KR20180058929A - Antimicrobial peptide from Bacillus tequilensis K1R and the use thereof - Google Patents

Abstract

The present invention relates to an antibacterial peptide derived from Bacillus tequilensis K1R and use thereof. According to the present invention, peptide K1R exhibits better results than vancomycin and bacitracin with respect to various kinds of multi-drug resistant (MDR) pathogenic bacteria, in particular, living organisms of a vancomycin-resistant Enterococcus group. An antagonistic effect of the antibacterial peptide against MDR pathogenic bacteria such as Salmonella typhimurium and Enterococcus sp. has been identified to have potential applicability thereof when K1R treats cases of MDR pathogenic bacteria. Antioxidant activity of K1R equals to antioxidant activity of standard ascorbic acid. Accordingly, the antibacterial peptide derived from Bacillus tequilensis K1R of the present invention has excellent antibacterial properties compared to a conventional antibacterial agent and thus can be advantageously used as an active ingredient of a pharmacological composition for antibacterial and antioxidant purposes.

Description

Antimicrobial peptides derived from the novel strains of Bacillus tequilensis and their uses {Antimicrobial peptide from Bacillus tequilensis K1R and the use thereof}

The present invention relates to a novel antimicrobial peptide exhibiting efficient activity against multidrug-resistant bacteria from Bacillus tequirences K1R strain isolated from kimchi, which is a Korean traditional food, and its use.

Antimicrobial peptides constitute a unique class of naturally occurring antimicrobial molecules with activity against a variety of pathogenic microorganisms. For the time being, conventional antibiotics are under strong pressure from developed tolerance. AMPs are an exciting leading group in the development of new disinfectants. They are also an alternative to chemical food preservatives. Despite persistent efforts, the prevalence of increasing resistance to conventional antibiotics among pathogenic bacteria has become the most important concern in modern medicines. Significantly reduced investments in major antibiotics research and development by major pharmaceutical companies have kept pace with the emergence of tolerance and the supply pipeline has become dangerously close to depleting, as new alternatives to existing treatment strategies are not being produced at a sufficient rate .[One]. In the natural world, antimicrobial peptides act as important defense weapons throughout the animal and plant system against a broad spectrum of bacterial and fungal pathogens [2]. Sources range from single-celled microbes such as bacteria (bacteriocins) [3] to invertebrates [4]. Antimicrobial peptides not only have direct effects on microorganisms, but also promote the accumulation of immune cells, including macrophages, neutrophils and lymphocytes [5]; Neutralize lipopolysaccharide endotoxin from gram-negative bacteria [6]; Help wound healing; It has been found that resistance development to stimulate angiogenesis and control the action of congenital and adaptive immune responses is rarely reported [7]. Antimicrobial peptides exert their bactericidal effect through the destruction of cell membranes of microorganisms as well as intercellular action [8-9]. Antimicrobial peptides are considered as one of the candidates for the development of novel therapeutic agents that are an alternative to conventional antibiotic therapy. AMPs have been extensively evaluated as new antibacterial drugs by destroying the membrane integrity of microorganisms and thus causing almost no resistance to [10].

Overall, these peptides describe a broad spectrum of antiviral and antimicrobial actions and modes of action, and it is important to distinguish between direct sterilization and indirect action against such pathogens. Structural requirements of peptides for antiviral and antimicrobial actions are evaluated in terms of various sets of primary and secondary structures described for host defense peptides [11]. The production of antimicrobial peptides by the Bacillus strain has been rapidly characterized in recent years, and many peptides produced by this group of bacteria have been widely used in fermentation techniques for producing antibiotics. Bacillus and related species have been of special interest because of their safety, their widespread distribution in a wide variety of habitats, and their remarkable ability to survive hostile conditions due to the development of endospores. Bacillus spp. Derived AMPs include antibacterial surface active biosurfactants such as cyclic peptides synthesized by various classes of bacteriocins [12], fatigue peptides, glycopeptides, and non-ribosomes [13-14].

Korean Patent Publication No. 10-2015-0087868 (Jul. 31, 2015)

Falagas, M. E., K. N. Fragoulis, and I. A. Karydis (2006) Comparative study on the cost of new antibiotics and other therapeutic categories. PLoS ONE. 1: 1-4.

The inventors of the present invention have conducted intensive studies to solve the problem of multidrug resistance which is a difficulty in the development of antimicrobial agents. As a result, it has been found that Bacillus tequilensis K1R strain isolated from Kimchi, a Korean traditional food, The present invention has been completed by suggesting an alternative that can control multidrug-resistant bacteria by purifying a novel peptide.

Accordingly, an object of the present invention can be achieved by an antimicrobial peptide characterized by comprising the amino acid sequence of SEQ ID NO: 1 and its use.

The peptide K1R according to the present invention showed better results than the vancomycin and bacitracin against various multidrug resistance (MDR) pathogens, especially against the organisms of the vancomycin resistant enterococcus group. Salmonella typhimurium and Enterococcus sp. Antagonistic effects on MDR pathogens have confirmed that K1R has its potential applicability in treating infectious MDR cases. The antioxidant activity of K1R was also comparable to that of standard ascorbic acid. Therefore, the antimicrobial peptide derived from bacillus tenuciensis K1R of the present invention has excellent antibacterial properties as compared with the conventional antimicrobial agent, and thus can be effectively used as an effective ingredient of a pharmaceutical composition for antibacterial use.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a phylogenetic tree of Bacillus strains according to the present invention.
Figure 2 is a graphical representation of the optimization of carbon, nitrogen and metal ions quantitatively.
3 is a graph showing the results of a) gel permeation chromatography using a) Sephadex G-50 column and b) gel permeation chromatography using a Sephadex G-25 column as the elution profile of K1R.
4 is a photograph showing the result of Tricine SDS-PAGE of K1R.
5 is a photograph showing the results of active staining of K1R peptide.
6 is a graph showing the effect of temperature on the antibacterial activity of K1R.
7 is a graph showing the effect of pH on the antibacterial activity of K1R.
Figure 8 is a graph showing the DPPH radical scavenging activity of K1R at different concentrations.

In one aspect, the present invention provides an antimicrobial peptide characterized by comprising the amino acid sequence of SEQ ID NO: 1, and uses thereof.

The main object of the present invention is to find the efficacy and activity of antimicrobial peptides which may contribute to the field of pharmacology, microbiology and biotechnology for human development. The present inventors have continued to find an alternative to antibiotic therapy that is useful for the removal of multidrug-resistant bacteria. Antimicrobial peptides are naturally synthesized compounds that have an antagonistic effect on other species. The germicidal effect generated by the microorganisms provides a means for them to exist around him as a means of survival. These naturally occurring compounds can be an alternative to useful antibiotic therapy. Antimicrobial peptides for therapeutic use still have some problems faced with low stability, toxicity and high manufacturing costs.

The present inventors have made efforts to utilize the newly isolated antimicrobial peptide K1R. K1R isolated from fermented food called kimchi, a Korean traditional food, showed a wide range of stability under the test conditions. To protect them from microorganisms, the microorganisms are part of the innate immune system, and when challenged by pathogens, the rapid increase in concentration in the host secretes the antimicrobial peptide. Bacillus is a very interesting genus of studies on the antimicrobial activity of these peptides because they produce a large number of peptides with biological activity. Kimchi is a traditional Korean food fermented by various microorganisms such as Bacillus and Lactobacillus , and has been found to be complete for the production of antimicrobial peptides.

It was an object of the present invention to study the therapeutic potential of antimicrobial substances produced by Bacillus species. The Bacillus strain K1R was fermented in a culture medium containing carbon and nitrogen sources, which was optimized for the specific strain, followed by ammonium sulfate precipitation, membrane filtration, dialysis, and continuous chromatography. An antimicrobial peptide (AMP) was obtained. A protein band of about 4.6 kDa was obtained by tricine-SDS-PAGE, which was confirmed by the in situ inhibitory activity of the gel. K1R was stable over a wide range of pH (6.5-9), was heat resistant below 60 ° C, and did not change activity at low temperatures of 0 ° C and 4 ° C. K1R showed better results than vancomycin and bacitracin for various multidrug resistant (MDR) pathogens, especially for organisms of the vancomycin resistant enterococcus group. The entire amino acid sequence of K1R was AVQGTLEDALNLSKGALNQVQKAIQNGDXLTVXGXLGTIXLA VSX (SEQ ID NO: 1). Salmonella typhimurium and Enterococcus sp. Antagonistic effects on MDR pathogens have confirmed that K1R has its potential applicability in treating infectious MDR cases. The antioxidant activity of K1R was also comparable to that of standard ascorbic acid.

The antimicrobial peptide derived from Bacillus tacheensis K1R of the present invention has excellent antibacterial properties as compared with the conventional antimicrobial agent, and thus can be effectively used as an effective ingredient of a pharmaceutical composition for antibacterial use.

The peptide of the present invention can be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration may mean administration through an administration route other than oral, such as rectal, intravenous, peritoneal, muscular, arterial, transdermal, nasal, inhalation, ocular and subcutaneous. That is, the antimicrobial peptide of the present invention can be administered in various formulations of actual parenteral formulations. For formulation, the antimicrobial peptide is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, 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 suspending agent. As a suppository base, witepsol, macrogol, tween 61, cacao paper, Liu ling, glycerogelatin and the like can be used.

In addition, the antimicrobial peptide of the present invention can be used in combination with various carriers that are accepted as medicines 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 may be used as pharmaceuticals.

The effective dose of the antimicrobial peptide of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and can be administered once to three times a day.

The total effective amount of the novel antimicrobial peptide of the present invention in the pharmaceutical composition of the present invention may be administered to a patient in a single dose by bolus or by infusion for a relatively short period of time, , And multiple doses may be administered by a fractionated treatment protocol administered over a prolonged period of time. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, Lt; RTI ID = 0.0 > antimicrobial < / RTI > peptide as a pharmaceutical composition.

Hereinafter, the present invention will be described in detail with reference to examples. only. The following examples are illustrative of the present invention, but the present invention is not limited to the following examples.

Sephadex G-50 and Sephadex G-25 were obtained from Pharmacia (Uppsala, Sweden). All reagents were of analytical grade.

Example 1: Isolation and Screening

The bacterial strain K1R was isolated from the traditional Korean kimchi. For isolation, 1 gm of kimchi was mixed with 9 ml of 0.85% NaCl and incubated at 37 DEG C for 24 hours. After dilution to 10 ^-7 using distilled water, the diluted samples were inoculated into Mueller-Hinton agar plates. After growth, plates were screened and identified according to Bergey's manual of systematic bacteriology. Many researchers have proved kimchi as the source of many bacterial strains capable of producing AMPs [15-17].

The strains selected from colonial morphology, biochemical characteristics and utilization of nutrients were identified as Bacillus. The refined product from the fermented food changes the dietary item to a pre-consuming form and changes our own intestinal microflora profile in a way that fermentation-enriched chemicals act [30]. The functional activities of such products include anti-mutation / anti-cancer [31], anti-obesity [32], anti-arteriosclerosis and immunomodulating effects [33].

Example 2: Sequence similarity

The degree of DNA similarity was assessed using the BLAST program ( www.ncbi.nlm.nih.gov/blast ).

For molecular phylogenetic analysis, 16S rRNA sequences of isolated microorganisms were compared with sequences in 10 Bacillus species. To determine the relationship of local isolates to Bacillus strains, multiple sequence alignments were performed between the 16S rRNA gene sequences of various Bacillus species and local isolates. Computer assisted RNA probes for bacterial databases were also shown to be more than 99% identical for the 10 species of Bacillus strains of the 16S rRNA sequence. The 16S rRNA sequence of K1R was deposited with GenBank under accession number AYTO01000043. The phylogenetic tree is shown in Fig.

The strain was deposited at Korea Institute of Bioscience and Technology as KCTC 18513P on November 22, 2016.

Example 3: Optimization of culture medium

Optimization of the culture medium is of great importance for the good production of the antimicrobial peptide. Various media were analyzed by various researchers to increase bacteriocin production. Bacteriocins are usually produced in complex media [18-19]. Physicochemical factors have dramatic effects on the production of bacteriocins [20]. Enhanced media as well as standard culture media are not always efficient in preparing peptides [21-22]. Media optimization is different carbon sources (lactose, sorbitol, sucrose, glucose, mannitol, starch, fructose, maltose), a nitrogen source (tryptone, yeast extract, peptone, oatmeal, beef X, malteu, soy meal) and metal ions (KH ₂ PO ₄ , FeSO ₄ , NaCl, Na ₂ HPO ₄ , CaCl ₂ , NaH ₂ PO ₄ , MgSO ₄ , ZnSO ₄ , MgCl ₂ ). MRS was used as a control medium and the amount of the three sources varied.

Many bacteria grow in a laboratory culture medium, which is designed to provide all essential nutrients as solutions for bacterial growth, but nutrients and growth factors must be optimized for maximum production of antimicrobial compounds [21]. Figure 2 is a graphical representation of the quantitative optimization of carbon, nitrogen and metal ions (MH: Mueller Hinton). a) carbon source, b) nitrogen source, c) metal ion, d) different amounts of beef x and maltose.

3-1: Optimization of carbon, nitrogen and metal ion sources

For the carbon source, Bacillus strain K1R was inoculated on a culture medium containing different concentrations of carbon source and incubated for up to 108 hours and samples were taken every 12 hours. A disk diffusion assay was performed to check the activity of the antimicrobial peptide produced by the Bacillus strain K1R against the indicated organism to create a graft. We plotted the incubation time versus the inhibition zone, indicating that maltose was the best carbon source for this strain. Again, this process was repeated to select the best source of nitrogen and metal ions. In case of nitrogen source, beef extract was found to be the best, but none of the metal ions showed good antimicrobial activity. Therefore, maltose and beef extracts were selected as the best carbon source and nitrogen source.

3-2: Final optimization

Complete optimization was carried out by varying the amount of beef extract and maltose. The peptides produced by Bacillus strain K1 were cultured for 30 hours after inoculation when 2% maltose (MAL) and 2% beef extract (BE) And it was confirmed that it showed the maximum activity. In this study, Mycobacterium smegmatis ATCC 9341 was selected as the indicator organism.

Finally, 2% beef x 2% maltose was set as the optimal medium and fermentation was carried out at 37 ° C with continuous shaking at 160 rpm on a 250 ml Erlenmeyer flask containing 20% medium. The effect of metal ions was not seen.

Example 4 Peptide Production and Purification

The seed culture was incubated at 160 rpm at 37 DEG C for 16 hours. It was transferred to a mass culture medium containing optimized medium (MRS medium) and incubated for 30 hours. After 30 hours, they were harvested by centrifugation at 10,000 x g for 35 minutes in a cold centrifuge.

Peptides were obtained from the harvested broth using the ammonium sulfate precipitation method [23] and then centrifuged at 4O < 0 > C at 10,000 x g for 50 minutes. The resulting precipitate was dialyzed against 10 mM Tris / HCl (pH 7.5). The desalting process was then performed using ultrafiltration membranes (Millipore Corp.) of 30 kDa and 10 kDa. After dialysis and filtration, the same buffer was used to purify on a Sephadex G-50 column (1.5 cm x 65 cm). The active fractions were pooled, concentrated and further purified on a Sephadex G-25 column (1.4 cm x 25 cm) using the same buffer.

The antimicrobial peptides secreted by the strain K1R showed antibacterial activity against Mycobacterium smegmatis ATCC 9341, Enterococcus faecalis ATCC 29212 and Micrococcus luteus ATCC 9341.

4-1: Ammonium precipitation method

After growing for 30 hours, the broth of the strain K1R was harvested. The cell-free culture medium was precipitated with ammonium sulfate (80% w / v) and stored at 4 ° C overnight under constant agitation. The pellets were pooled, suspended and dialyzed into Tris-HCl buffer (pH 7.5).

4-2: Ultrafiltration Technology

After the ammonium sulfate precipitation method was used, the pellet suspended in the buffer was purified using Amicon ultrafiltration technology. 30 kDa and 10 kDa filter paper were used in this procedure, respectively.

4-3: Column chromatography

The crude extract was applied to a Sephadex G-50 column and eluted with 10 mM Tris-HCl buffer (pH 7.5) as mobile phase. Fractions were collected and the antimicrobial activity against the indicator bacteria Mycobacterium smegmatis ATCC 9341 was monitored. The active fractions were lyophilized, concentrated, loaded onto a Sephadex G-25 column and eluted with the same buffer before use. Fractions that were positive for antimicrobial activity were pooled and stored at 4 < 0 > C. Protein content was assessed by the Bradford Method [24]. Figure 3 shows the concept of understanding of the two chromatographic steps involved during the purification and selection of the active fractions by observing the antimicrobial activity vs. protein content, i.e. absorbance at 595 nm vs. inhibition band.

Example 5: Protein evaluation, molecular weight measurement

Protein concentrations were assessed by the Bradford method [24] using bovine serum albumin as a standard. Molecular weight determination was performed by Tricine SDS-PAGE [25].

Example 6: In-situ analysis

In-situ analysis was performed on the indicator organism Mycobacterium smegmatis ATCC 9341 (1.5 × 10 ⁸ cfu / ml) with a 50 mM Tris-HCl buffer (pH 7.5) containing 2.5% Triton X-100, followed by Tricine SDS-PAGE Was superimposed on 0.6% agar on Mueller-Hinton medium and incubated at 37 < 0 > C.

The antimicrobial peptides were isolated by Tricine SDS-PAGE. The ultrahigh molecular weight marker protein (1.7 to 42 kDa) was used to determine the approximate molecular weight of the active protein band. After electrophoresis, one gel was stained with Coomassie Biolent Blue and the other was stored for identification of unstained gel-like AMPs bands for in-situ testing.

The antimicrobial compound was identified by the presence of a single band in the initial gel with a molecular weight near 4.6 kDa and the in situ test results showed a distinct band of inhibition points for the indicator organisms (see FIGS. 4 and 5). 4 is a photograph showing the result of Tricine SDS-PAGE of K1R. Lane 1, protein size marker; Lane 2 and 3, purified K1R peptide.

Example 7 Effect of Temperature and pH on Antimicrobial Activity

The thermal stability of K1R was determined by exposing the residual activity to 0, 10, 20, 37, 50, 60, 70, 80 ° C for 30 minutes and standard autoclave conditions before analysis. Likewise, pH stability was measured at various pH values (4.0-13.5) using 100 mM pH buffer: citric acid / phosphate (4-5.5), tris / hydrochloric acid (6.5-9.5) and potassium chloride / sodium hydroxide (10-13.5) Respectively.

The effects of temperature and pH on the antimicrobial activity of the peptides are shown in Figures 6 and 7. K1R was found to be the most stable at 30-60 ° C and lost about 25% activity at 70 ° C. The autoclave sample completely lost its activity. Analysis of the residual activity revealed that the AMP of K1R was most stable near pH 6.5-9. When the pH was increased to 12, 50% of the activity was lost. Finally, 35% residual activity was observed at pH 13.5.

Example 8 Effect of Chemical on Antimicrobial Activity

(Hydrogen peroxide and sodium perborate), reducing agent (? -Mercaptoethanol), chelating agent (EDTA and EGTA), detergent (SDS, CHAPS, Triton X-100, Tween 20, Tween 80, (Ca ⁺⁺ , Mg ⁺⁺ , Co ⁺⁺ , Cu ⁺⁺ , Ni ⁺⁺ , Zn ⁺⁺ , Mn ⁺⁺ and Ba ⁺⁺ ) and a solvent (acetone, chloroform, dimethylsulfoxide , Methanol, ethanol, 2-propanol, 1-butanol, toluene, diethyl ether, TCA, sodium chloride and potassium chloride).

The effects of various chemicals in terms of residual activity are shown in Table 1.

From the results in Table 1, it can be seen that the intact activity of the antimicrobial peptides produced by the Bacillus strain K1R was stably maintained in the presence of oxidants such as hydrogen peroxide and sodium perborate, and detergents such as deoxycholic acid and SDS. Triton X-100 and toluene had a similar type of effect, and metal ions such as calcium inhibited activity.

Example 9: Antibacterial activity of K1R

9-1. Minimal inhibition concentration test

Agar dilution was used to determine the minimum inhibitory concentration [26]. Bacitracin and vancomycin were used as reference antibiotics.

The indicator organisms chosen for the test are:

1. Gram's negative bacteria: Escherichia coli KCTC 1923, Salmonella typhimurium KCTC 1925, Pseudomonas aeruginosa KCTC 1637, Alcaligenes faecalis ATCC 1004.

2. Gram's positive bacteria: Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Mycobacterium smegmatis ATCC 9341, Vancomycin Resistant Enterococci 4 (VRE 4), Methicillin resistant Staphylococcus aureus 5-3 (MRSA 5-3), Methylenic resistant Staphylococcus aureus 4- , Vancomycin Resistant Enterococci 89 (VRE89), Vancomycin Resistant Enterococci 82 (VRE 82), Staphylococcus aureus KCTC 1928, Micrococcus luteus ATCC 5 (MRSA 4-5), Vancomycin resistant Satphylococcus aureus (VRSA), Vancomycin Resistant Enterococci 98 9341.

As a control, inoculated test organisms (1.5 x 10 < ⁸ > cfu / ml) in various concentrations of antibiotic plates with plates not containing antibiotics were incubated at 37 < 0 > C. Results were observed after 12 hours compared to the control plate

The antibacterial activity of K1R in terms of minimum inhibitory concentration is shown in Table 2.

The emergence of multidrug resistance has created a challenge to pharmaceutical research. In the present study, the present inventors conducted an antibacterial activity study to compare the efficacy of the newly extracted antimicrobial peptide with those of known donors such as vancomycin and bacitracin. K1R showed antagonistic action against various multidrug resistant pathogens. K1R showed a stronger potency against pathogens such as Vancomycin Resistant Enterococci 4, Vancomycin Resistant Enterococci 82, Vancomycin Resistant Enterococci 89, and Vancomycin Resistant Enterococci 98 than vancomycin and bacitracin. Its effect was similar to vancomycin for Enterococcus faecalis ATCC 29212 and was better than bacitracin for the same pathogen. The MIC value of K1R for Micrococcus luteus ATCC 9341 was found to be more efficient than that of basitracin.

The resulting effect of K1R compared to MDR pathogens represents a good impact on Gram-positive bacteria with a resistance pattern to vancomycin. Strains of staphylococcus and enterococcus strains resistant to vancomycin showed promising results. K1R has been found to be more efficient than our previous studies [34].

9-2. Aga's Diffusion Essay

The antimicrobial activity of the peptide K1R was detected by the Agadisc diffusion assays [27] and tested for all indicators as described at the minimum inhibitory concentration. Aliquots of 50 [mu] l K1R were added to discs (8 mm) on agar plates previously inoculated with each individual indicator strain.

The suppression spectra of K1R based on the agar disc diffusion assay are shown in Table 3. The results are consistent with the results of the MIC test.

Example 10 Amino acid sequence analysis

The amino acid sequence of K1R was determined by Edman degradation using a Procise Model 491 HT Protein Sequencer (Applied Biosystems, USA). The molecular weight of this peptide was also confirmed using the same sequence [28].

The amino acid sequence of K1R was found to be AVQGTLEDALNLSKGALNQVQK AIQNGDXLTVXGXLGTIXLAVSX (SEQ ID NO: 1). This sequence does not show significant homology with reported peptides of a similar type in the NCBI protein database using the BLAST (basic local alignment search tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi ) (Table 4) of the Bacillus genus.

Based on the amino acid sequence and the use of protein identification and analysis tools on the ExPASy Server, the molecular weight of the peptides was found to be ~ 4578.23 dalton, consistent with the results from Tricine SDS-PAGE and in situ analysis.

Example 11: Antioxidant activity of K1R

The DPPH method was slightly modified and the antioxidant activity was checked using the procedure described by Zhang et al [29]. DPPH is a stable free radical containing a hole-electron in its structure and is typically used for the detection of radical scavenging ability in chemical analysis. 100 쨉 l of various concentrations of the peptide solution was added to 100 쨉 l of DPPH (0.3 mM) in the methanol solution. The mixture was kept in a dark room at room temperature for 30 minutes and absorbance was measured at 517 nm by a spectrophotometer. Methanol was used as a blank and distilled water was used as a voice control. Ascorbic acid was used as a positive control. DPPH radical scavenging activity was determined by the following formula:

% = (1-Abs _sample / Abs _blank ) (where Abs _sample and Abs _blank are the absorbance of the sample and blank, respectively)

To obtain information on the mechanism of antioxidant effect of AMP, the radical scavenging activity was examined by measuring the change in absorbance of the DPPH radical at 517 nm. Both ascorbic acid and K1R showed concentration dependent abilities of DPPH radicals. The efficacy of K1R was similar or comparable to that of ascorbic acid at all concentrations tested (1 - 1000 / / ml). Figure 8 shows the results of the free radical scavenging activity expressed as% inhibition. K1R showed 70.97% inhibition at 1000 ㎍ / ml, whereas standard ascorbic acid showed 75.36% inhibition at the same concentration. In both cases, a progressive increase in percent inhibition was observed with increasing ascorbic acid concentration. Antioxidant activity was comparable to other peptides [35]. All determinations were performed 3 times and mean values were obtained.

Example 12: Preparation of pharmaceutical preparation (capsule)

The components of the peptide of the present invention (20 mg), crystalline cellulose (10 mg), lactose (25 mg) and magnesium stearate (1 mg) were mixed and filled in gelatin capsules according to the conventional preparation method.

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Institution name: Korea Biotechnology Research Institute

Access number: KCTC18513P

Checked on: 20161122

<110> Industry-Academic Cooperation Foundation, Chosun University <120> Antimicrobial peptide from Bacillus tequilensis K1R and the use          the <130> 1552 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 45 <212> PRT <213> Bacillus sp. <400> 1 Ala Val Gln Gly Thr Leu Glu Asp Ala Leu Asn Leu Ser Lys Gly Ala   1 5 10 15 Leu Asn Gln Val Gln Lys Ala Ile Gln Asn Gly Asp Xaa Leu Thr Val              20 25 30 Xaa Gly Xaa Leu Gly Thr Ile Xaa Leu Ala Val Ser Xaa          35 40 45

Claims (6)

1. An antimicrobial peptide comprising the amino acid sequence of SEQ ID NO: 1. The antimicrobial peptide according to claim 1, wherein the peptide is derived from a strain of Bacillus tequilensis K1R . The peptide of claim 1, wherein the peptide has a molecular weight of ~ 4.6 kDa, is stable over a pH of 6.5 to 9, is heat resistant at 60 캜 or lower, and does not change its activity at low temperatures of 0 캜 and 4 캜. Peptides. [Claim 3] The antimicrobial peptide according to claim 1, wherein the peptide has an antibacterial activity against multidrug-resistant pathogens. A pharmaceutical composition for antimicrobial use, which comprises the antimicrobial peptide of claim 1 as an active ingredient. An antioxidant composition comprising the antimicrobial peptide of claim 1 as an active ingredient.

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