KR20180052181A - An anti-microbial peptide, Protaetiamycine 2 isolated from Protaetia brevitarsis seulensis and its synthetic composition - Google Patents

Abstract

The present invention relates to a novel peptide protaetiamycine 2 isolated from Protaetia brevitarsis and to a composition thereof. The chemically synthesized peptides in the present invention exhibit strong antibacterial action to gram-positive bacteria, gram-negative bacteria or fungi, but show little cytotoxicity, thereby being usefully used as antimicrobial agents, antifungal agents, antibiotics, food preservatives, cosmetic preservatives, pharmaceutical preservatives, enteritis therapeutic agents and the like.

Description

An antimicrobial peptide proteetiamycin 2 derived from white spotted flowers and its composition {An anti-microbial peptide, Protaetiamycin 2 isolated from Protaetia brevitarsis seulensis and its synthetic composition}

The present invention relates to novel peptides derived from white spotted flowers and their compositions, and more particularly to novel peptides and peptides derived from white spotted flowers and exhibiting excellent antibacterial and antifungal activity without cytotoxicity. ≪ / RTI >

Infection with pathogenic microorganisms is one of the most common and fatal causes of human disease, unfortunately the antibiotic resistance of pathogenic microorganisms has been caused by the abuse of antibiotics. In fact, 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 can pose a life threat, I have developed resistance to all antibiotics.

Tolerance to antibiotics is distinct from resistance to antibiotics, which was first discovered in the 1970s by Pneumococcus sp. And provided important clues to the mechanism of action of penicillin. Resistant species stop growing in the presence of the usual concentration of antibiotics but do not eventually die. This resistance occurs because antibiotics inhibit the cell wall synthetase when autolytic enzymes such as autolysin do not activate, which makes penicillin an endogenous hydrolytic enzyme. Activation kills bacteria and bacteria also inhibit their activity, resulting in the survival of antibiotics.

It is clinically very important that pathogenic microorganisms have resistance to antibiotics, because the inability to eradicate the resistant microorganisms is ineffective in antibiotic treatment in clinical infections. In addition, tolerance is considered to be a prerequisite for resistance to antibiotics, which is due to the survival of strains despite antibiotic therapy. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics. Since virtually all pathogenic microorganisms showing resistance are known to have resistance, development of new antibiotics capable of killing pathogenic microorganisms resistant to such antibiotic resistance is urgent.

As described above, it is necessary to develop a new antibiotic to prevent damage caused by pathogenic microorganisms showing resistance to antibiotics, and to develop a new antibiotic that acts independently of autolysin activity. In addition, there is a need to provide pharmaceutical compositions for effectively treating such new antibiotics with an infection of pathogenic microorganisms.

On the other hand, some of the substances that play an important role in maintaining the homeostasis of organisms are physiologically active substances derived from various organisms. Many researches have been carried out on a number of biologically active substances, among which antimicrobial peptides isolated from various organisms are known to act in various ways, ranging from bacteria, fungi and viruses. Antimicrobial peptides are also known to play an important role in host defense and the innate immune system. Korean Patent No. 10-1062975 (antibiotic peptide derivative having high bacterial selectivity designed from protactiamycin antibiotic peptide) and Korean Patent No. 10-1420849 (a prophylactic agent showing high antimicrobial activity against multi-drug resistant bacteria) Thiamycin antimicrobial peptide derivatives and uses thereof) are known.

Korean Patent No. 10-1062975, entitled "Antibiotic Peptide Derivative Having High Bacterial Selectivity Designed from Protactamycin Antibiotic Peptide," Applicant: Industry-University Collaboration Foundation, Konkuk University, Registered on August 31, 2011) Korean Patent No. 10-1420849, entitled PROTATIAMIC ANTIBACTERIAL PEPTIDE DERIVATIVES HAVING HIGH ANTIBACTERIAL PROBLEMS IN MULTI-DRUG RESISTANCE AND USE THEREOF, APPLICANT: Industry & Academy Collaboration Foundation, Konkuk University, Registered on July 11, 2014)

It is an object of the present invention to provide an amino acid sequence of an antimicrobial peptide proteetiamycin 2 isolated from a white spotted flower which can be usefully used for the prevention or treatment of diseases induced by Gram-negative bacteria, Gram-positive bacteria and fungi, and compositions thereof .

The present invention relates to a peptide represented by the following SEQ ID NO: 1 synthesized from a gene of an antimicrobial or antifungal peptide isolated from a white spotted flower ( Protaetia brevitarsis seulensis).

SEQ ID NO: 1: MNWKKNKKWVVMKFF-NH ₂

The peptide may exhibit an antimicrobial activity against at least one of Staphylococcus aureus and Escherichia coli 0-157. Alternatively, the peptide may exhibit antifungal activity against Candida albicans .

The peptide is characterized by being non-toxic to red blood cells.

The present invention provides a pharmaceutical composition, antibiotic, food preservative, cosmetic preservative or pharmaceutical preservative for antifungal or antifungal preparations containing the peptide as an active ingredient.

In addition, the present invention can provide a composition for treating food poisoning or enteritis, containing the peptide as an active ingredient. The food poisoning or enteritis is caused by at least one of Staphylococcus aureus and Escherichia coli 0-157 ( Escherichia coli 0-157).

Hereinafter, the present invention will be described in detail.

The present invention relates to a peptide represented by SEQ ID NO: 1 synthesized from the gene of an antibacterial or antifungal peptide isolated from white spotted flower ( Protaetia brevitarsis seulensis). Accordingly, the present invention may relate to an antimicrobial or antifungal peptide directly isolated from a white spotted flower having the same sequence as that of SEQ ID NO: 1. Accordingly, the present invention may relate to an antimicrobial or antifungal peptide directly isolated from a white spotted flower having the same sequence as that of SEQ ID NO: 1, wherein the peptide at the carboxyl end may be in the form of -NH ₂ or -OH.

In the present invention, in order to identify the gene for the white spotted flower, the first step is to rapidly freeze white spotted flowers with liquid nitrogen to isolate total RNA, and then, using RNA seq analysis, And based on the sequence of the translated amino acid (peptide) from each transcript, it is possible to select the unicin, which is presumed to be a peptide showing antimicrobial or antifungal activity, using the properties of the existing antimicrobial peptide have.

The amino acid sequence of the peptide is MNWKKNKKWVVMKFF-NH ₂ , which is composed of 15 amino acids and is named as Protaetiamycine 2.

The present invention also provides a pharmaceutical composition for antibiotic or antifungal preparation comprising the peptide as an active ingredient, an antibiotic, a food preservative, a cosmetic preservative or a pharmaceutical preservative.

More specifically, the proteythamicin 2 peptide is a strain of Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157) which is an antifungal activity target, And exhibits antibacterial or antifungal activity in an RDA assay (radial diffusion assay) against Candida albicans (see FIG. 1).

In addition, in the test for confirming the erythrocyte cell destructive ability, melitin, a bee venom known as an antifungal or antimicrobial effect, is highly cytotoxic, whereas the novel proteythamicin 2 peptide of the present invention is found to have little cytotoxicity (See Table 2). That is, the peptide is characterized in that it is less toxic to red blood cells of the rat.

As described above, the pharmaceutical composition containing the novel proteythamicin 2 peptide of the present invention as an active ingredient exhibits antifungal and antibacterial effects as well as almost no cytotoxicity, and thus can be effectively used as a safe antifungal or antifungal agent.

The pharmaceutical composition containing the proteythamicin 2 peptide of the present invention can also be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. The peptide or the composition containing the peptide may be administered in various forms of parenteral administration. In the case of formulation, the peptide may be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In addition, the novel peptide of the present invention can be used in combination with various carriers permitted as pharmaceutical agents, such as physiological saline or an organic solvent, and can be used in combination with carbohydrates such as glucose, sucrose or dextran, Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals.

The total effective amount of the novel peptide of the present invention or a peptide having more than 95% homology thereto in the above pharmaceutical composition can be measured in a single dose by a bolus form or by infusion for a relatively short period of time, And may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. The concentration is determined by taking into consideration various factors such as the route of administration and the number of treatments of the pharmaceutical composition as well as the age and health condition of the patient. Thus, in view of this point, It will be possible to determine an appropriate effective dose depending on the particular use as a pharmaceutical composition containing the novel peptide of the present invention.

In addition, the present invention provides antibiotics, food preservatives, cosmetic preservatives, and pharmaceutical preservatives containing the novel peptide as an active ingredient. In this case, food preservative, cosmetic preservative or pharmaceutical preservative is an additive used to prevent deterioration, decay, discoloration and chemical change of food or medicine. It includes antiseptic and antioxidant and inhibits the growth of microorganisms such as bacteria, fungi and yeast And also includes functional antibiotics such as inhibiting the growth of microorganisms or sterilizing the microorganisms in foods or medicines. Ideal conditions for these food preservatives, cosmetics or pharmaceutical preservatives should not be toxic and should be effective in trace amounts.

As shown in FIG. 1 and Table 2, the novel proteythamicin 2 peptide of the present invention exhibits antibacterial or antifungal activity and is insignificant in toxicity, so that it can be usefully used as a food preservative, a cosmetic or a medicine preservative.

In addition, the present invention can provide a composition for treating food poisoning or enteritis, containing the peptide as an active ingredient. Since the peptide has an antifungal effect against Staphylococcus aureus or Escherichia coli 0-157, the food poisoning or enteritis is caused by Staphylococcus aureus , And Escherichia coli 0-157 ( Escherichia coli 0-157).

The present invention relates to an antimicrobial peptide proteetiamycin 2 and a composition thereof derived from a white spotted flower. The antimicrobial peptide has little cytotoxicity and exhibits excellent antimicrobial or antifungal activity. Therefore, the antimicrobial peptide is useful as an antibiotic, a food preservative, a cosmetic preservative, Preservatives, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing antimicrobial activity and antifungal activity of the novel proteythamicin 2 peptide of the present invention derived from white spotted flower.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

≪ Example 1: Selection of antimicrobial peptide gene derived from white spotted flower &

In order to identify the peptide gene showing antimicrobial or antifungal activity from white spotted flowers, firstly, white spotted flowers were rapidly frozen in liquid nitrogen to isolate total RNA. RNA seq analysis was used to identify white spotted flowers Based on the sequence of the translated amino acid from each transcript, the unicin was filtered based on the characteristics of the existing antimicrobial peptides. As a result, genes presumed as new antimicrobial peptides were selected. The selected amino acid sequence was named MNWKKNKKWVVMKFF-NH ₂ and consisted of 15 amino acids and was named as Protaetiamycine 2 (Table 1).

Peptides Amino acid sequence Proteomycin 2 MNWKKNKKWVVMKFF-NH ₂

≪ Example 2: Synthesis and separation and purification of proteiamycin 2 >

The proteythamicin 2 peptide derived from the white spotted flower of Example 1 was synthesized, isolated and purified.

First, in order to synthesize proteythamicin 2 peptide, the present inventors used Merrifield's liquid phase method (Merrifield, RB., J. Am. Chem. Soc., 85, 2149, 1963) was used to prepare peptides. Method of the peptide synthesis the Fmoc (9-fluorenylmethoxycarbonyl) amino acids of the N _α were synthesized and used as a protecting group (protecting group) of the amino group (amino group) to the conventional method (solid phase method).

Concretely, the peptide having carboxyl terminal in the form of -NH ₂ used Rink Amide MBHA-Resin as the starting material, and the peptide having carboxyl terminal in the form of -OH used Fmoc-amino acid-Wang Resin as a starting material. Elongation of the peptide chain by coupling of Fmoc-amino acid was performed by DCC ( N -hydroxybenzo-triazole (HOBt) -dicyclo-hexylcar-bodiimide) method.

After the Fmoc-amino acid of the amino terminal of each peptide was coupled, the Fmoc group was removed with 20% piperidine / N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM) It was dried with gas.

The mixture of trifluoroacetic acid (TFA): phenol: thioanisole: H ₂ O: triisop ropylsilane (85: 5: 5: 2.5: 2.5, v: v: v: v: v) Removal and separation of the peptide from the resin followed by precipitation of the peptide with diethyl ether. The crude peptide thus obtained was purified by HPLC on a reverse phase HPLC column (Delta Pak, C ₁₈ 300 Å, 15 袖 m, 19.0 mm ×) using an acetonitrile gradient containing 0.1% TFA 30 mm, Waters).

After the synthetic peptide was hydrolyzed with 6N HCl at 110 ° C for 24 hours, the obtained residue was concentrated under reduced pressure, dissolved in 0.02N HCl, and the amino acid composition was measured with an amino acid analyzer (Hitachi 8500 A). The molecular weight was calculated based on the sequence of the synthesized peptides, and the exact molecular weight was measured using a MALDI mass spectrometer (MALDI).

As a result, it was confirmed that the antimicrobial peptide having the correct amino acid sequence was synthesized because the calculated molecular weight and the calculated molecular weight were identical.

<Example 3: Antimicrobial activity against pathogenic microorganisms of proteythamicin 2 antimicrobial peptide>

A radial diffusion assay (RDA) was performed to investigate the antimicrobial activity against the proteythamicin 2 peptide. Melittin, a powerful antimicrobial 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 distilled water.

Example 3-1. Antimicrobial activity measurement

Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157), 3% (w / v) TSB (trypticase soy broth) was used to measure the antimicrobial activity of the antimicrobial peptide ) At 37 ° C and 200 rpm for 18 hours, and then cultured for 2 hours and 30 minutes at a concentration of 4 × 10 ⁶ CFU (colony forming units) / ml under the same conditions.

The cells were then incubated with a citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type I (low electroendosmosis) agarose and 0.03% (w / Bacteria (4 × 10 ⁶ colony forming units / ml) were added to the underlay gel, mixed and poured into a square plate. When the underlay gel was hardened, a hole with a diameter of 3 mm was pierced, Were added to the wells in 5 μl aliquots.

10 mL of an overlay gel (6% TSB, 1% agarose) was poured and cultured at 37 ° C again to confirm formation of a clear zone (CLEAR ZONE) The results are shown in Fig.

Example 3-2. Antifungal activity measurement

Measurement of antifungal activity of antimicrobial peptides, the pathogenic fungi of Candida albicans (Candida albicans) a YPD medium (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) dextrose) At 30 ° C and 200 rpm overnight, inoculated on fresh medium and grown for 2 hours to become logarithmic phase.

Bacteria cultured on sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type agarose (low electroendosmosis) ⁶ colony forming units / ㎖). After mixing, the mixture was poured into a square plate. When the underlay gel was hardened, a hole having a diameter of 3 mm was punched out and 5 μl of peptide was added per concentration.

10 mL of overlay gel (6% TSB, 1% agarose) was poured and cultured at 37 ° C. to confirm the formation of a clear zone. Respectively.

Example 3-3. Results of antibacterial activity

As shown in Fig. 1, the proteythamicin 2 peptide has activity against the pathogenic bacteria and positive bacteria, which is increased in a concentration-dependent manner. This indicates that there is a possibility of treating the existing harmful strains as a therapeutic agent. However, when compared with melittin, a potent antimicrobial peptide, all of the pathogenic bacteria have lower activity than melittin.

Example 3-4. Antifungal Activity Results

As shown in FIG. 1, it was confirmed that a clear zone (CLEAR ZONE) was formed as a result of measuring the antifungal activity of the proteythamicin 2 peptide, and it was confirmed that the activity was increased in a concentration-dependent manner as in the case of a negative control strain. But exhibit lower antifungal activity than melittin, a potent antifungal agent used as a control.

<Example 4: Cytotoxicity measurement on the red blood cells of protethymaic 2 peptide>

The measurement of cytotoxicity against Rat red blood cells was carried out as follows.

Rat erythrocytes were washed three times with phosphate-sodium chloride buffer (PBS, pH 7.4) and then diluted with phosphate-sodium chloride buffer (PBS, pH 7.4) to prepare a 5.0% red blood cell solution. Thereafter, 100 μl of the 5.0% red blood cell solution was dispensed into 96-well plates, and then a stepwise diluted solution of the antimicrobial peptide was mixed. Then, phosphoric acid-sodium chloride buffer (PBS, pH 7.4) was added to all the tubes so that the total amount became 200 쨉 l, and cultured in a 37 째 C incubator for 30 minutes. After the incubation, the supernatant was transferred to a 96-well plate by centrifugation at 1000 rpm for 10 minutes in a centrifuge.

The destructive capacity of the red blood cells was measured by measuring the absorbance at a wavelength of 550 nm using an ELISA reader. 100% destructive ability was calculated by treating with 0.1% (w / v) Triton X-100 as a control, and 0% destructive ability when only red cell was added. At this time, the destructive ability of the peptide was calculated by the following equation (1), and the results are shown in Table 2 below.

[Equation 1]

% Destructive ability = (absorbance of solution treated with peptide: 550 nm-absorbance of phosphoric acid-sodium chloride buffer: 550 nm) / (absorbance of solution treated with Triton X-100: 550 nm-absorbance of phosphoric acid-sodium chloride buffer:

Condition Percent red cell destructive ability ^a (占 퐂 / ml) 0 12.5 25 50 100 200 Proteomycin 2 0 1.36 3.28 2.2 2.91 1.26 Melitin 0 75.8 83.1 89.8 88.3 91.4 [Note] a: Cytotoxicity is determined by the ability of the antimicrobial peptide to destroy red blood cells

As shown in Table 2, it is confirmed that proteythamicin 2 peptide has almost no destructive ability of red blood cells. In contrast, melittin, a potent antimicrobial peptide, is highly cytotoxic even at low concentrations.

These results indicate that the antimicrobial peptide found in the present invention has no cytotoxicity and exhibits excellent antimicrobial and antifungal activity, and thus can be used as safe antibiotics, food preservatives, cosmetic preservatives, and medicines.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> An anti-microbial peptide, Protaetiamycine 2 isolated from          Protaetia brevitarsis seulensis and its synthetic composition <130> <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 15 <212> PRT <213> Unknown <220> <223> Protaetia brevitarsis <400> 1 Met Asn Trp Lys Lys Asn Lys Lys Trp Val Val Met Lys Phe Phe   1 5 10 15

Claims (10)

The peptide represented by SEQ ID NO: 1, which is synthesized from the gene of an antibacterial or antifungal peptide isolated from white spotted flower ( Protaetia brevitarsis seulensis).
SEQ ID NO: 1: MNWKKNKKWVVMKFF-NH ₂ The method according to claim 1,
Wherein said peptide exhibits an antimicrobial activity against any one or more of Staphylococcus aureus and Escherichia coli 0-157 ( Escherichia coli 0-157). The method according to claim 1,
Wherein said peptide exhibits antifungal activity against Candida albicans . The method according to claim 1,
Wherein the peptide is harmless to the red blood cells. A pharmaceutical composition for an antimicrobial or antifungal agent comprising the peptide of any one of claims 1 to 4 as an active ingredient. An antibiotic characterized by containing the peptide of any one of claims 1 to 4 as an active ingredient. A food preservative characterized by containing the peptide of any one of claims 1 to 4 as an active ingredient. A preservative for cosmetics characterized by containing the peptide of any one of claims 1 to 4 as an active ingredient. A pharmaceutical preservative agent containing the peptide of any one of claims 1 to 4 as an active ingredient. A composition for treating food poisoning or enteritis, which comprises the peptide of any one of claims 1 to 4 as an active ingredient.

Images