calculated by Equation 1 below, and the obtained results are shown in Table 2 below.

[Equation 1]

% Destructive ability = (absorbance of the solution treated with the peptide 550 nm-absorbance of the phosphate-sodium chloride buffer 550 nm) / (absorbance of the solution treated with Triton X-100 550 nm-absorbance of the phosphate-sodium chloride buffer 550 nm) × 100

Measurement of the cytotoxicity of an antibacterial peptide of tereogrlysine 3 on rat erythrocytes
% Rat red blood cell destruction ability ^a (㎍/ml) 0 12.5 25 50 100 200 Teleogryllusine 3 0 0 0.09 0.28 0 1.22 Melittin 0 75.8 83.1 89.8 88.3 91.4 [Note] a: Cytotoxicity is measured by the ability to destroy antibacterial peptides against red blood cells.

As shown in Table 2 above, it was confirmed that the tereogrlysine 3 peptide had minimal destruction ability of red blood cells when treated with a concentration of up to 200 μg/ml. On the other hand, it can be seen that melittin, a strong antibacterial peptide as a control, has high cytotoxicity even at a low concentration.

These results suggest that the antimicrobial peptides found in the present invention can be used as safe antibiotics, food preservatives, cosmetic preservatives, and medicines, as they exhibit not only minimal cytotoxicity but also antibacterial and antifungal activity.

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> An anti-microbial peptide, Teleogryllusine 3 isolated from Teleogryllus emma and its synthetic composition <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 13 <212> PRT <213> Unknown <220> <223> Teleogryllus emma <400> 1 Met Phe Trp Lys Asn Lys Leu Phe Phe Ile Val Lys Lys 1 5 10

Claims (9)

Antibacterial or antifungal peptide, characterized by consisting of the amino acid sequence of SEQ ID NO: 1, derived from the king cricket ( Teleogryllus emma ).
SEQ ID NO: MFWKNKLFFIVKK-NH ₂ According to claim 1,
The peptide is a peptide characterized in that it has antibacterial activity against any one or more of Staphylococcus aureus and Escherichia coli 0-157. According to claim 1,
The peptide is a peptide characterized by having antifungal activity against Candida albicans . According to claim 1,
The peptide is characterized in that the toxicity is minimal for rat red blood cells. A pharmaceutical composition for an antibacterial or antifungal agent, comprising the peptide of any one of claims 1 to 4 as an active ingredient. An antibiotic comprising the peptide of any one of claims 1 to 4 as an active ingredient. A food preservative comprising the peptide of any one of claims 1 to 4 as an active ingredient. Cosmetic preservative comprising the peptide of any one of claims 1 to 4 as an active ingredient. A pharmaceutical preservative comprising the peptide of any one of claims 1 to 4 as an active ingredient.

Images