KR101249771B1 - ＨＰＡ３ＮＴ３ peptide having antialgae activity against organism induced red tide and the use of the same - Google Patents

Abstract

The present invention relates to a natural tide peptide derived from an organism, and more specifically, the HPA3NT3 peptide according to the present invention exhibits excellent tide activity, and this tide activity is selectively shown only in red tide-causing organisms, and is naturally degraded. It is free of cytotoxicity and can be used as a safe and useful wake preparation for marine ecosystems.

Description

{HAPA3NTT peptide having antialgae activity against organism induced red tide and the use of the same}

The present invention relates to an anti-red tide peptide having an activity of inhibiting the growth of red tide organisms and a composition for controlling red tide containing the same.

Red tide is called collectively red tide when the color of the seawater turns red, green, or brown due to the variety of pigments it encounters and is suitable for plankton. During the rainy season or rainy season in the southern coast of Korea, the color of the sea turns reddish brown, causing fish, such as oysters, shellfish, and mussels, to live in the sea. In addition, when people eat fish and shellfish that eat toxic phytoplankton, it causes various paddock phenomenon.

Seed. Polycricoides was first discovered on the coast of Puerto Rico (Margalef, 1961), and then on C. heterolobatum (Silva), which caused red tide in the Bay of Bayegat, New Jersey, and the coast of California, USA. , 1967). This species has been reported to have caused red tide in Harima Nada and Japan's Yatsushiro Bay, where it caused fisheries damage (Yuki and Yoshimatsu, 1989), as well as in central and western Japan. (Fukuyo et al., 1990). It has recently been reported to cause red tide in Canada (Whyte et al., 2000), the Gulf of California in Mexico (Carate Lizarraga et al., 2000), and the Pacific coast of Guatemala in Latin America. Blood also belonging to coccyx. Minimum (P. minimum), and f. H. belonging to P. micans and needle-jointed steel. H. akshiwo , Chattonella sp .) and seeds. Marina ( C. marina ) is also known to cause red tide. Chattonella Esp In case of sp .), when the red tide density is over 50, it occurs over the 2 ~ 5Km radius of water, and the red tide advisory is issued due to fear of fishery damage. A red tide alarm is issued as expected.

To date, several methods have been studied to prevent red tide or reduce damage caused by red tide. Spraying chemicals, using ultrasonic grinder, ozone generator, or centrifugal separator to remove red tide organisms chemically and physically. In addition, a method of adsorbing red tide causative organisms using clay to settle to the bottom has also been developed. However, these methods can have another adverse effect on the ecosystem, and there are many problems to be used in a wide range of red tides. Currently, biological methods for removing red tide causative organisms are also being studied. This is how the predator-predator relationship is used to eliminate unwanted organisms within the ecosystem. However, this method may change the food chain of the ecosystem and cause other problems. In another field, methods for killing red tide by marine bacteria are also being actively researched. Recently, attempts have been made to control red tide organisms using protein synthesis inhibitors, cell wall inhibitors, and antibiotics that act on cell membranes. Or by using these materials as models, artificially synthesize low-molecular substances, which produce foods produced in marine ecosystems or at sea, such as problems caused by pesticides that accumulate in the sea and eventually accumulate in the sea. Will accumulate in the human body.

Considering these problems, peptides or protein preparations can be decomposed naturally and can be used as a pharmaceutical preparation that is harmless to the human body or the natural ecosystem. They will also be able to reduce the production cost by mass expression through genetic manipulation, as well as express peptide activity on the surface of bacteria or viruses that specifically attach to red tide organisms.

To date, the antibacterial peptide Mastoparan B, which has an amphipathic alpha-helix structure purified from bee venom, contains Alexandrium tamarense and chattonella catenium ( Chattonella). catenatum ) has been shown to have a killing effect. However, the peptides have been reported to show high cytotoxicity at low concentrations.

Therefore, the present inventors cut the N-terminal part having an irregular structure of an analog (HPA3) peptide and an HPA3 peptide having an increased hydrophobic part by using the HP (2-20) peptide derived from Helicobacter piori, and then increased the cation. HPA3NT3 peptide in the analogue of HPA3NT3 peptide has excellent anti-tanking activity in red tide organisms and has stability against cytotoxicity (hemolytic activity), thus completing the present invention as a non-toxic anti-drop preparation.

It is an object of the present invention to provide a composition for controlling red tide containing HPA3NT3 peptide having excellent anti-tide activity, a preparation method thereof and an active ingredient containing the peptide.

The present invention provides HPA3NT3 peptides having the amino acid sequence set forth in SEQ ID NO: 3.

The present invention also provides a method for preparing an HPA3NT3 peptide having the amino acid sequence set forth in SEQ ID NO: 3.

In addition, the present invention provides a composition for controlling red tide containing HPA3NT3 peptide having an amino acid sequence set forth in SEQ ID NO: 3 as an active ingredient.

The HPA3NT3 peptide of the present invention selectively shows excellent anti-tanking action on red algae-induced algae, and does not show cytotoxicity, so it can be usefully used as a safe anti-tanking agent for marine ecosystems.

Figure 1 is a micrograph showing the trough activity of the HPA3NT3 peptide according to the present invention:
A of Figure 1 is a needle-like imitation steel. Micrographs showing wake activity against C. marina ;
1: control group not treated with peptide;
2: HP (2-20) treated group;
3: HPA3 treatment group;
4: HPA3NT3 peptide treatment group;
FIG. 1B is a micrograph showing wake tide activity against H.akashiwo ;
1: control group not treated with peptide;
2: HP (2-20) treated group;
3: HPA3 treatment group; And
4: HPA3NT3 peptide treatment group.
2 is a seed of the flagella algae. C. polykrikoides and blood. Here is a micrograph showing the activity of the peptide's activity against P. minimum :
FIG. 2A is a micrograph showing the trough activity of a peptide in a control group not treated with the peptide; FIG.
1: Seed as a flagella algae. C. polykrikoides ;
2: blood. Minimum ( P. minimum );
2B is a micrograph showing the activity of trough activity in the HPA3 peptide treated group;
1: Mr. C. polykrikoides ;
2: blood. Minimum ( P. minimum );
FIG. 2C is a micrograph showing banding activity in the HPA3NT3 peptide treated group;
1: Mr. C. polykrikoides ; And
2: blood. Minimum ( P. minimum ).
3 is S. The figure shows the non-inhibitory activity of the tide peptides in the growth of S. costatum :
A: control group not treated with peptide;
B: group treated with 128 micromolar (μM) HPA3 peptide; And
C: group treated with 128 micromolar (μM) of HPA3NT3 peptide.
FIG. 4 shows the selective activity of HPA3NT3 peptides:
A: When treated with 2 micromoles (μM) of HPA3NT3 peptide, S. incubation time was determined. A graph showing cell density of S. costatum ;
B: When treated with 8 micromole (μM) of HPA3NT3 peptide, S. incubation time was determined. A graph showing cell density of S. costatum ;
C: Mr. C. marina and S. The co-cultivation of S. costatum, followed by 4 μM treatment of HPA3NT3 peptide;
D: H. H. akashiwo and S. The co-cultivation of S. costatum, followed by 1 μM treatment of HPA3NT3 peptide; And
E: Blood. P. minimum and S. The co-cultivation of S. costatum followed by 8 μM treatment of HPA3NT3 peptide.
FIG. 5 is a plot showing the position in the red tide focusing sequentially the fluorescently labeled HPA3NT3 peptide:
Figure 5A shows the H according to the fluorescently labeled HPA3NT3 peptide treatment concentration. A picture of the position of peptides in H. akashiwo ;
Figure 5B is seed according to the fluorescently labeled HPA3NT3 peptide treatment concentration. A picture of the position of peptides in C. marin a; And
FIG. 5C is labeled according to the fluorescently labeled HPA3NT3 peptide treatment concentration. Figure shows the position of the peptide in the minimum ( P. minimum ).
6 is H. After treatment with HPA3NT3 in H. akashiwo RA-020 ( H. akashiwo RA-020) is a graph showing the change in chlorophyll A with incubation time:
■: control group not treated with peptide;
▲ group treated with HPA3 peptide; And
●: group treated with HPA3NT3 peptide.
7 is a graph showing the penetration of HPA3NT3 peptides through the cell membranes of red tide:
●: H. Group treated with 0.25 μM of HPA3NT3 peptide to H. akashiwo ;
○: H. Group treated with 0.5 μM HPA3NT3 peptide to H. akashiwo ;
▲: S. Costatium ( S. costatum) treated with 256 μM HPA3NT3 peptide; And
△: S. A group treated with 512 μM of HPA3NT3 peptide to S. costatum.
8 is a graph showing erythrocyte hemolysis of fish erythrocytes of HPA3 and HPA3NT3 peptides:
■: group treated with HPA3 peptide;
◆: group treated with HPA3NT3 peptide; And
▲: You've treated melittin.

Hereinafter, the present invention will be described in detail.

The present invention provides HPA3NT3 peptides having the amino acid sequence set forth in SEQ ID NO: 3.

The HPA3NT3 peptide preferably has an amino acid sequence set forth in SEQ ID NO: 3, but is not limited thereto. The HPA3NT3 peptide is preferably a sequence in which the ninth glutamic acid (Glu) and the thirteenth serine (Ser) of the HPA3 peptide described in SEQ ID NO: 2 are substituted with lysine (Lys), and the HPA3 peptide is set forth in SEQ ID NO: 1 It is preferable to have a sequence in which the 16th glutamine (Gln) and the 18th amino acid aspartic acid (Asp) of the HP (2-20) peptide derived from Helicobacter piori are replaced with tryptophan (Trp), but is not limited thereto. .

In addition, the composition for controlling red tide is seed. C. polykrikoides , C. C. marina and H. It is preferable to have anti-algae activity against at least one red-induced algae selected from the group consisting of H. akashiwo , S. akashiwo . It is preferable not to exhibit the tide activity against costium ( S. costatum ), but is not limited thereto.

In a specific embodiment of the present invention, the present inventors, in order to synthesize an amphiphilic tide peptide, HP (2-20) peptide derived from Helicobacter pylori and glutamine (16th amino acid of the HP (2-20) peptide) Gln) and the analog HPA3 peptide substituted with the 18th amino acid aspartic acid (Asp) with tryptophan (Trp), and the ninth glutamic acid (Glu) to increase the cation after cleaving structurally irregular N-terminal sites in HPA3. An HPA3NT3 peptide was prepared having an amino acid sequence in which the 13th serine (Ser) was substituted with lysine (Lys) (see Table 1).

In addition, in order to measure the activity of the HPA3 peptide and HPA3NT3 peptide prepared in the above embodiment, the inventors of the present invention, 50% of inhibitory concentration (50% of inhibitory concentration, IC ₅₀ ) Was measured. As a result, seeds used in the experiment. C. polykrikoides , p. P ( minimum ), p. P. micans , H. H. akashiwo , h. H. akashiwo RA- 020 ), H. H. akashiwo RA -018 ), Chattonella Esp sp ), seed. Marina ( C. marina ), and S. Costa lithium (S. costatum) with respect to the red tide organisms, compared to the peptide HPA3 HPA3NT3 peptides was confirmed to inhibit the growth of algae in a lower concentration (see Table 2). In other words, the HPA3NT3 peptide was confirmed to have superior anti-tanking activity compared to HPA3.

Also in a specific embodiment of the invention, seeds that do not have the cellulose cell wall of the HPA3NT3 peptide. Marina (C. marina) and HI. A tide activity against A. akashiwo was observed. In red tide treated with HP (2-20) peptides, HPA3 peptides, and HPA3NT3 peptides (32 μM / well), almost 2 minutes after peptide treatment, almost all organisms burst, causing material or organelles to leak out, especially HPA3NT3 The activity of the peptide was the best (see Figure 1).

In addition, in a specific embodiment of the present invention, a seed having a cellulose cell wall of the HPA3NT3 peptide according to the present invention. C. polykrikoides and blood. Track activity against the minimum ( P. minimum ) was observed. Cultured seeds. Polycrycoides and blood. In the medium, HPA3 peptide and HPA3NT3 peptide were added and cultured and observed, compared to the red tide without cell wall, the peptide showed about 1 hour later, but the HPA3NT3 peptide was untreated and HPA3 peptide. Compared to the group treated with, showed good wake activity (see Fig. 2).

In addition, in a specific embodiment of the present invention, by treating the beneficial diatoms with the HPA3NT3 peptide according to the present invention, it was confirmed that the anti-tide activity of the HPA3NT3 peptide selectively acts on the red tide organisms. s. After culturing the Costatium, after treatment with HPA3NT3 peptide and HPA3 peptide, and observed with an optical microscope, as shown in Figure 3, no change in the diatoms was observed even after incubation for 3 days (see Figure 3) . In other words, the HPA3NT3 peptide shows selective activity against the tide in organisms that cause red tide. It can be seen that it does not affect the harmless diatoms such as Costatium.

In addition, in a specific embodiment of the present invention, in order to confirm the selective wake-up activity of the HPA3NT3 peptide according to the present invention, after co-cultivating the beneficial diatoms and the organism causing the red tide phenomenon, HPA3NT3 peptide and HPA3 peptide As a result of treatment, S. a beneficial diatom after two days of treatment. Costatium did not show any changes, but it was a harmful red tide creature. Marina, H. Akashius, and blood. The minimum could observe the disappearance of matter and organelles in the organism. In other words, the selective activity of the tide activity against the red tide organisms of the HPA3NT3 peptide was confirmed (see FIG. 4).

In addition, in a specific embodiment of the present invention, in order to confirm whether the HPA3NT3 peptide according to the present invention binds to the cell membranes of red tide organisms and exhibits anti-tide activity, the fluorescent material is labeled on the HPA3NT3 peptide and then added to the culture medium. It was. As a result, seeds with cellulose walls as shown in FIG. 5. Marina and H. In Akashwoo, fluorescently labeled peptides were observed on the outermost cell membranes. Blood, a red tide organism without cellulosic walls. In the minimum, the peptide was located just inside the cellulose layer. That is, the HPA3NT3 peptide according to the present invention was able to confirm that the red tide organisms bind to the cell membrane and show the effect of destroying the red tide organisms (see FIG. 5).

In addition, in a specific embodiment of the present invention, in order to determine whether the activity of the tide activity of the HPA3NT3 peptide according to the present invention is induced by inhibiting photosynthesis of red tide organisms, to determine the content of chlorophyll a (red) It was. As a result, as shown in FIG. After treatment of HPA3NT3 peptide and HPA3 peptide to Akashwoo A-020 red tide organisms, the chlorophyll A content was measured. Significant decreases were observed compared to one group and no treatments treated with nothing. Therefore, it was confirmed that the HPA3NT3 peptide of the present invention inhibits photosynthesis of red tide organisms and can induce the death of red tide organisms (see FIG. 6).

In addition, in a specific embodiment of the present invention, to determine whether the HPA3NT3 peptide according to the present invention penetrates the cell membrane, red tide biological H. After culturing Akashus, the reaction was performed by adding SYTOX green, which stains the nucleus, to the medium, and adding 0.25 and 0.5 μM of HPA3NT3 peptide, and measuring the intensity of fluorescence using a fluorescence spectrometer. As a result, as shown in Figure 7, it was observed that the intensity of the fluorescence increases as the concentration of HPA3NT3 peptide increases. On the other hand, S is a non-harmful diatom. After adding 256 and 512 [mu] M of HPA3NT3 peptide to the Costatium, the intensity of fluorescence was close to zero. In other words, the HPA3 peptide has the property of selectively permeating cell membranes of red tide biomass, and thus the fluorescence intensity of Cytox Green is H. An increase only in Akash was observed (see FIG. 7).

In addition, in order to confirm the cytotoxicity of the HPA3NT3 peptide according to the present invention in a specific experimental example of the present invention, after diluting the red blood cells of the fish, after adding the peptide, the degree of destruction of the red blood cells Triton X-100 (Triton X- Based on the erythrocyte destructive capacity of 100), the erythrocyte destructive ability of the peptide of the present invention was compared. As a result, as shown in Figure 8, when the addition of melittin (melittin) known as a toxic component of the bee was observed hemolysis of 100% from 20 μM or less, but when the HPA3 peptide is added Although rarely occurred below 20 μM, about 80% of hemolytic reactions were observed when 64 μM or more were treated. However, when the HPA3NT3 peptide was added, it was observed that the hemolysis was less than 10% even at 128 μM concentration, which hardly occurred. Therefore, it could be confirmed that the HPA3NT3 peptide of the present invention has little cytotoxicity (see FIG. 8).

Therefore, the HPA3NT3 peptide according to the present invention has little cytotoxicity, has no effect on non-hazardous diatoms, and shows selective tide activity only to red tide organisms. Owing to permeation and permeation, and also by reducing the content of chlorophyll-A in red tide organisms, causing destruction of red tide organisms, HPA3NT3 peptide can be usefully used as a composition for red tide control.

The present invention also provides a method for preparing an HPA3NT3 peptide having an amino acid sequence set forth in SEQ ID NO: 3 comprising the following steps.

1) The 16th glutamine (Gln) and the 18th amino acid aspartic acid (Asp) of the HP (2-20) peptide derived from Helicobacter piori described in SEQ ID NO: 1 were replaced with tryptophan (Trp) to SEQ ID NO: 2 Preparing an HPA3 peptide having the amino acid sequence described; And

2) replacing ninth glutamic acid (Glu) and thirteenth serine (Ser) of the HPA3 peptide having the amino acid sequence set forth in SEQ ID NO: 2 prepared in step 1) with lysine (Lys).

The HPA3NT3 peptide prepared by the above method has no cytotoxicity, and showed selective activity of red tide in algae causing algae, and the activity of this tide activity after the HPA3NT3 peptide according to the present invention binds to the cell membrane of red tide organisms. It was confirmed that it is possible to control red tide by inducing the death of red tide organisms and inhibiting photosynthesis of red tide creatures.

In addition, the present invention provides a composition for controlling red tide containing HPA3NT3 peptide having an amino acid sequence set forth in SEQ ID NO: 3 as an active ingredient.

The HPA3NT3 peptide preferably has an amino acid sequence set forth in SEQ ID NO: 3, but is not limited thereto. The HPA3NT3 peptide is preferably a sequence in which the ninth glutamic acid (Glu) and the thirteenth serine (Ser) of the HPA3 peptide described in SEQ ID NO: 2 are substituted with lysine (Lys), and the HPA3 peptide is set forth in SEQ ID NO: 1 It is preferable to have a sequence in which the 16th glutamine (Gln) and the 18th amino acid aspartic acid (Asp) of the HP (2-20) peptide derived from Helicobacter piori are replaced with tryptophan (Trp), but is not limited thereto. .

In addition, the composition for controlling red tide is seed. C. polykrikoides , C. C. marina and H. It is preferable to have anti-algae activity against at least one red-induced algae selected from the group consisting of H. akashiwo , S. akashiwo . It is preferable not to exhibit the tide activity against costium ( S. costatum ), but is not limited thereto.

HPA3NT3 peptide according to the present invention has little cytotoxicity, has no effect on non-harmful diatoms, and showed selective tide activity only to red tide organisms. It penetrates into, and also decreases the content of chlorophyll-A in the red tide organisms, causing destruction of the red tide organisms, so that the HPA3NT3 peptide can be usefully used as a composition for controlling red tide.

As a component that can be added to the composition for controlling red tide, ocher already used for red tide control is preferable. However, the present invention is not limited thereto, and the red tide control peptide according to the present invention may be added to the composition as long as the effect thereof may be enhanced by dispersing it in an appropriate state in the ocean or in an appropriately mixed state in the polymer.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Synthesis and Separation of Peptides

In order to synthesize an amphiphilic tide peptide, the present inventors used an HP (2-20) peptide derived from Helicobacter pylori, an analog HPA3 peptide substituted with tryptophan with glutamine (16th amino acid) and aspartic acid (18th amino acid), HPA3. In order to increase the cation after cutting the structurally irregular N-terminal site, HPA3NT3 peptide was prepared having an amino acid sequence substituted with 9th glutamic acid and 13th serine with lysine (Table 1).

Peptide was synthesized using the liquid phase solidification method of Merrifield using Fmoc (9-fluorenylmethoxycarbonyl) as an amino group protective container. In the peptide of the present invention, the peptide having the carboxyl terminus of the -NH ₂ form was used as a starting material, Link Amide MBHA-Resin. The extension of the peptide chain by coupling of Fmoc-amino acids is based on the N-hydroxybenzo triazole-dicyclo-hexycarbodiimide (HOBt-DCC). ).

After coupling the Fmoc-amino acid of each peptide amino terminal, the Fmoc group was removed with 20% piperidine / dimethyl formamide (hereinafter abbreviated as 'DMF') solution, and DMP and dichoromethane, DCM) several times and dried with nitrogen gas. Trifluoroacetic acid: phenol: thioanisole: water: trifluoroacetic acid (TFA): phenol: thioanisole: H ₂ O: triisopropylsilane = 85: 5: 5: 2.5: 2.5 (vol ./vol.)), the solution was reacted for 4 hours to remove the protecting group, the peptide was separated from the resin, reacted with cold diethyl ether for 30 minutes, and the peptide was precipitated by centrifugation. Again, the peptide precipitated with diethyl ether is washed once more, precipitated, and dried in air. Crude form of the peptide obtained by the above method was dissolved in ammonium bicarbonate (pH 8) and acetonitrile was used as a gradient (reverse phase-HPLC column) (Delta Pak) , C ₁₈ 300 mm 3, 19.0 mm x 30 cm, Waters). The amino acid composition was measured by the amino acid analyzer (Hitachi 8500 A) by the Edman degradation method of the purified synthetic peptide, and the molecular weight of the peptide was measured by using the Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. .

As a result, all the peptides synthesized in the present invention showed a purity of 95% or more, and it was confirmed that the peptides having the correct amino acid sequence were synthesized by matching the molecular weights calculated from the amino acid sequences.

Amino acid sequence of the peptide used in the test Peptide Name order Molecular Weight  HP (2-20) AKKVFKRLEKLFSKIQNDK-NH2 (SEQ ID NO: 1) 2320.0  HPA3 AKKVFKRLEKLFSKIWNWK- NH2 (SEQ ID NO: 2) 2449.2  HPA3NT3 FKRLKKLFKKIWNWK- NH2 (SEQ ID NO: 3) 2062.6

< Experimental Example  1> Peptide Trace  Active measurement

<1-1> H PA3NT3 Measurement of 50% Inhibitory Concentration of Peptides on Red Tide

The tide activity of the peptide synthesized in <Example 1> was measured. Specifically, seeds that have been sold by the Korea Maritime Microalgae Bank as red tide organisms to measure activity in wake tide. Marina (C. marina), HI. H. akashiwo , mr. C. polykrikoides , p. P. minimum , and S. S. costatum was used.

Each of the algae in F2 medium [150 mg NaNO ₃ , 8.69 mg NaH ₂ PO 4 · 9H ₂ O, 10 mg ferric EDTA, 0.22 mg MnCl ₂ , 0.11 mg CoCl ₂ , 0.0196 mg CuSO ₄ · 5H ₂ O, ZnSO ₄ · 7H ₂ O, 50 mg Na ₂ SiO ₃ · 9H ₂ O, 0.012 mg Na ₂ MoO ₄ · 2H ₂ O, 1 μg Vitamine B ₁₂ , 1 μg Biotin, 0.2 μg ThiamineHCl / 1 L filtered seawater) The salinity was 34 ‰, and the illuminance was 3,000 lux, and the cells were incubated at regular intervals of 14 hours / 10 hours. Precultured red tide biological cells were diluted to a concentration of 10 ⁴ / ml of cells and seeded in 24 well micro titrate plates. The peptide synthesized in <Example 1> was added to the plate inoculated with the strain by diluting 1/2 fold starting from the concentration of 256 micromolar (μM) / well (well). Incubated for 36 hours under the above-mentioned culture conditions, the number of red tide organisms was measured every 6 hours using a hemoytometer, abbreviated as 50% of inhibitory concentration (hereinafter referred to as 'IC ₅₀ '). ) Value was determined.

As a result, HP (2-20), HPA3, and HPA3NT3 peptides all showed activity against red tide, but among them, the peptide of the present invention, HPA3NT3, showed the strongest tide activity (Table 2). Therefore, the HPA3NT3 peptide prepared in <Example 1> of the present invention was found to be a peptide having excellent anti-tanking activity.

Measurement of Tide Activity for Red Tide-Induced Marine Life (Seaweed) Strains
Inhibitory concentration (μM)
HP (2-20) HPA3 HPA3NT3 IC ₅₀ IC ₉₀ IC ₅₀ IC ₉₀ IC ₅₀ IC ₉₀ Waxy imitation Seed. C. polykrikoides 64 128 4 8 4 8 blood. Minimum (P. minimum D-120) 128 > 256 16 32 4 8 blood. Micans (P. micans D-077) 128 > 256 16 32 4 8 Needle knitted steel H. Akashiu (H. akashiwo RA-020) 32 128 2 2-4 0.5 One H. Akashiu (H. akashiwo RA-018) 64 128 2 2-4 0.5 One H. H. akashiwo 64 128 2 4 0.5 One Chattonella sp. RA-005 64 128 2 4 One 2 Seed. Marina (Chattonella marina) 32 64 4 8 2 4 Diatoms s. Costatium (S. costatum B-796) 256 > 256 128 > 256 128 > 256

<1-2> HPA3NT3 Peptide Cellulose  In red tide without cell walls Trace  Active observation

In order to visualize on a light microscope that the HPA3NT3 peptide according to the present invention exhibits a tide activity, C. imitation steel. C. marina and H. H. akashiwo , a single-sided rhinoceros. C. polykrikoides and blood. P. minimum and diatoms S. S. costatum was incubated in F2 medium (medium composition described in Test Example 1 above). Cultivated red tide was inoculated into a 24-well micro titrate plate at a concentration of 10 ⁴ / ml followed by inoculation of the red tide by HP (2-20), HPA3, and HPA3NT3 peptide at a concentration of 32 μM / well. It was added to the prepared plate and incubated for 24 hours at 22 ℃. After incubation, it was observed on an optical microscope for visualization.

As can be seen in Figure 1, a seed that is a red tide-inducing organism that does not have a celluose cell wall. C. marina and H. In the untreated group treated with nothing in H. akashiwo , the number of organisms was maintained and there was no change in appearance, but in red tide treated with HP (2-20), HPA3, and HPA3NT3 peptides, two minutes after peptide treatment Nearly all organisms were destroyed, and substances and organelles in the organisms leaked out, especially the HPA3NT3 peptide. As a result, it was possible to confirm the anti-tanking activity of the HPA3NT3 peptide of the present invention.

<1-3> HPA3NT3 Peptide Cellulose  For red tide with cell walls Trace  Active observation

In order to observe the activity of the tide against red tide with cellulose cell wall of the HPA3NT3 peptide according to the present invention, Mr. C. C. polykrikoides and blood. The minimum ( P. minimum ) was inoculated in F2 medium (medium composition described in Experimental Example 1), followed by incubation. Cultivated red tide was inoculated into a 24-well micro titrate plate at a concentration of 10 ⁴ / ml followed by inoculation of the red tide by HP (2-20), HPA3, and HPA3NT3 peptide at a concentration of 256 μM / well. It was added to the prepared plate and incubated for 36 hours at 22 ℃. After incubation, it was observed on an optical microscope for visualization.

In addition, the red tide seed having a cellulosic cell wall as shown in FIG. C. polykrikoides and blood. In P. minimum , the HPA3NT3 peptide was active for about 1 hour later than the red tide without cellulose cell wall, but similar to the results of FIG. 1, the HPA3NT3 peptide was HP (2-20), HPA3 peptide. It showed the best activity compared to (FIG. 2).

<1-4> HPA3NT3 Peptide  For beneficial diatoms Peptide Trace  Active observation

In order to confirm the effect of the tide activity of the HPA3NT3 peptide according to the present invention on marine life not causing red tide, S. Costatum ( S. costatum ) was inoculated in F2 medium and then cultured. The cultured red tide was inoculated into a 24-well micro titrate plate at a concentration of 10 ⁴ / ml, followed by the HP (2-20), HPA3, and HPA3NT3 peptides. Add to the plate inoculated with the red tide at a concentration of 256 μM / well Incubated at 22 ° C. for 72 hours. After incubation, it was observed on an optical microscope for visualization.

As a result, as shown in Figure 3, S. diatom, which does not harm marine ecosystems. As a result of confirming the activity of HPA3NT3 peptide against S. costatum, S. costatum was treated with S. costatum, which was diatoms even after 3 days of treatment with HPA3 and HPA3NT3 peptides. It was confirmed that it does not inhibit the Costatium at all (Figure 3).

<1-5> HPA3NT3 Peptide  On coculture of red tide and diatoms Trace  Active observation

In order to confirm that the peptide of the present invention exhibits anti-tide activity only to red tide organisms, the present inventors have identified a beneficial diatom. S. costatum and imitation needles. C. marina and H. H. akashiwo , and blood. The minimum (minimum P.) was co-cultured respectively. Diatoms and red tide organisms were inoculated into 24-well micro titrate plates at a concentration of 10 ⁴ / ml and then s. S. costatum and Mr. Plates inoculated with C. marina had 4 μM HPA3NT3 peptide concentrations, S. costatum and H. c. Plates inoculated with H. akashiwo had a concentration of 1 μM HPA3NT3 peptide and S. akashiwo . S. costatum and blood. Add 8 μM HPA3NT3 peptide concentration to the plate inoculated with P. minimum Incubated at 22 ° C. for 48 hours.

As a result, as shown in Figure 4, after co-culture of harmful species causing red tide and harmless diatoms in the marine ecosystem, when treated with HPA3NT3 peptide, B treated 8 μM than A treated 2 μM I was able to see better activity in both cases. The survival rate of Costatium (square) is high while H. Akashuu (won), p. It was found that the number of minimums (triangles) decreased. Similarly when viewed through a microscope, S. diatoms. Costatium maintains its shape while the red tide-generating seed. Marina (C), H. Akashwoo (D), and blood. Minimum (E) confirmed that the organism was bursting. As a result, it was confirmed that the peptide selectively inhibits only harmful red tide (FIG. 4).

< Experimental Example  2> HPA3NT3 Peptide  Locate in red tide cells

After reacting the tether HPA3NT3 peptide of the present invention with a fluorescent material called 10 mmol Tamra (TAMRA) (Molecular probe, Eugene, OR) at room temperature for 1 hour, only the labeled peptide was isolated through HPLC. 0.5 μM of the labeled TAMRA-HPA3NT3 peptide thus isolated was seeded with red cellulose without cellulosic cell walls at a concentration of 10 ⁴ / mL. Marina (C. marina), HI. H. akashiwo , and red tide blood with cellulose cell walls. After reacting with minimum ( P. minimum ) for 5 minutes at 22 ℃, washed twice with F2 medium. To confirm the peptide position in the red tide organisms, the red tide organisms were observed using confocal microscopy, with the red tide organisms continuously focused from top to bottom.

As a result, as shown in Figure 5, it can be confirmed that the HPA3NT3 peptide labeled Tamra is located on the red cell membrane. Especially red seed without cellulosic cell wall. C. marina and H. In H. akashiwo , a blood that is located on the outermost cell membrane but with cellulose. In the minimum ( P. minimum ) it was confirmed that the peptide is located just inside the cellulose layer (Fig. 5).

< Experimental Example  3> HPA3NT3 Peptide  Photosynthesis inhibition experiment

The present inventors attempted to confirm the anti-tidal activity of the HPA3NT3 peptide according to the present invention by measuring the content of chlorophyll a of red tide organisms. H. Akashwoo AL-020 was inoculated in F2 medium and cultured. The cultured red tide was inoculated into a 12 well micro titrate plate at a concentration of 10 ⁵ / mL and HPA3 peptide and HPA3NT3 peptide were inoculated at 2 micromolar (μM) and 1 micromolar (μM) / well, respectively. After the addition was incubated for 6, 12, 24, 36, 48, 72 hours at 22 ℃.

After incubation, the samples were centrifuged, and then crushed with a homogenizer while adding 90% acetone as an extraction solvent. In order to liberate the chlorophyll-A in the crushed sample, it was left for 4 hours after blocking the light at -20 ° C. After 4 hours the samples were centrifuged and the supernatant carefully taken. In addition, the amount of chlorophyll A in the supernatant was briefly measured using the absorbance of the whole cells and the following Equation 1.

In Equation 1, A ₆₆₃ is absorbance at a wavelength of 663 nm, A ₆₂₀ is absorbance at a wavelength of 620 nm, and A ₇₅₀ represents absorbance at a wavelength of 750 nm.

As a result, H. As the treatment time of HPA3NT3 peptide on Akashwoo A-020 increased, the amount of chlorophyll-A was significantly decreased compared to the group treated with HPA3 peptide. That is, the HPA3NT3 peptide according to the present invention was found to inhibit the growth of red tide.

< Experimental Example  4> HPA3NT3 Peptide  Red Tide Membrane Permeation Experiment

Red tide H. After washing three times with H. akashiwo using a buffer, the amount of cells was 5 × 10 ⁵ / well I set it to be. Red Tide Creature H. After the concentration of SYTOX green (Molecular probe, Eugene, OR), which stains the nucleus in Akash, was added to 1 μM, 90 μl was dispensed into a 96 well plate and reacted at 22 ° C. for 15 minutes. . HPA3NT3 peptide was added to 0.25 and 0.5 micrometers, respectively, in the reaction red tide plate, and the intensity of fluorescence was measured using the fluorescence spectrometer. S, a non-harmful diatom. In the case of Costatium, after adding Cytox Green to the medium and reacting in the same manner as above, HPA3NT3 peptide was treated with 256 μM and 512 μM, and then the intensity of Cytox-Green fluorescence was measured for 30 minutes.

As a result, as shown in Figure 7, Cytox Green is a fluorescent material that emits light must bind to the nucleus without entering itself into the cell. In H. akashiwo , the fluorescence intensity increased as the concentration of HPA3NT3 peptide increased. This means that the more peptides, the more the red tide cell membrane acts to enter Cytox green into the cell and bind to the nucleus to emit more light. On the other hand, S is a non-harmful diatom. After adding 256,512 μM of HPA3NT3 peptide to S. costatum, the intensity of fluorescence was close to zero. In other words, the HPA3NT3 peptide has a property of selectively penetrating the cell membrane of the red tide organisms, thereby allowing Cytox Green to enter the red tide organisms through the destroyed cell membranes and bind with the nucleus to increase the fluorescence intensity. It was only observed to increase in A. akashwo (see FIG. 7).

< Experimental Example  5> HPA3NT3 Peptide  Cytotoxicity experiment

In order to confirm whether the HPA3NT3 peptide according to the present invention is cytotoxic, the present inventors investigated the erythrocyte destroying ability of the HPA3NT3 peptide. Specifically, the red blood cells of the fish (flatfish, Wando) washed three times with phosphate buffer (PBS, pH 7.0) to a concentration of 8% and then diluted, and here the HPA3 peptide, HPA3NT3 peptide and melittin ( Antibiotic peptides (Melittin) were serially diluted 1/2 fold from 128 micromolar (μM) / well concentration and reacted at 37 ° C for 1 hour. The reaction solution was centrifuged at 1,000 x g and the amount of hemoglobin contained in the supernatant was measured for absorbance at 414 nm. To investigate the degree of cell destruction, absorbance was measured by adding 1% Triton-X100 (Triton X-100), and the cell destruction capacity of 1% Triton X-100 was 100%, which was compared with the erythrocyte destruction capacity of each peptide. It was calculated according to the following <Equation 2>.

In Equation 2, absorbance A is an absorbance when the peptide solution is treated at a wavelength of 414 nm, and absorbance B is an absorbance when only PBS having a destructive power of 0% is treated at a wavelength of 414 nm, and absorbance C is 414. Absorbance of 1% Triton X-100 at nm wavelength.

As a result, the HPA3NT3 of the present invention shows little cytotoxicity, whereas melittin, a bee venom used as a positive control, was highly cytotoxic. In addition, as the HPA3 peptide increased to 20 micromolar (μM) or more, it was confirmed that the level of the hemolytic response was significantly increased (FIG. 8).

As described above, the HPA3NT3 peptide prepared in the present invention has no cytotoxicity, did not affect harmful diatoms, and showed an excellent anti-tanking activity in red algae-induced algae, and the membrane of HPA3NT3 peptide. Since the permeability and the content of chlorophyll A of red tide organisms are reduced to cause the killing of red tide-causing organisms, it can be usefully used as a safe track preparation for marine ecosystems.

<110> Industry Academy Cooperation Foundation Chosun University <120> HPA3NT3 peptide having antialgae activity against organism          induced red tide and the use of the same <130> 10p-07-52 <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> HP (2-20) peptide <400> 1 Ala Lys Lys Val Phe Lys Arg Leu Glu Lys Leu Phe Ser Lys Ile Gln   1 5 10 15 Asn Asp Lys             <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> HPA3 peptide <400> 2 Ala Lys Lys Val Phe Lys Arg Leu Glu Lys Leu Phe Ser Lys Ile Trp   1 5 10 15 Asn Trp Lys             <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> HPA3NT3 peptide <400> 3 Phe Lys Arg Leu Lys Lys Leu Phe Lys Lys Ile Trp Asn Trp Lys   1 5 10 15

Claims (9)

HPA3NT3 peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3.
The sequence of claim 1, wherein the HPA3NT3 peptide deletes the first to fourth amino acids of the HPA3 peptide represented by SEQ ID NO: 2, and replaces the ninth glutamic acid (Glu) and the thirteenth serine (Ser) with lysine (Lys). Peptide, characterized in that.
3. The HPA3 peptide is characterized in that the tryptophan (Trp) is the 16th glutamine (Gln) of the HP (2-20) peptide derived from Helicobacter piori as shown in SEQ ID NO: 1 and the aspartic acid (Asp), which is the 18th amino acid. Peptides having a sequence substituted with.
The method of claim 1, wherein the HPA3NT3 peptide is seed. C. polykrikoides , C. C. marina and H. Peptides characterized by having anti-algae activity against at least one red tide-induced algae selected from the group consisting of A. akashiwo .
The method of claim 1, wherein the HPA3NT3 peptide is S. a. Peptides, characterized in that it does not exhibit anti-tanking activity against costium ( S. costatum ).
1) The 16th glutamine (Gln) and the 18th amino acid aspartic acid (Asp) of the HP (2-20) peptide derived from Helicobacter piori described in SEQ ID NO: 1 were replaced with tryptophan (Trp) to SEQ ID NO: 2 Preparing an HPA3 peptide having the amino acid sequence described; And
2) The 1st to 4th amino acids of the HPA3 peptide having the amino acid sequence set forth in SEQ ID NO: 2 prepared in step 1) were deleted, and the 9th glutamic acid (Glu) and the 13th serine (Ser) were lysine (Lys). A method for preparing an HPA3NT3 peptide having an amino acid sequence as set forth in SEQ ID NO: 3 comprising the step of replacing.
Red tide control composition containing the peptide of claim 1 as an active ingredient.
8. The composition of claim 7, wherein the composition for controlling red tide is seed. C. polykrikoides , C. C. marina and H. A composition for controlling red tide, characterized by having anti-algae activity against at least one red tide-induced algae selected from the group consisting of H. akashiwo .
According to claim 7, wherein the red tide control composition is S. Red tide control composition characterized in that it does not show the activity of the bath tide against Costatium ( S. costatum ).

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