KR20080044689A - Novel bacterium bacillus subtilis s1, novel peptides from it and use thereof - Google Patents

Abstract

A novel peptide isolated from a novel Bacillus subtilis S1 is provided to show excellent antibiotic effect on harmful fungi and bacteria, thereby being usefully used as an antibacterial and anti-fungal pharmaceutical composition, a pesticide, or a preservative of food, cosmetics or medicines. A Bacillus subtilis S1 having the antibiotics activity is deposited as a deposition no. KCTC 10888BP. A method for purifying a peptide from the Bacillus subtilis comprises the steps of: (a) culturing the Bacillus subtilis S1; (b) after filtering a culture solution obtained from the step(a), loading the filtrate into RP-HPLC(reverse phase-high performance liquid chromatography); (c) eluting proteins to a column of the step(b) using an eluent with gradually increased acetonitrile concentration; and (d) recovering a peak fraction from the eluent and investigating the antibacterial activity thereof. A peptide purified by the method and showing anti-bacterial and anti-fungal activities is characterized in that it is secreted from the Bacillus subtilis S1, has a molecular weight of 1.4-1.6 KDa, and shows antibacterial activity on Rhizoctonia solani and Colletotrichum coccodes. An anti-bacterial and anti-fungal pharmaceutical composition, a biological pesticide or a preservative of foods, cosmetics, or medicines comprises the peptide as an effective ingredient. Further, the concentration of the acetonitrile increases from 5% to 65%.

Description

Bacillus subtilis S1, a new strain, a novel peptide purified therefrom and its use {Novel bacterium Bacillus subtilis S1, novel peptides from it and use}

1 is a picture showing the results of screening the antifungal activity of bacteria,

A: Aspergillus species vs S-10

B: Aspergillus fumigatus vs S-10

C: Aspergillus species vs D-2

D: Fusarium moniliforme var. subglutinans vs S-1 , F: test fungi, S: screening bacteria.

2 is a diagram showing the result of aligning the nucleotide sequence of Bacillus subtilis and S-1 colony,

Figure 3 shows the Fusarium moniliforme var. The figure shows antifungal activity against subglutinans ,

A: the supernatans (40 μg)

B: Mes buffer

C: Mes buffer + BSA.

4 is a graph showing the results of separating peptides by performing reverse phase-high performance liquid chromatography (RP-HPLC) first;

5 is a graph showing the results of separating and purifying new peptides S1-14, S1-19, and S1-23 by performing reverse phase-high performance liquid chromatography (RP-HPLC) in a second manner;

6 is a protein electrophoresis picture of the novel peptides S1-14, S1-19 and S1-23 isolated and purified from Bacillus subtilis ,

Figure 7a is a new peptide S1-14, S1-19, S1- by culturing the fungi Colletotrichum coccodes in the medium to which the new peptides S1-14, S1-19 and S1-23 are added Figure 23 visualizes the antifungal activity of

Figure 7b is a new peptides S1-14, S1-19, S1-23 by culturing the fungi Rhizoctonia solani in a medium to which the new peptides S1-14, S1-19 and S1-23 are added The picture visualizes the antifungal activity of

8 is a diagram showing the results of CD (Circular Dichroism) analysis for secondary structure analysis for the novel peptides S1-14, S1-19, S1-23,

9 is a diagram showing the results of the structural analysis of S1-14.

The present invention relates to a novel isolate, identified Bacillus subtilis S1 ( Bacillus subtilis S1 ), a novel peptide isolated therefrom and its use, and specifically, Bacillus subtilis S1 , a new strain isolated and identified from soil ( Bacillus subtilis S1 ), and anti-fungal novel peptides S1-14, S1-19, S1-23, and their use, which are separated and purified therefrom.

Bacterial infection is one of the most common and deadly causes of human disease. Since Fleming's first separation of penicillin from blue mold in 1928, antibiotic development is considered one of the greatest achievements of modern medicine in contributing to treating infections and reducing mortality.

The term antibiotic refers to selman a. In the United States, where microorganisms were isolated from soil samples to find streptomycin. It was born when Waksman proposed to the American Society of Bacteriology to call "antibiotics" for substances that inhibit the development or metabolism of other microorganisms. Common antibiotics are chemical substances produced by microorganisms, and are defined as substances that inhibit the growth of other microorganisms even at low concentrations.In addition to separation from microorganisms, they can be synthesized chemically or chemically from microbial products. Include as long as possible to make derivatives. Furthermore, it includes the case of inhibiting the growth or metabolism of cancer cells as well as the growth or metabolism of microorganisms.

On the other hand, in the case of antibiotics used in the treatment of diseases, many kinds of antibiotics have been discovered and developed so far, but are widely used, but the number of pathogenic microorganisms that have become resistant to antibiotics has appeared and the number thereof has increased. In other words, antibiotics have been used to treat diseases of humans and animals, and the use of agricultural and marine products and increased productivity has led to the emergence of resistant bacteria. In addition, super bacteria that are resistant to vancomycin, the world's strongest antibiotic, have appeared and are shocking. Even more problematic is that the time for bacteria to develop resistance to antibiotics that are already developed is incomparably faster than the time to develop new antibiotics.

Tolerance to antibiotics is a distinct phenomenon from antibiotic resistance and was first discovered in Pneumococcus sp. In the 1970s (Tomasz et al., Nature , 227; 138-140, 1970). Resistant species stop growing but do not die in the presence of the usual concentration of antibiotic, which does not cause the activity of autolytic enzymes, such as autolysin, in the bacteria itself when the antibiotic inhibits cell wall synthase. Because.

In terms of the mechanism of action, resistance is largely divided into two pathways, the first being a phenotypic resistance that occurs when growth rate decreases in all bacteria (Tuomanen E., Revs. Infect. Dis. , 3; S279-S291, 1986), the second is genetic resistance by mutations in certain bacteria. In both cases, the down regulation of autolysine activity occurs in common, which is temporary for outward resistance and permanent for mutational genetic resistance. In the case of the simplest genetic resistance, a defect occurs in the autolysine enzyme, but no clinically resistant strain has been found due to the deletion of the enzyme. Rather, a case of resistance by regulating the activity of the autolysine is mainly reported. (Tuomanen et al., J. infect. Dis. , 158; 36-43, 1988).

It is clinically very important for bacteria to be resistant to various antibiotics, because the inability to eradicate resistant bacteria makes antibiotic treatment less effective in clinical infections (Handwerger and Tomasz, Rev. Infec. Dis ., 7; 368-386, 1985). In addition, resistance is a prerequisite for resistance to antibiotics, since strains that survive despite antibiotic treatment are obtained and these strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics. .

Currently, VANCOMYCIN and teicoplanin have been developed as potent antibiotics against penicillin and methicillin-based antibiotic resistant strains, such as MRSA (methicillin resistant staphylococcus) and MRSE (Methicillin Resistant Staphylococcus epidermidis). Its use has continued to increase. However, due to excessive use, the pathogenic bacterium entererococci, which is resistant to even vancomycin, the last antibiotic, is found, Enterococcus faecalis and Mycobacterium tuberculosis. ) And Pseudomonas aeruginosa have shown resistance to all antibiotics known to date (Stuart B. Levy, Scientific American , 46-53, 1998). Therefore, there is an urgent need to develop new antibiotics capable of killing bacteria that have antibiotic resistance and resistance.

Accordingly, the present inventors have isolated and identified a novel bacterium Bacillus subtilis S1 , and isolated and purified new peptides S1-14, S1-19 and S1-23 from the culture of the strain. As a result of observing whether the activity shows strong antibiotic activity, it was confirmed that the present invention can be usefully used as a preservative of biopesticides, functional cosmetics, cosmetics, foods or pharmaceuticals as well as antibacterial and antifungal agents.

It is an object of the present invention to provide a novel strain Bacillus subtilis S1, a novel peptide having antibacterial and antifungal activity isolated therefrom and its use.

The present invention provides a novel strain Bacillus subtilis S1.

The present invention also provides a peptide exhibiting antibacterial and antifungal activity obtained by culturing and purifying Bacillus subtilis and a method of producing the same.

In addition, it provides a pharmaceutical composition for antibacterial and antifungal with the peptide as an active ingredient.

The present invention also provides a biopesticide comprising the peptide as an active ingredient.

In addition, the present invention provides a preservative for food, cosmetics or pharmaceuticals using the peptide as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel strain Bacillus subtilis S1.

The present inventors first obtained several kinds of strains from soil, and then separated them into a single colony state, and cultured media of these colonies, pathogenic fungi, and Fusarium moniliforme var. Subglutinans in solid medium. S1 with strong activity were selected by comparing their degree of antibiotic activity. The strains were identified by 16S rRNA gene sequence and confirmed to be 100% Bacillus subtilis. In order to identify the conditions that produce the most active peptides in this strain, activity was determined in various culture media (LB (Luria Broth), BHI (Brain Hart Infusion), NB (Nutrient broth), TSB (Trypticase Soy Broth) medium). As a result of the measurement, the highest activity was observed when cultured in TSB medium.

The Bacillus subtilis S1 was deposited on January 11, 2006 to the Korea Biotechnology Research Institute Gene Bank, an international depository institution (accession number KCTC 10888BP).

The present invention also provides a peptide exhibiting antibacterial and antifungal activity obtained by culturing and purifying Bacillus subtilis and a method of producing the same.

The present inventors cultured the Bacillus subtilis S1 strain, and then subjected to chromatography to separate peptides from Bacillus subtilis culture.

First, the Bacillus subtilis was incubated with shaking and the culture solution was filtered through a filter paper, and then loaded into Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). In order to elute the protein bound to the column, the protein was separated by adding an eluent gradually increasing the acetonitrile concentration from 5 to 65%. The maximum fraction was recovered and subjected to SDS-PAGE to obtain three peptides. The isolated three peptides were identified as peptides having a molecular weight of about 2 kDa or less as shown in FIG. The inventors have named the novel peptides 'S-1-14, S1-19, S1-23 (S1-14, S1-19, S1-23)'.

The sequencing was attempted by the conventional method for sequencing of the peptide, but the exact sequencing was impossible. Therefore, Maldi-MS was used to specify the molecular size of these given peptides, and then the structure search process centered on databases similar to molecular size. Was performed. Using the exact molecular size of these peptides, the electronic database CCD (The Combinded Chemical Dictionary: Dictionary of Natural Products) search showed that S1-14 was identified as a peptide having the structure shown in FIG. The structures of S1-23 are considered to be similar in the case of S1-14, S1-19, and S1-23 since the maldi MS / MS data of the masses are almost similar.

In order to measure the antifungal activity of S-1-14, S1-19, S1-23 (S1-14, S1-19, S1-23), the present inventors have identified plant pathogenic fungi. Antifungal tests were performed on Rhizoctonia solani and Colletotrichum coccodes . As a result, it was visually confirmed that the novel peptides S-1-14, S1-19, S1-23 (S1-14, S1-19, S1-23) of the present invention exhibit potent antifungal activity (see FIGS. 7A and B). .

In order to confirm whether the novel peptides of the present invention, S1-14, S1-19, S1-23 (S1-14, S1-19, S1-23), are cytotoxic, S1-14, S1-19, As a result of investigating the erythrocyte destructive ability of human of S1-23 (S1-14, S1-19, S1-23), S1-14, S1-19, S1-23 (S1-14, S1-19, S1) of the present invention -23) showed no cytotoxicity, whereas melittin, a bee venom used as a positive control, showed very high cytotoxicity (see Table 2 ).

From the above results, the pharmaceutical composition containing the novel peptides S-1-14, S1-19, S1-23 (S1-14, S1-19, S1-23) of the present invention as an active ingredient shows excellent antifungal effect. As it is not cytotoxic, it was confirmed that it can be usefully used as a safe antifungal agent to the human body.

In addition, the present invention provides a pharmaceutical composition for antibacterial and antifungal comprising the peptide.

The novel peptides S-1-14, S-1-19, S1-23 (S1-14, S1-19, S1-23) of the present invention can be administered in various parenteral dosage forms, and when formulated, Diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like, are prepared. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The novel peptides of the present invention can be used in admixture with a number of carriers that are pharmaceutically acceptable, 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, small molecule proteins or other stabilizers can be used as medicaments.

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

In the pharmaceutical composition of the present invention, the total effective amount of the novel peptides of the present invention may be administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time, and multiple administration. Multiple doses may be administered by a fractionated treatment protocol that is administered for a long time. Since the concentration is determined by taking into consideration various factors such as the age and health status of the patient as well as the route of administration and the number of treatments of the drug, it is considered by those of ordinary skill in the art in view of this point. Appropriate effective dosages may be determined depending upon the particular use of the novel peptides of the invention as pharmaceutical compositions.

In addition, the present invention provides a biological pesticide, food preservative, cosmetic preservative or pharmaceutical preservative containing the novel peptides as an active ingredient.

Food preservatives, cosmetic preservatives and pharmaceutical preservatives are additives used to prevent the deterioration, decay, discoloration and chemical change of foods and medicines. These include fungicides and antioxidants, and inhibit the growth of microorganisms such as bacteria, fungi and yeasts. Functional antibiotics, such as inhibiting the development or disinfection of decay microorganisms in food and pharmaceuticals, are included. The ideal conditions for these food preservatives, cosmetics and pharmaceutical preservatives should be nontoxic and effective in trace amounts.

Pesticides to eradicate pests of crops must also inhibit the growth of harmful microorganisms and harm the human body so that humans can safely consume pesticide-spread crops. As the novel peptides of the present invention show strong antibiotic activity and no toxicity as shown in Table 1 and Table 2 , it was confirmed that they can be usefully used as biological pesticides, food preservatives, cosmetics and pharmaceutical preservatives.

The biopesticides of the present invention may add one or more other pesticides in addition to the peptide component and may be mixed with suitable carriers and / or additives commonly used in pesticides such as hydrating agents, emulsion concentrates, water soluble powders, powders, granules, suspension concentrates, and the like. It can be prepared by. As the solid carrier, minerals such as grains such as soy flour and flour, diatomaceous earth, apatite, gypsum, talc, bentonite, and clay may be used, but are not limited thereto. Vegetable oils, mineral oils, petroleum (e.g. kerosene and naphtha), xylenes, cyclohexane, cyclohexanone, dimethylformamide, dimethylsulfoxide, tricholoethylene, methylisobutylketone and water may be used as liquid carriers. However, it is not limited thereto. If necessary, surfactants are added to form a homogeneous and stable preparation.

The biopesticide of the present invention may be used an aqueous suspension or an aqueous emulsion for spraying plants by diluting the hydrated powder, the emulsifying thickener, the water-soluble powder and the suspending thickener to a specific concentration with water.

In the biopesticide of the present invention, the effective concentration for controlling the novel peptide is 0.1 μg / ml to 1 mg / ml, preferably 1 μg / ml to 100 μg / ml.

The biopesticides of the present invention may be used for the protection of fields, forests, plantations, greenhouses, orchards or vineyards, for cultivation of crops or forests such as cereals (eg corn, wheat, rice or sugar cane), cotton, tobacco, vegetables ( Eg, cucumbers, peppers, soybeans, lettuce, onions, tomatoes or peppers, field crops (e.g. strawberries, potatoes, beets, peanuts or beans), sugarcane, pasture or forage (e.g. corn or sugar cane), cultivation Crops (e.g. tea, coffee, cocoa, banana, palm oil, coconut, rubber or spices), fruit trees or small vineyards (e.g. citrus fruits, kiwi, avocado, mango, olives or hodo), vineyards, decorative plants, flowers or vegetables Or for the protection of shrubs (deciduous and evergreen), plantations or amniotic fluids in shrubs, gardens and parks or in forests.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Strain Isolation and Identification

The present inventors first isolate several strains from the soil and isolate them into a single colony state, followed by culture of these colonies, pathogenic fungi, and Fusarium moniliforme var. Subglutinans (KCTC 16909). S1 with strong activity were selected by comparing the degree of their antibiotic activity by raising them in a solid medium. The strain was identified by 16S rRNA gene sequence at Biotechnology Research Center Bioventure Co., Ltd. Probionic and confirmed that it is 100% Bacillus subtilis .

Table 1: Identification Results of S-1 Strains

Strain  Blast Survey Results 1 S-1 Bacillus subtilis (100%)

The Bacillus subtilis S1 was deposited on January 11, 2006 to the Korea Biotechnology Research Institute Gene Bank, an international depository institution (accession number KCTC 10888BP).

And in order to identify the conditions that produce the most active ingredient in this strain, the temperature of 30 ℃ in various culture medium, LB (Luria Broth), BHI (Brain Hart Infusion), NB (Nutrient broth), TSB (Trypticase Soy Broth) medium As a result of measuring the activity by incubation at 37 ℃, it was found that the highest activity when incubated for 24 hours at 37 ℃ in TSB medium.

Table 2: Antifungal Activity of S1 Culture Supernatants Obtained in Various Media

 badge  Growth temperature (℃)  Antifungal activity LB 30 － 37 － NB 30 － 37 － BHI 30 － 37 + TSB 30 + 37 +

<Example 2> Bacillus subtilis ( Bacillus subtilis Protein extraction from the medium

The inventors performed Revers Phase (HPLC) -HPLC to isolate proteins from Bacillus subtilis culture.

First, Bacillus subtilis was incubated for 24 hours in a TSB (Trypticase Soy Broth) liquid medium, and the culture medium shaken with a culture temperature of 37 ° C. was filtered through a filter paper (Whatman, No. 2). It was loaded in Reverse Phase (RP) -HPLC (High Performance Liquid Chromatography) equipped with a Shim-pack VP-ODS C18 column (4.6 × 250 mm, particles; 12.0 ± 1.0 nm pore, Shimadzu). In acetonitrile buffer solution containing 0.1% Trifluoroacetic Acid, the acetonitrile concentration was increased from 5% to 65% for 2 minutes at 2% per minute (linear gradient) to bind the protein bound to the column. After elution, the peak fraction was recovered (FIG. 4). As a result, the first reverse phase (RP) -HPLC (High Performance Liquid Chromatography) was able to obtain a myriad of protein peaks. To further subdivide the fractions, the fractions were randomly divided into subdivisions (0-30 minutes, 31-60 minutes, 61-90 minutes), and again, Reverse Phase (RP) -HPLC (High Performance Liquid Chromatography) was performed. As in the first test, the active parts were screened to obtain the final three peaks (FIG. 5).

Example 3 Protein Electrophoresis

The inventors prepared a 16.5% slab gel and obtained a fraction obtained in <Example 1> according to the method of Laemmli (Laemmli et al . 1972), and 16.5% Tricine-SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). Was carried out.

The protein sample and the standard protein obtained in Example 2 were loaded on a 16.5% acrylamide gel and subjected to electrophoresis, and then the gel was stained with Coomassie brilliant blue R-250. In addition, a band of protein was identified by destaining with a destaining buffer (MeOH: glacial acetic acid: distilled water = 30: 10: 60).

Molecular weight standard proteins include Triosephosphate Isomerase, 17 kD Myoglobin (Horse Heart Myoglobin), 14.2 kDa alpha- isolated from muscle of 26.6 kDa mice belonging to the standard protein category upon Tricine protein electrophoresis. Molecular weight of protein using α-Lactaalbumin from bovine milk, 6.5 kDa Aprotinin from bovine lung, 3.5 kDa Insulin Chain B, and 1 kDa Bradykinin Was measured ( FIG. 6 ). As a result, it was confirmed that the molecular weight of the new peptide isolated from the Bacillus subtilis culture was about 2 kDa or less.

Example 4 Confirmation of Molecular Weight of New Peptide

The peptide isolated in Example 2 was dissolved in 6N-HCl, and then hydrolyzed and dried at 110 ° C. for 22 hours to confirm molecular weight by mass spectra (Voyager DE RP instrument (Perseptive Biosystems, Framingham, MA)). was as a result, the novel peptides of the invention SI-14 as shown in Figures 4 and 5:. 1462.79, SI-19 : 1490.81, SI-23:. it was confirmed in three peptides having a molecular weight of 1504.83 inventors They named the peptides S1-14, S1-19, S1-23, respectively.

Example 5 Antifungal Activity Measurement

<5-1> MTT assay and microscopic observation

For the determination of antifungal activity of the peptides of the present invention, plant pathogenic fungi After adding S1-14, S1-19 and S1-23 to Rhizoctonia solani and Colletotrichum coccodes (KACC, Korean Agricultural Culture Collection), MTT assay was performed. .

In a 96-well microtiter plate, Rhizoctonia solani (KACC, Korean Agricultural Culture Collection) and Colletotrichum coccodes (KACC, Korean Agricultural Culture Collection) Each of the YPD medium containing the diluted to 100μl per well to be 2 X 10 ³ cell / well. Here, S1-14, S1-19, S1-23 of the present invention and melittin peptide (Anigen synthesis) known to have strong antifungal activity were added at various concentrations, respectively. After leaving it at 30 ° C. overnight, the absorbance was measured at 620 nm with an Enzyme-Linked Immunosorbent Assay (ELISA), the absorbance graph was prepared for each concentration, and the minimum growth inhibitory concentration (MIC) was measured. Decided.

As a result, as shown in Tables 3 and 4 , S1-14, S1-19, and S1-23 of the present invention have a minimal inhibitory concentration (MIC) with melittin (3.13 μM) having strong antifungal activity. It was confirmed that the same or twice as strong antifungal activity (1.56 ~ 3.13μM).

Table 3: Determination of antifungal activity against the Collector Coconut Cocords of S1-14, S1-19, S1-23

Colletotrichum coccodes S1 14 S1 19 S1 23 Melittin 3.13-1.56 μM 3.13-1.56 μM 1.56 μM 3.13 μM

Table 4: Determination of antifungal activity against the Collector Coconut Cocords of S1-14, S1-19, S1-23

Rhizoctonia solani S1 14 S1 19 S1 23 Melittin 3.13-1.56 μM 3.13-1.56 μM 3.13-1.56 μM 3.13 μM

<5-2> microscopic observation

Well in which nothing is added to Rhizoctonia solani and Colletotrichum coccodes in Example <5-1> , S1-14, S1-19, S1-23 of the present invention Each well and melittin added wells were set as negative control, experimental and positive control, respectively, and visualized by observing them under a microscope.

As a result, as shown in Figures 7a and b, in the negative control group to which nothing was added, Rhizoctonia solani and Colletotrichum coccodes grew vigorously and S1-14 of the present invention. In the experimental group to which S1-19 and S1-23 were added, there was little growth of Rhizoctonia solani and Colletotrichum coccodes , similar to the positive control group. This visually confirmed that it exhibits strong antifungal activity of S1-14, S1-19, S1-23 of the present invention.

Example 6 Cytotoxicity Measurement of S1-14, S1-19, S1-23

In order to confirm whether S1-14, S1-19, and S1-23 of the present invention show cytotoxicity, the present inventors examined the erythrocyte destructive ability of S1-14, S1-19, and S1-23, respectively.

PBS buffer solution was added to human blood at a ratio of 1: 1 and centrifuged at 2000 rpm three times. After washing three times, pure erythrocytes were obtained to obtain erythrocytes at a concentration of 8% (PBS, pH 7.0). Dilution). The S1-14, S1-19, S1-23 of the present invention was continuously diluted to a concentration of 12.5 μM / well to 1/2 and reacted at 37 ° C. for 1 hour, followed by centrifugation at 1,000 g of the supernatant. The amount of hemoglobin contained in the stomach was investigated by measuring absorbance at 414 nm wavelength. In order to investigate the degree of cell destruction, 1% Triton X-100 was added to human erythrocytes and the absorbance of the supernatant was measured. It was calculated according to the following Equation 1 compared with the erythrocyte destructive ability.

Where Absorbance A is the absorbance of S1-14, S1-19 and S1-23 solutions at 414 nm, absorbance B is the absorbance of PBS at 414 nm and absorbance C is 1% of Triton X-100 at 414 nm. Absorbance is shown.

The results of erythrocyte destructive ability according to the above formula are shown in Table 5 below.

Table 5: Determination of hemolytic activity of S1-14, S1-19, S1-23

0.10 (μM) 0.20 0.39 0.78 1.56 3.13 6.25 12.5 S1-14 0 0 0 0 0 0 0 0 S1-19 0 0 0 0 0 0 0 0 S1-23 0 0 0 0 0 0 0 0 Melittin 0 0 34 100 100 100 100 100

As a result, the S1-14, S1-19, S1-23 of the present invention shows little cytotoxicity, whereas the melittin peptide used as a positive control is very high cytotoxicity.

Example 7 Peptide Sequence and Structural Analysis

<7-1> Secondary Structure Analysis of S1-14, S1-19, S1-23

CD spectra of S1-14, S1-19, and S1-23 were measured using a J 720 spectropolarimeter (Jasco, Japan). All S1-14, S1-19, S1-23 samples were maintained at 25 ° C. during the analysis. A pathlength cell of 1 mm was used and four consecutive measurements were made at 190-250 nm at 0.1 nm intervals per sample. In order to measure the secondary structure under the conditions of mimicking the cell membrane, it was measured in 50% TFE and 30 mM SDS including 10 mM phosphate buffer (pH 7.0). The concentrations of S1-14, S1-19, and S1-23 for measuring the CD spectrum were set to 100 µg / ml. Mean residue ellipticity (θ) is expressed as deg · cm ² · dmol ⁻¹ , and is expressed as [θ] = [θ] _obs (MRW / 10ℓ c ). [θ] _obs is the ellipticity measured in milligrees, and MRW is the mean residue molecular weight of S1-14, S1-19, and S1-23. c represents the concentration of the sample (mg / ml) and l represents the optical pathlength (cm) of the cell. % Α-helicity of S1-14, S1-19, S1-23 was calculated by the following equation.

% Α-helicity = 100 ([ θ] 222 - [θ] 0 222) / [θ] 100 222

([θ] ₂₂₂ = the observed mean residue ellipticity per residue at 222nm in deg · cm ² ㆍ dmol ^-1 . [θ] ⁰ ₂₂₂ = -3,000 deg · cm ² ㆍ dmol ^-1 (the estimated ellipticity corresponding to a random coil structure) [θ] ¹⁰⁰ ₂₂₂ = -33,000 degcm ² dmol ^-1 (the estimated ellipticity corresponding to 100% α-helical peptide)

As a result of the secondary structure analysis, the structures of S1-14, S1-19, and S1-23 were expected to be a straight β-SHEET structure instead of the α-HELIX structure.

<7-2> Tertiary Structure Analysis of S1-14

The present inventors attempted a conventional sequencing method for structural analysis of the peptide, but since it was impossible to accurately determine the sequence, it was possible to specify the molecular size of these given peptides using Maldi-MS (AXIMA-QIT., Shimadzu, Japan). In addition, the structure search process was performed based on databases of similar molecular size. Using the exact molecular size of these peptides, an electronic database CCD (The Combinded Chemical Dictionary: Dictionary of Natural Products) search was conducted. Once the mass is approximately equal to the margin of error, the amino acid sequence and the N-terminal blocking were included. This is also consistent with the results of this protein sequencing (Harvey DJ et al., Rapid Commun Mass Spectrom. 2004; 18 (24): 2997-3007.

In the case of S1-14, S1-19, and S1-23, the maldi MS / MS data of the masses are almost similar, so it was predicted that the basic structure of the three molecular weights was almost the same and there was a part with mass difference. Comparing with MS / MS data, some agreement was found. Therefore, S1-14 was found to be a peptide having the structure shown in FIG. 9, and the structures of the remaining S1-19 and S1-23 are considered to be similar.

As described above, the novel peptides S1-14, S1-19, S1-23 of the present invention exhibit excellent antibiotic effects in harmful fungi and bacteria, so as well as pesticides, antimicrobial and antifungal pharmaceutical compositions using the same Food preservatives, cosmetic preservatives can be usefully used as a pharmaceutical preservative.

Claims (10)

Antimicrobial Bacillus subtilis S1 KCTC 10888BP. 1) culturing the Bacillus subtilis S1 of claim 1; 2) filtering the culture solution of step 1, and then loading it into Reverse Phase-High Performance Liquid Chromatography (RP-HPLC); 3) eluting the protein with eluent with increasing acetonitrile concentration in the column of step 2; 4) A method for purifying peptides from Bacillus subtilis comprising recovering the peak fraction of the eluate of step 3 and examining their antimicrobial activity. 3. The method of claim 2, wherein in step 4 the acetonitrile concentration is increased from 5% to 65%. Peptides purified by the method of claim 2 and exhibiting antimicrobial and antifungal activity having the following properties: 1) is secreted from Bacillus subtilis S1 of claim 1, 2) the molecular weight is 1.4 to 1.6 KDa as measured by mass spectra. 3) It exhibits antimicrobial activity against Lizathenia solani and Collector trigun Cocords. The SI-14 peptide according to claim 4, represented by the following structural formula (I).       (Ⅰ)

The S-23 peptide according to claim 4, wherein the molecular weight is 1.5 to 1.6 KDa. The SI-19 peptide according to claim 4, wherein the molecular weight is greater than SI-14 and less than SI-23. Antimicrobial and antifungal pharmaceutical composition comprising the peptide of claim 4 as an active ingredient. A biopesticide comprising the peptide of claim 4 as an active ingredient. A preservative for food, cosmetics or pharmaceuticals using the peptide of claim 4 as an active ingredient.

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