KR102516550B1 - Antibacterial peptide derived from Haliotis madaka - Google Patents

Abstract

The present invention relates to an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from royal abalone, and the antibacterial peptide according to the present invention is effective against Bacillus subtilis , Pseudomonas aeruginosa and Vibrio parahaemolyticus . Since it has antibacterial activity against the drug, there is an advantage that it can be used as a lead material and candidate material for new drug development in the pharmaceutical and bioindustry.

Description

Antibacterial peptide derived from Haliotis madaka}

The present invention relates to an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from Haliotis madaka and its use.

Representative abalones that live in Korea are round abalone (Black abalone, Haliotis discus discus ), northern abalone ( H. discus hannai ), king abalone ( H. madaka ), and horse abalone ( H. gigantea ). It lives in the entire coast from the coast of Jeju Island with the 12℃ isotherm as the boundary. Among them, the northern abalone and round abalone are the main breeding species in Korea, and as of 2019, the shallow sea aquaculture production volume was 18,436 M/T, and the production amount was 591,741,551 thousand won, and the aquaculture scale is on the rise every year. 98% of abalone farming production is produced in Jeollanam-do, and the remaining 2% is produced in Gyeongsangnam-do, Chungcheongnam-do, Jeju-do, and Ulsan Metropolitan City.

Although numerous types of antibiotics have been developed and used since penicillin, strains having antibiotic resistance are increasing in recent years. According to the US Centers for Disease Control (CDC) antimicrobial resistance (AMR) hospital antibiotics report, about 100,000 people were infected with methicillin-resistant Staphylococcus aureus (MRSA) in 2006 alone due to misuse of antibiotics. About 20,000 of them died. In 2010, Carbapenem-resistant Enterobacteriaceae (CRE) containing the NDM-1 (New Delhi metalo-beta-lactamase) gene, which is called a super bacterium, was discovered in Korea, and the risk due to resistant bacteria is increasing. . Currently, most of the research on infectious diseases conducted worldwide focuses on the development of antibiotics that selectively inhibit and control the metabolic or biosynthetic pathways of pathogens and the development of vaccines by culturing attenuated pathogens. In the case of the host's metabolic and biosynthetic pathways, enzymes that control the enzymes are also inhibited at the same time, resulting in side effects and toxicity in many cases, and their use is limited due to resistance problems, and there is a problem that it is impossible to quickly cope with new pathogens only with vaccine development. Therefore, it is urgent to develop a new paradigm of infection treatment that can effectively control infectious diseases in the early stages, and recently, domestic and foreign researchers are paying attention to antimicrobial peptides (AMP) as a way to solve the existing problems.

Antimicrobial peptide (AMP) is a small peptide protein expressed by living organisms and is known to be in charge of immunity or anti-inflammatory action against pathogens, fungi, parasites, viruses, etc. that have entered the body. It is characterized by a very short length of 10 to 50 amino acids, mainly positively charged (+2 to +9), and containing more than 30% of hydrophobic amino acid residues. AMP contacts the negatively charged bacterial cell membrane to form an α-helix or β-sheet, forming a 'barrel-stave', 'carpet', or It is known to kill bacteria by piercing the cell membrane with the 'toroidal-pore' mechanism. Since AMP is a protein derived from living organisms, it has effective antibacterial activity and is known to have no side effects or toxicity, and is therefore used as a raw material for pharmaceuticals and cosmetics.

In the present invention, in order to search for abalone-derived active substances based on the genetic resources of royal abalone ( Haliotis madaka ), antibacterial peptide (AMP) candidates were searched for based on transcriptome data of royal abalone ( Haliotis madaka ), and their antibacterial activity was confirmed did

Republic of Korea Patent No. 10-1668229

An object of the present invention is to provide an antimicrobial peptide derived from abalone ( Haliotis madaka ) and an antibacterial pharmaceutical composition comprising the same, an antibacterial cosmetic composition, an antibacterial feed additive, hygiene products, and a pharmaceutical composition for preventing or treating food poisoning.

In order to achieve the object of the present invention as described above, in one aspect, an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from abalone ( Haliotis madaka ) is provided.

The permanent antibacterial peptide may have antibacterial activity against at least one selected from the group consisting of Bacillus subtilis , Pseudomonas aeruginosa and Vibrio parahaemolyticus .

It also provides an antibacterial pharmaceutical composition comprising the antimicrobial peptide as an active ingredient.

It also provides an antibacterial food composition comprising the antimicrobial peptide as an active ingredient.

It also provides an antibacterial cosmetic composition comprising the antimicrobial peptide as an active ingredient.

It also provides an antibacterial feed additive containing the antimicrobial peptide as an active ingredient.

In addition, sanitary products containing the antimicrobial peptide as an active ingredient are provided.

It also provides a pharmaceutical composition for preventing or treating food poisoning containing the antimicrobial peptide as an active ingredient.

Since the antibacterial peptide according to the present invention has antibacterial activity against Bacillus subtilis , Pseudomonas aeruginosa and Vibrio parahaemolyticus , it can be used as a leading material and candidate for new drug development in the pharmaceutical and bioindustry. There is an advantage to being

1 is a schematic diagram of an antimicrobial peptide (AMP) candidate search process (workflow).
Figure 2 shows the results of the antibacterial activity test according to the sample concentration of the abalone ( Haliotis madaka )-derived peptide (HM07215_3) (1 to 10; sample concentration, mg / ml).

In order to solve the above problems, the present invention provides an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from Haliotis madaka .

In the present invention, the antimicrobial peptide consists of the amino acid sequence of SEQ ID NO: 1.

In the examples of the present invention, it was confirmed that the HM07215_3 peptide had an amino acid sequence of SEQ ID NO: 1, a molecular weight of 1657.02 Da, an isoelectric point (pI) of 10.5057, and a charge of +4.

Therefore, the antimicrobial peptide of the present invention can be characterized as having a molecular weight of 1657.02 Da, an isoelectric point (pI) of 10.5057, and a charge of +4.

In an embodiment of the present invention, RNA was extracted from Haliotis madaka , a library was prepared to secure transcripts, and antimicrobial peptide candidates were searched for by converting the royal abalone transcript sequences into protein sequences. Specifically, it is less than 80 amino acids, isoelectric point (pI) ranges from 8 to 12, alpha helix propensity ranges from 0 to 25, and beta sheet propensity ranges from 25 To 100, an in vitro aggregation (AGG) of 500 or less, and an in vivo aggregation (Na4vSS) of -40 to 60, antimicrobial peptides were selected. Of these, 33 novel antimicrobial peptides were selected. Among the selected antimicrobial peptides, it was confirmed that the antibacterial peptide of SEQ ID NO: 1 (HM07215_3) has an antibacterial effect against Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Pseudomonas aeruginosa and Vibrio parahaemolyticus .

Therefore, the peptide consisting of SEQ ID NO: 1 of the present invention has antibacterial activity.

In the present invention, "antibacterial" refers to inhibiting the growth or growth of all microorganisms having harmful properties, and in the present invention, the microorganisms may be gram-positive bacteria and gram-negative bacteria, and the microorganisms are specifically Bacillus subtilis and Pseudomonas aeruginosa. ( Pseudomonas aeruginosa ) and Vibrio enteritis ( Vibrio parahaemolyticus ) It may be any one or more from the group consisting of.

Also, in an embodiment of the present invention, the peptide having the amino acid sequence of SEQ ID NO: 1 was derived from royal abalone ( Haliotis madaka ), and the antibacterial peptide of the present invention may be isolated from royal abalone ( Haliotis madaka ).

The antimicrobial peptide of the present invention can be used as an antimicrobial composition.

The antimicrobial composition may have the same meaning as antibiotics, which is a generic term for antimicrobial agents, and may have the same meaning as antibacterial agents, bactericides, preservatives, preservatives, or disinfectants.

The antimicrobial composition may be used for various purposes for antibacterial purposes, examples thereof include pharmaceutical compositions, food compositions, cosmetic compositions, feed additives, sanitary products, quasi-drugs, and the like.

In addition, the antibacterial peptide can prevent or treat diseases such as food poisoning, which is a disease typically caused by Bacillus subtilis , Pseudomonas aeruginosa and Vibrio parahaemolyticus , which exhibit antibacterial activity. It can be used as a pharmaceutical composition for preventing or treating food poisoning by containing an antimicrobial peptide as an active ingredient.

In one embodiment of the present invention, the HM07215_3 peptide derived from royal abalone of SEQ ID NO: 1 does not show antibacterial activity against Escherichia coli, but the minimum inhibitory concentration (MIC) against Bacillus subtilis is 0.5 mg / ml, the minimum bactericidal concentration (MBC) represents 1mg/ml, the minimum inhibitory concentration (MIC) for Pseudomonas aeruginosa is 4mg/ml, the minimum bactericidal concentration (MBC) is 6mg/ml, the minimum inhibitory concentration (MIC) for Vibrio enteritis is 1mg/ml, the minimum bactericidal concentration It was confirmed that the concentration (MBC) was 6 mg/ml.

Therefore, when the antibacterial peptide of the present invention is used for antibacterial purposes, it can be added at 0.5 mg / ml or more, which is confirmed as the minimum inhibitory concentration for Bacillus subtilis , Bacillus subtilis, Pseudomonas aeruginosa ) and Vibrio parahaemolyticus enteritis ), an antimicrobial peptide may be added at a concentration of 4 mg/ml to simultaneously exhibit an antibacterial effect.

In addition, the present invention provides an antibacterial pharmaceutical composition comprising the antimicrobial peptide as an active ingredient.

Description of the antimicrobial peptide in the present invention is as described above.

In the present invention, the antibacterial pharmaceutical composition may be aimed at preventing or treating diseases caused by microbial infection, specifically treating infectious diseases caused by gram-positive bacteria or gram-negative bacteria.

In the present invention, the infectious disease may be caused by one type of microorganism selected from the group consisting of Bacillus subtilis and Gram-negative bacteria, Pseudomonas aeruginosa and Vibrio parahaemolyticus .

The pharmaceutical composition may further include suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil; and the like.

The pharmaceutical composition may be administered through conventional intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. It can be administered to a subject in a manner.

A preferable dosage of the antimicrobial peptide may vary depending on the condition and body weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention provides an antibacterial food composition containing the antimicrobial peptide as an active ingredient.

Description of the antimicrobial peptide in the present invention is as described above.

The food composition according to the present invention can be prepared in the form of a composition by mixing with known active ingredients known to have antibacterial effects, and can be used as functional food, nutritional supplement, health food, and food additive. (food additives). Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

In addition, the present invention provides an antibacterial feed additive containing the antimicrobial peptide as an active ingredient.

The antibacterial peptide of the present invention has an antibacterial effect and can be used as a substitute for antibiotics added to feed.

The feed additive may be a feed additive that is added and mixed into a formulated feed for one or more animals of chicken, duck, goose, pheasant, pig, cow, goat, dog, and cat.

In addition, the feed additive may further include an antibiotic in addition to the peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides sanitary products containing the antimicrobial peptide as an active ingredient.

Description of the antimicrobial peptide in the present invention is as described above.

In the present invention, sanitary products include soap, detergent, wet tissue, hand sanitizer, mouthwash, and mouthwash.

In addition, the present invention provides a pharmaceutical composition for preventing or treating food poisoning containing the antimicrobial peptide as an active ingredient.

Description of the antimicrobial peptide in the present invention is as described above.

In the present invention, food poisoning means an infectious or toxin-type disease that is determined to be caused or caused by microorganisms or toxic substances harmful to the human body associated with the intake of food.

In the present invention, the food poisoning may be caused by at least one selected from the group consisting of Bacillus subtilis , Pseudomonas aeruginosa and Vibrio parahaemolyticus .

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

1. Search and synthesis of abalone-derived antibacterial peptide candidates

King abalone ( H. madaka ) was provided by Jeju Special Self-Governing Province Maritime and Fisheries Research Institute. Tissues such as the tentacle were excised and rapidly frozen in liquid nitrogen. Total RNA was extracted from 50 to 100 mg of frozen tissue according to RNAiso plus (Total RNA extraction reagent, TaKaRa) method.

Total RNA extracted from each tissue was mixed in equal amounts, and an RNA library including a library adapter with a size of about 150 bp was prepared by requesting Labgenomics. Among the prepared RNA libraries, base sequences with a length of 500 bp or more, excluding adapters, were sequenced with the Miseq platform provided by Insilicogen, and the transcriptome for each abalone was obtained through a de novo assembly process.

From the royal abalone transcripts, AMP candidates specifically expressed in royal abalone were searched by requesting Insilicogen Co., Ltd.

The abalone transcript sequence was converted into a protein sequence, and AMP candidate substances were searched for using analysis methods such as PEPSTATS, TANGO, and AGGRESCAN. AMP candidates are less than 80 amino acids in size, have an isoelectronic point (pI) range of 8 to 12, an alpha helix propensity range of 0 to 25, and a beta sheet propensity. Among sequences with a range of 25 to 100, an in vitro aggregation (AGG) of 500 or less, and an in vivo aggregation (Na4vSS) of -40 to 60, only new peptides that have not yet been published were selected. (Fig. 1).

As a result, 27 novel antimicrobial peptide (AMP) candidates were selected from royal abalone. Table 1 shows the results of AMP analysis of royal abalone.

One (HM07215_3) was selected from the selected AMP candidates and synthesized by requesting to Anygen. It was confirmed that the HM07215_3 peptide had an amino acid sequence of SEQ ID NO: 1, a molecular weight of 1657.02 Da, an isoelectric point (pI) of 10.5057, and a charge of +4 (Table 2).

origin Peptide ID amino acid sequence length
(mer) Molecular Weight
M.W.
(Da) pI majesty royal abalone
( H. madaka ) HM07215_3 RISFISCKKSGCTVK
(SEQ ID NO: 1) 15 1657.02 10.5057 +4

2. Analysis of antibacterial effect

In the antimicrobial activity test, gram-positive Bacillus subtilis , gram-negative bacteria Escherichia coli , Pseudomonas aeruginosa , and Vibrio parahaemolyticus were tested at the Korea Culture Center of Microorganisms (KCCM). It was distributed and used (Table 3).

Gram Species accession number Positive Bacillus subtilits KCCM 11779 Negative Escherichia coli KCTC 1116 Pseudomonas aeruginosa KCCM11266 Vibrio parahaemolyticus KCCM 11965

The antibacterial activity test was conducted by modifying the disk diffusion method of CLSI (Clinical and Laboratory Standards Institute). Purely cultured colonies were suspended in LB (Luria Bertani) liquid medium to have McFarland 0.5. The bacterial solution was smeared with a cotton swab, spread evenly on the surface of the LB solid medium, and then allowed to stand for 10 to 15 minutes at room temperature in an aseptic state. After 1 drop of the AMP to be tested, it was incubated at 30° C. for 18 hours, and then the antibacterial activity was determined by checking the presence or absence of inhibition rings.

AMP was completely dissolved in distilled water, and the concentration of the sample was gradually diluted from 10 mg/ml to 1 mg/ml to confirm antibacterial activity. The approximate antibacterial activity concentration was confirmed through the test, and based on this, the minimum inhibitory concentration (MIC, Minimal Inhibitory Concentration) and the minimum bactericidal concentration (MBC, Minimal Bactericidal Concentration) were measured according to the CLSI standard.

After inoculating the bacteria in LB liquid medium and culturing at 30 ° C, a bacterial suspension was prepared by adjusting the concentration with McFarland 0.5 (1-5 × 10 ⁶ CFU / ml). After diluting the AMP step by step from 10 to 0.125 mg/ml, each of the bacterial suspensions was inoculated and the MIC value was determined after 18 hours of incubation. Each culture medium treated for MIC determination was smeared on LB solid medium, cultured for 18 hours, and then the concentration of the maximum dilution factor that kills 99.9% of the number of bacteria initially inoculated was determined as MBC.

As a result of the analysis, antibacterial activity was shown at concentrations of 1 to 10 mg/ml against Bacillus subtilis, and antibacterial activity was shown at concentrations of 6 mg/ml or higher for Pseudomonas aeruginosa and Vibrio enteritis. did not appear (Fig. 2, Table 4).

Peptide ID bacteria Sample concentration (mg/mL) One 2 3 4 5 6 7 8 9 10 HM07215_3 B. subtilis + + + + + + + + + + E. coli - - - - - - - - - - P. aeruginosa - - - - - + + + + + V. parahaemolyticus - - - - - + + + + +

Next, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each strain of the HM07215_3 peptide derived from royal abalone were analyzed. As a result of the analysis, the HM07215_3 peptide does not exhibit antibacterial activity against Escherichia coli, but the minimum inhibitory concentration (MIC) for Bacillus subtilis is 0.5 mg / ml and the minimum bactericidal concentration (MBC) is 1 mg / ml, and the minimum inhibitory concentration for P. aeruginosa ( MIC) was 4 mg / ml, the minimum bactericidal concentration (MBC) was 6 mg / ml, the minimum inhibitory concentration (MIC) for Vibrio enteritis was 1 mg / ml, and the minimum bactericidal concentration (MBC) was 6 mg / ml (Table 5) .

That is, it was confirmed that the HM07215_3 peptide derived from royal abalone had antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa and Vibrio enteritis.

Peptide ID B. subtilis E. coli P. aeruginosa V. parahaemolyticus MIC MBC MIC MBC MIC MBC MIC MBC HM07215_3 0.5 One - - 4 6 One 6

<110> Jeju National University Industry-Academic Cooperation Foundation <120> Antibacterial peptide derived from Haliotis madaka <130> DP20200174 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> PRT <213> artificial sequence <220> <223> HM07215_3 <400> 1 Arg Ile Ser Phe Ile Ser Cys Lys Lys Ser Gly Cys Thr Val Lys 1 5 10 15

Claims (8)

An antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from royal abalone. According to claim 1,
The peptide is Bacillus subtilis ( Bacillus subtilis ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and enteritis Vibrio bacteria ( Vibrio parahaemolyticus ) Which has an antibacterial effect against at least one selected from the group consisting of peptides. A pharmaceutical composition for antibacterial use against harmful microorganisms comprising the antimicrobial peptide of claim 1 as an active ingredient,
The harmful microorganism is Bacillus subtilis ( Bacillus subtilis ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and enteritis Vibrio bacteria ( Vibrio parahaemolyticus ) At least one member selected from the group consisting of, antibacterial pharmaceutical composition. An antibacterial food composition comprising the antimicrobial peptide of claim 1 as an active ingredient. An antibacterial cosmetic composition comprising the antimicrobial peptide of claim 1 as an active ingredient. An antibacterial feed additive comprising the antimicrobial peptide of claim 1 as an active ingredient. Sanitary products containing the antibacterial peptide of claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating food poisoning caused by harmful microorganisms, comprising the antimicrobial peptide of claim 1 as an active ingredient,
The harmful microorganism is Bacillus subtilis ( Bacillus subtilis ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and enteritis Vibrio bacteria ( Vibrio parahaemolyticus ) At least one member selected from the group consisting of, a pharmaceutical composition.

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