KR101271157B1 - Method for controlling harmful algae using bio nano capsid - Google Patents

Abstract

The present invention relates to a harmful algae control method using a bio-nanocapsid, more specifically
1) harvesting and culturing harmful algae causing red and green algae;
2) selecting a virus specific for the cultured harmful algae;
3) cloning the capsid gene of the selected virus;
5) mass-producing nanocapsids by preparing a recombinant yeast library of the cloned genes; And
6) mounting the algae on the mass-produced nano-capsid; And
7) characterized in that it comprises the step of spraying the nano-capsid on which the algae material is mounted. The harmful algae control method according to the present invention uses a virus specific to a specific algae causing red algae or green algae to selectively affect only a specific algae, thereby preventing marine algae or other red algal phenomena. The effect of fundamentally eliminating adverse effects on the ecosystem is expected.

Description

Method for controlling harmful algae using bio nano capsid

The present invention relates to a technique in the field that can prevent environmental pollution, in particular marine pollution as an invention relating to a method for controlling harmful algae.

The present invention also relates to a sterilizing material effective for controlling harmful algae, a loading technology thereof, and a spraying method for preventing marine pollution.

On the other hand, the present invention is related to the technology in the field of biotechnology because it relates to the technology utilizing the nano-level biocapsid.

Red tide is a phenomenon in which seawater discolors and marine life is damaged by the explosive growth of marine microorganisms (plankton, protozoa, bacteria). The most common causes of red tide are phytoplankton, which is found in Korea, including diatoms, dinoflagellates, blue-green algae, microalgae, and ciliates. . Depending on the type of plankton, the color of the seawater is not limited to red, but may be dark brown, blue, or white.

In the seawater ecosystem of temperate regions like Korea, eutrophication is beneficial to the growth of phytoplankton, the primary producer, as the water layer is formed in the summer and the amount of light is increased, and the long rainy season and the water temperature rise. As the environment is created, the outbreak of algae proceeds. In particular, species that produce toxins which directly lethal to fish and shellfish of the species that cause the red tide phenomenon in the country coast kokeulrodinium (Cochlodinium) in (屬), Jim nodi nium (Gymnodinium) in, Giro di nium (Gyrodinium), A It is a species belonging to dinoflagellate, such as genus Heterosigma .

Looking at the biological cause of red tide phenomenon in the country as a seasonal, spring, the skeletal Loreto nematic (Skeletonema), A cake Chitose Los Centrum (Prorocentrum) to speed, the Pro is there, and summer hetero Sigma (Heterosigma), such as diatoms belonging to the genus (Chaetoceros) such as single mothers in the small bird, and a single mother birds in the summer months from late autumn until the large Serra tium (Ceratium) in Jim nodi nium (Gymnodinium), a kokeulrodinium (Cochlodinium). By year since 1995 in the South Coast and East Coast waters south kokeulrodinium Poly Cree Koh Death (Cochlodinium polykrikoides ) species frequently cause red tide. In order to control such red tide, the yellow soil spray method, ozone treatment method, chemical spray method, biological treatment method and the like have been conventionally used. The control of red tide by ocher spray is to coagulate and settle the red tide microorganisms in the ocher by using the characteristics of the ocher colloid particles to aggregate and adsorb suspended solids such as nutrients and microplankton in seawater. The removal effect of toxic red algae by ocher spray was different depending on the species, but the removal effect of Cochlodinium red algae was 70 ~ 80% by indoor and field survey. In the control of red algae microorganisms in acidic clay in Japan, experiments with 14 red tide-causing organisms including Cochlodinium confirmed the effects of adsorption, sedimentation and cell destruction. However, when the soil is sprayed, side effects are caused by the temporary increase of suspended solids, and the gills of the fish are blocked, causing a problem of death. When spraying raw ocher to the ocean, loess and impurities are sedimented as it is, so there is no time for adsorption and aggregation of red tide organisms, thus reducing red tide removal efficiency and causing secondary environmental pollution of the seabed sediment.

The ozone treatment method has the advantage of minimizing the disturbance of the marine ecosystem without causing secondary pollution, but since the generator must be mounted on the ship separately with the ozone generator, which has a large power requirement and a large scale, the construction of a dedicated ship is required. Since red tide occurs mainly during the summer, there is an excessive amount of idle facilities, or it is difficult to mount or dismantle a heavy ozone generator and generator on a ship in accordance with the red tide time. In particular, since ozone is impossible to store, when red tide occurs excessively and the amount of ozone temporarily takes a large amount of oxygen, the amount of oxygen in the seawater is excessively increased, adversely affecting the ecosystem and insufficient red tide control ability.

Chemical spraying has been used since the past as a method of spraying copper sulfate (CuSO ₄ ), chlorine dioxide (ClO ₂ ), simazine (Simazine), etc. Among them, copper sulfate, which is the most inexpensive and widely used, is harmful Because it is not specific, it may affect other marine organisms besides the red tides, which may cause problems in terms of toxicity and corrosion to other organisms in the water. In addition, the copper sulfate is unstable under the high alkaline environmental conditions accompanying the occurrence of algae red tide, which is disadvantageous because it must be processed in large quantities.

Many biological methods have been tried as another way to control red tide. In particular, the use of natural zooplankton and marine bacteria or nutritional competition with harmful or toxic red tide organisms artificially induces less harmful red tide species to promote the consumption of nutrients. Biological method research is being actively conducted.

As a specific document, reference may be made to Patent Publication No. 10-0758997. The document relates to a method for inhibiting flagella algae of Heterosigma genus using Pseudomonas genus microorganisms, specifically, Pseudomonas fluorescin strain HAK-13 capable of inhibiting genus hetero sigma and flagella algae using the same It is about a method to suppress. The method

1) selecting Pseudomonas genus microorganisms having the ability to inhibit flagella algae in hetero sigma;

2) culturing the Pseudomonas genus microorganism of step 1) to obtain a culture;

3) It is composed of the step of spraying the culture of step 2) in the red tide generation area.

Pseudomonas fluorescein HAK-13 strain was cultured in nutrient agar medium (Nutrient agar; 0.8% (w / v) Nutrient broth, 1.5% agar powder) at a culture temperature of 18-35 ° C. for 1-5 days, As a result of inoculation into the heterologous growth medium of Heterosigma akashiwo D-075, the growth of the cells of the heterosigma Akashio D-075 strain was rapidly inhibited 12 hours after the inoculation, resulting in a decrease in cell count of 90% or more. In addition to complete death in 48 hours, when the Pseudomonas fluorescein HAK-13 strain treated group and the untreated control group were visually observed, the Pseudomonas fluorescein untreated control group was reddish brown, and a large amount of heterosigma acacio D-075 strain was used. Reproduction status was shown. Since the Pseudomonas fluorescein has the ability to inhibit flagella algae, the algae using the strain can be usefully used to control the red tide phenomenon caused by flagella algae.

However, this has not yet been commercialized in that it is required to find and mass produce microorganisms that affect certain algae that cause red tide.

Therefore, the present inventors have studied the algae with excellent harmful algae control effect and the loading and spreading technology of the algae that can prevent marine pollution, and as a result, the capsid gene of the virus that is specific to the specific harmful algae causing red algae or green algae phenomenon By mounting and spraying algae material, the harmful algae can be selectively controlled, thereby revealing the effects of fundamentally preventing side effects such as pollution of marine ecosystems, which is a problem of the existing yellow soil spraying method or other methods of preventing red tide. The present invention has been completed.

Patent Registration 10-0758997 Published Patent Publication 2002-0015344 JP11098979A2

In the present invention, the main problem to be solved in the above-described conventional red tide removing method.

Specifically, the above-mentioned problem is achieved by selecting a specific virus that specifically adsorbs / infects red tide-inducing alga and mass production of only its external capsid protein in the form of nanoparticles.

In addition, the present invention can be more effectively achieved by providing a method for mounting a natural or novel algae on the nano-capsid produced by the above method.

In order to solve the above problems,

1) harvesting and culturing harmful algae causing red and green algae;

2) selecting a virus specific for the cultured harmful algae;

3) cloning the capsid gene of the selected virus;

4) mass-producing nanocapsids after expressing the cloned genes in recombinant E. coli or yeast; And

5) It provides a harmful algae control method using the nano-capsid comprising the step of mounting the algae on the mass-produced nano-capsid.

In addition, the present invention has a specificity to the harmful algae in order to solve the above problems more effectively, there is no nucleic acid inside the capsid, there is no proliferative force and no human hazard, the capsid protein is used only as a carrier of the algae It provides a harmful algae control method using a nano-capsid.

In addition, the present invention is characterized in that the algae material mounted on the virus nanocapsid is any one selected from the group consisting of quinone compounds, thiazolidinedione compounds and magnesium organoclays. The method of mounting on the nanocapsid may be performed by coexpression using a genetic recombination method or by a biological method performed by self assembly in a buffer solution for over-dissolution or recombination. It provides a method for controlling harmful algae using nano capsid.

The present invention uses a virus specific to a specific algae causing red tide or green algae to selectively affect only a specific algae, thereby fundamentally affecting the side effects on the marine ecosystem, which is a problem of the conventional red clay spraying or other red tide preventing methods. It is effective to get rid of.

1 is a view showing a mass production process of virus capsid using a yeast expression system.
2 is specifically infected with the harmful algae, hepatic algae heterocapsa ( Hterocapsa circularisquama ) Figure showing the capsid gene sequence of wild-type HcRNAV virus (first line), the genes modified to be expressed in yeast Pichia (second line), and their amino acid sequence (third line) information. .
3 is a diagram showing the results of electrophoresis confirming the size of the cloned modified gene.
4 is a diagram showing the expression results of the HcRNAV capsid gene expressed in yeast.
5 is a diagram taken with an electron microscope of HcRNAV virus capsid expressed in yeast.
Figure 6 is a diagram showing a schematic diagram of attacking and destroying harmful algae mounted on the bio-nanocapsid material.
7 is a diagram illustrating the host-specific binding of the capsid protein loaded with the fluorescent material FITC in C. salsugineum .
8 is a view showing a phenomenon in which harmful algae are destroyed with time after treatment of the TD-based compound with Cochlodinium polykrikoides ( Cochlodinium polykrikoides ):
(a): C not treated with compound 17. polykrikoides ; And
(b) to (f): C treated compound 17 for 1 hour (b), 2 hours (c), 3 hours (d), 4 hours (e), or 5 hours (f). polykrikoides .
FIG. 9 is a diagram illustrating a phenomenon in which harmful algae are destroyed over time after treating a TD-based compound with Heterosigma akashiwo :
(a): H not treated with compound 17. akashiwo ; And
(b)-(f): 1 μM of compound 17 for 1 hour (b), 2 hours (c), 4 hours (d), 6 hours (e), 8 hours (f), 10 hours (g) or H treated more than 10 hours (h). akashiwo .
FIG. 10 shows FT-IR and ¹ H-NMR spectra of magnesium organoclays used as algae.
FIG. 11 shows electron micrographs (SEM and TEM images) of the synthesized magnesium organoclay. FIG.
12 is a view showing the killing effect of magnesium organic clay.

Hereinafter, the present invention will be described in detail.

The present invention

1) harvesting and culturing harmful algae causing red and green algae;

2) selecting a virus specific for the cultured harmful algae;

3) cloning the capsid gene of the selected virus;

4) mass-producing nanocapsids after expressing the cloned genes in recombinant E. coli or yeast; And

5) It provides a harmful algae control method using the nano-capsid comprising the step of mounting the algae on the mass-produced nano-capsid.

In the method, the harmful algae causing red algae and green algae is Heterocapsa circular caps ( Heterocapsa) circularisquama ), chitoceros salinium salsugineum), poly kokeulrodinium Cri Koh des (Cochlodinium polykrikoides), heteroaryl Sigma Oh Casio (Heterosigma akashiwo ), Chattonella marina marina and Protoceratium reticulatum ) is preferably any one selected from the group consisting of, but is not limited thereto. The harmful algae often appear on the south coast or west coast around June-September, when it can be harvested and cultured.

After culturing the harmful algae, it is possible to select and isolate a virus that specifically infects only the harmful algae. The virus has no nucleic acid inside the capsid, so there is no proliferative ability and no human hazard, and the capsid protein may be used only as a carrier of the algae. The virus may be a naked icosahedral nucleocapsid, preferably a 55 nm sized envelope, and a heterocapsa circularis RNA virus virus ( Heterocapsa). circularisquama RNA Virus (HcRNAV), ketoseros salmonium intranuclear inclusion virus ( Chaetoceros) salsugineum Nuclear Inclusion Virus (CsNIV) and Heterosigma RNA Virus ( Heterosigma) akashiwo RNA virus, HaRNAV) is more preferably any one selected from the group consisting of, but is not limited thereto.

Among the viruses, HcRNAV has a gene corresponding to the capsid of the protein constituting the virus having a size of 1,080 bp (SEQ ID NO: 1), and the protein encoded by the gene is preferably composed of about 360 amino acids, but is not limited thereto. no.

In the above method, the algae material is a material that reacts specifically to harmful algae, and thus it can minimize the destruction of marine ecosystems because it does not harm other organisms at all. Specifically, the algae is preferably a quinone compound, a thiazolidinedione compound, or a magnesium organoclay, but is not limited thereto.

The magnesium organic clay is Al ₂ Si ₂ O ₅ (OH) ₄ , Mg _s Si ₂ O ₅ (OH) _4, Al ₂ Si ₄ O ₁₀ (OH) _2, Mg ₃ Si ₄ O ₁₀ (OH) _2, KAl _{2 (AlSi 3) O 10 (} OH) 2, Ca 0 .25 (Si 3 .5 Al 0 .5) Al 2 O 10 (OH) 2 and _{(Mg, Fe, Al) 6} (Si, Al) 4 O ₁₀ (OH) ₈ can be selected from any one selected from the group consisting of, or a mixture thereof. In addition, the quinone-based compound and thiazolidinedione-based compound is any one of the following formulas 1 and 2 (quinone-based compound having harmful algae killing ability), and formula 3 (thiazolidinedione with harmful algae killing ability) One, or mixtures thereof.

Wherein R is substituted or unsubstituted alkyl, substituted or unsubstituted (hetero) cycloalkyl, (hetero) cycloalkenyl or (hetero) aryl.

Wherein R ₁ is hydrogen, nitro group amine, alkyl, methoxy, trifluoromethyl, carboxyl or halogen and R ₂ is hydrogen methyl ethyl, or unsubstituted (hetero) cycloalkyl, (hetero) cycloalkenyl or (Hetero) aryl and n is an integer of 0-5.

The algae can be encapsulated on the virus capsid by itself or in combination with the coating material. The bio-nanocapsid loaded in this way is a target algae ( Heterocapsa). by binding to circularisquama, etc., it can inhibit the growth of harmful algae and kill them. Algae loading method is a physical mounting method using the swelling of the virus according to the change of heat or pH, coexpression using a genetic recombination method, or self-assembly using a buffer solution for the breakdown or recombination Biological loading methods such as self assembly can be used.

We screened and isolated HcRNAV in the virus, cloned the virus's capsid gene and expressed it in recombinant E. coli or yeast to mass produce capsid proteins. Specifically, the 1,080 bp HcRNAV capsid gene was cloned into the pPICZ vector and plasmids were expressed in yeast Pichia host. Glycerol was used as a substrate in the expression stage of the yeast for expression, and a large amount of capsid protein was made by inducing the expression of HcRNAV capsid protein by adding methanol at an appropriate microbial concentration. The mass produced plasmid was confirmed to be a gene having a size of 1,080 bp (see FIG. 2), and the final produced capsid protein was confirmed to be a protein having a size of 40 kDa (see FIG. 3). Therefore, the viral gene obtained by PCR was cloned into a shuttle vector available for Escherichia coli or Peachia, and the cloned gene was finally mass-produced as a capsid protein through mass fermentation of Peachia host (FIG. 1). Reference). In mounting the algae on the mass-produced nano-capsid, the present inventors specifically react to only harmful algae and do not cause any damage to other living organisms as a thiazolidinedione (TD) -based compound or Magnesium organoclay was used. As a result, most TD chemicals are Heterocapsa , a harmful algae. circularisquama , Heterosigma akasiwo And Cocholodium It showed high killing effect against polykrikodes , while showing very low toxicity against harmless algae (see Tables 1, 2, 8 and 9), and magnesium organoclay also selectively controlled harmful algae. Table 4, FIG. 12).

Therefore, the present invention is mounted in the capsid of a specific virus that specifically binds to the harmful algae, the algae material having the effect of selectively controlling only the harmful algae, it is possible to selectively control only the harmful algae without toxicity to other harmless algae This can be useful as a pest control method that can minimize the destruction of marine ecosystems.

Hereinafter, the present invention will be described in more detail with reference to the following examples.

However, the following examples are intended to illustrate the contents of the present invention, but the scope of the invention is not limited by the examples.

Of viruses specific to pests Capsid  Mass production of genes

Heterocapsa circularisquama , a type of coarse algae, was isolated and screened from Yeosu, Jeollanam-do, from August 2 to September 25, 2009. Selected birds were inoculated in f / 2 medium and incubated at 20 ° C. Wild type capsid gene information of HcRNAV109 virus can be found in Genbank No. Obtained from AB218609.1, the 1,080 bp gene was modified to be optimally expressed in yeast Pichia and then put into a pPICZA plasmid to finally prepare a pPICZA-CP vector. Cloned pPICZA-CP Vectors to Pichia Pastoris pastoris ) After insertion into the host, the screening was first screened on zeocin-YPD medium, and the isolated host was cultured in methanol-induced medium to confirm the expression of the HcRNAV gene and to mass produce a protein having a molecular weight of 40 kDa. Strains were obtained. The mass-proliferated plasmid was confirmed to be a gene having a size of 1,080 bp as shown in FIG. 2 (FIG. 2), and the final produced capsid protein was confirmed to be a protein having a size of 40 kDa (FIG. 2). 3). That is, the viral gene can be obtained by PCR, and the obtained gene can be cloned by inserting it into a shuttle vector usable for Escherichia coli or Peachia. It was confirmed that can be produced through. Fermentation proceeded to fed-batch culture using glycerol as a carbon source and induced protein expression by feeding 3.6 ml / h of methanol after 40 hours.

virus In capsid Algae  Mounted and sprayed

Hetero kaepsa using recombinant methods circulator La Surgical probably RNA viruses (Heterocapsa circularisquma RNA virus, HcRNAV) and Kane Chitose Los Guinea live Stadium nuclear inclusion virus (Chaetoceros The capsid genes of salsugineum nuclear inclusion virus (CsNIV) were each coexpressed in Peachia and purified by affinity chromatography using a Ni Sepharose column. Dialysis was performed in distilled water for 1 day to increase self assembly of the purified viral capsid proteins. In order to investigate the host specific binding of the self-assembled capsid protein and the loading capacity of the algae, the capsid protein was first dissolved in a dissociation buffer (10 mM Tris-HCl, 150 mM NaCl, 10 mM EGTA, pH 8.5). Dissociated by exposure to 1 h. To the dissociated capsid protein, the fluorescent substance FITC, which has similar characteristics to TD-type algae, was added and then dialyzed in reassociation buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl ₂ , pH 8.5) for 1 day. The loading capacity was examined by confirming that the fluorescent material was loaded. In addition, the capsid protein loaded with the fluorescent material was mixed with the harmful algae and incubated for 3 hours to examine the host specific binding of the capsid protein under a fluorescence microscope. In order to prevent non-specific binding to the algae of the FITC not loaded on the capsid protein, the dialysis was performed for 1 more day, followed by protein electrophoresis to confirm only the capsid protein loaded with the fluorescent material. In order to maintain the same conditions as the marine environment, the algae preserved in seawater was incubated for 3 hours by treating the capsid protein. Fluorescence microscopy with green (FITC) filter washes three times using filter sterilized seawater to remove capsid proteins that are not attached to algae and then sees whether FITC-loaded capsid proteins specifically bind to C. salsugineum Investigated under.

As a result, as shown in FIG. 7, the host-specific binding of the capsid protein loaded with the fluorescent material FITC was examined in C. salsugineum, and after treatment with the capsid protein loaded with FITC, the algae were subjected to the red filter condition of C. salsugineum . Fluorescence images (A ′) were observed under auto fluorescence (A) and FITC (green) filter conditions. That is, dark green fluorescence (FITC) was observed on the surface of C. salsugineum cells, and it was confirmed that the capsid protein specifically binds to the host. On the other hand, only the fluorescent material (FITC) as a control group was irradiated under the same conditions, as a result, the red fluorescent algae (B) was observed while FITC (green) fluorescence (B ') was not observed at all. This indicated that the capsid protein produced specifically binds to algae (FIG. 7).

Selective for harmful birds Algae effect

Algae screen experiment was performed using a thiazolidinedione (TD) -based compound of Formula 4 or 5 as the algae. The pesticide screening was performed by treating 135 selected TD compounds by concentration in 7 selected harmful algae. Put the same amount of cells in a microplate, and treated at concentrations of 0.05, 0.1, 1, 2, 5, 10, 20, 50, 100 μM, and after 3 days, Burker Turk hemacytometer and Sedgwick-Rafter blood cells Cell density was measured using a counting chamber. The percent reduction was calculated from the measured cell density to determine the correct IC ₅₀ value.

% Reduction = (1-Tt / Ct) × 100 (T: treated cell density, C: control cell density)

As a result, as shown in Table 1 and Table 2, most of the TD-based compounds showed a high killing effect against harmful algae, while showing very low toxicity to harmless algae. Among these compounds, Compound 17 showed the highest killing effect on harmful algae while not toxic to harmless algae (Tables 1 and 2). In addition, as shown in FIG. 8, after treatment with Cochlodinium polykrikoides of 1 μM TD-based compound 17, the change of cells was observed over time. As a result, the normal form in which eight cells were bound was observed. After 2 hours (c) after each treatment, each node began to loosen, and after 4 hours, each cell fell off, and after 5 hours (f), the separated cells burst and died (FIG. 8). . On the other hand, as shown in Figure 9, after treatment of 1 μM TD-based compound 17 in Heterosigma akashiwo ( Hterosigma akashiwo ) and observed the change in the cell as a result, after 2 hours after treatment the cell wall part bursts Intracellular material flowed out of the cell and showed a phenomenon of death (Fig. 9).

magnesium Organoclay  Synthesis and Analysis

Organic clay, another algae used in the present invention, used magnesium chloride hexahydrates (MgCl ₂ , 6H ₂ O, Junsei 98%) as a metal chloride reagent, and a silane compound having a functional group. Silver 3-aminopropyl-triethoxysilane (APTES) (Aldrich 98%) was used, the solvent was ethanol (95%, reagent), 1N-NaOH solution, ammonia water (30%, For reagents). In order to determine whether the organic clay, which is a product obtained from the reaction, was synthesized, the following equipment was analyzed. The solid samples were subjected to Si-O or Si-OH structure analysis using ²⁹ Si-HPDEC NMR (Avance 400, Bruker, Germany), and ¹ H-NMR (JEOL, of Metal-APTES compounds dissolved in D ₂ O). 300MHz, D ₂ O) analysis confirmed that the aminopropyl end (aminopropyl end) group was introduced into the clay. In addition, FT-IR (NICOLET6700, Thermoscientific, USA) analysis was carried out using potassium bromide (KBr) pellets and analyzed to confirm the introduction of a functional group, that is, aminopropyl (-(CH ₂ ) ₃ NH ₂ ). It was. X-ray diffraction (XRD) patterns were obtained using an X-ray diffractometer (X'Pert PRO, PANalytical BV), and solid (powder) samples were collected using CuKα radiation (20 mA). And 40 kV), 2 θ were measured at a rate of 2 ° / min from 2 ° to 70 ° to confirm the structure of the crystal. The morphology of the organic clay was confirmed from the analysis of transmission and scanning electron microscope, and the metal / Si component was analyzed through EDX to confirm the synthesis of the organic clay (FIG. 11). 1.68 g of magnesium chloride baggage (MgCl ₂ 6H ₂ O, 8.3 mmol) was dissolved in 50 ml of ethanol and 2.6 ml (11.1 mmol) of APTES was added slowly with vigorous stirring to precipitate a precipitate. The reaction was allowed to react at room temperature for 24 hours. I was. The white precipitate was washed three times or more with ethanol after centrifugation and dried. Yield is more than 96%, and analyzed by IR, NMR, SEM, TEM, XRD and the like to confirm the synthesis. In addition, a sodium hydroxide (NaOH) solution (1N NaOH, 10 mL) was synthesized according to the reported method, and the pH of the reactant was about 9.87, which maintained a higher pH value than the pH 8.15 of the reactant containing no aqueous NaOH solution. However, the difference in yield of the product was not large.

As a result, as shown in FIG. 10, it was confirmed that aminopropyl was introduced into Mg-APTES in FT-IR-spectrum and ¹ H-NMR (FIG. 10). Data of the structural properties are summarized in Table 3 below. Terminal introducer NH ₂ Extension vibration is 3600 ~ 3200 ㎝ ^-1 , NH ₂ It was confirmed that the organic clay was synthesized by showing a strong absorption band in bending vibration (1658 cm ^-1 ) and Si-O-Si stretching (1020 cm ^-1 ) (Table 3 and FIG. 11).

FT-IR ¹ H-NMR ²⁹ Si-NMR XRD 3600-3200 cm ^-1 (NH ₂ )
2950-2850 cm ^-1 (CH ₂ )
1658 cm ^-1 (NH ₂ )
1123 cm ^-1 (Si-C)
1020 cm ^-1 (Si-O-Si)
567 cm ^-1 (Mg-O-Mg)
484 cm ^-1 (Mg-O-Si) -OSi- CH ₂ -CH ₂ (2H)
; 0.55 to 0.60 ppm,
-CH ₂ - CH ₂ -CH ₂ (2H)
; 1.67-1.75 ppm,
-CH ₂ - CH ₂ -NH ₂ (2H)
; 2.88-2.93 ppm
R-SiO _3- (T3)
; -66.5 ppm,
R-SiO ₂ -OH (T2)
; -58.6 ppm,
R-SiO- (OH) _2- (T1)
; -48.4 ppm
basal distance; 1.44 nm (2 θ = 6.13)

magnesium Organoclay Algae effect

Magnesium organic clay three kinds of toxic algae (Heterocapsa circularisquama , Heterosigma akasiwo And Cocholodium polykrikodes ) when treated at a concentration of 0.2 mg / ㎖, it was confirmed that the killing ability of more than 80% compared to the control. On the other hand, four species of harmless algae { Navicula peliculosa pelliculosa), par dodak tilrum (Phaeodactylum) EPV, nanno claw drop system (Nannochloropsis) WT, Am nium in PD (Amphidinium sp .)} showed up to 20% killing ability. In addition, when the commercial mineral clay was treated in the same harmful algae, it was confirmed that the efficacy was lower than the organic clay. This confirmed that it is possible to selectively control only harmful algae using organic clay at a low concentration (Fig. 12). In addition, the IC ₅₀ values in Table 4 show that magnesium organoclays show excellent values, similar to ocher or ammonium chloride, which are similar compounds, and show low IC ₅₀ values even when compared to magnesium chloride used in the manufacture. It was proved to have (Table 4).

IC ₅₀ ^a (μM) Materials Harmful birds Harmless algae H. circularisquama H. akashiwo C. polykrikoides Amphidinium sp. Navicula pelliculosa Nanochloropsis oculata Phaeodactyl um EPV Mg-APTES
(Mg / ml) 0.162 ± 0.053 0.081 ± 0.006 0.056 ± 0.011 ND 0.905 ± 1.273 ND ND Purchased
Organoclay
(Mg / ml) ND ND 0.902 ± 0.846 ND ND ND NH ₄ Cl (pH 10)
(Mg / ml) 1.496 ± 0.025 1.968 ± 0.011 1.433 ± 0.931 ND ND ND ND MgCl ₂
(Mg / ml) ND ND ND ND ND ND ND ^{The a} value is the mean ± SD of three experiments.
ND, no detectable activity

Attach an electronic file to a sequence list

Claims (9)

1) harvesting and culturing harmful algae causing red and green algae;
2) selecting a virus specific for the harmful algae cultured in step 1);
3) cloning the capsid gene of the virus selected in step 2);
4) mass-producing nanocapsids after expressing the gene cloned in step 3) in recombinant E. coli or yeast; And
5) Harmful algae control method using the nano-capsid comprising the step of mounting the algae to the mass-produced nanocapsid in step 4).
According to claim 1, wherein the harmful algae causing red algae and green algae is Heterocapsa circular capacities ( Heterocapsa circularisquama ), chitoceros salinium salsugineum), poly kokeulrodinium Cri Koh des (Cochlodinium polykrikoides ), Heterosigma akashiwo ), Chattonella marina marina and Protoceratium reticulatum ) harmful algae control method using a nano-capsid, characterized in that any one selected from the group consisting of.
The method of claim 1, wherein the algae is any one selected from the group consisting of magnesium organoclay, quinone-based compound and thiazolidinedione-based compound.
4. The compound of claim 3, wherein the quinone compound is of Formula 1 or 2, wherein R of Formula 1 or 2 is unsubstituted alkyl, unsubstituted (hetero) cycloalkyl, (hetero) cycloalkenyl or (hetero) aryl Harmful algae control method using a nano-capsid, characterized in that.

[Formula 1]

(2)

The method of claim 3, wherein the thiazolidinedione-based compound is represented by Formula 3, wherein R ₁ of Formula 3 is hydrogen, nitro amine, alkyl, methoxy, trifluoromethyl, carboxyl or halogen R ₂ is hydrogen methyl ethyl, or unsubstituted (hetero) cycloalkyl, (hetero) cycloalkenyl or (hetero) aryl, and n is an integer of 0 to 5, wherein the harmful algae control method using nanocapsids .
(3)

The method of claim 1, wherein the virus specific to the harmful algae has no nucleic acid inside the capsid has no proliferative capacity and no human hazard, the harmful algae control using nano-capsid, characterized in that the capsid protein is used only as a carrier of the algae Way.
The method of claim 1, wherein the virus specific for the harmful algae is Heterocapsa Cycloriscuama RNA virus ( Heterocapsa circularisquama RNA Virus (HcRNAV), ketoseros salmonium intranuclear inclusion virus ( Chaetoceros) Salsugineum Nuclear Inclusion Virus (CsNIV) and heterosigma akashiwo RNA virus ( Heterosigma akashiwo RNA virus, HaRNAV) A method for controlling harmful birds using nano-capsids, characterized in that any one selected from the group consisting of.
The nanocapsid of claim 1, wherein the step of mounting the algae on the nanocapsid is performed by any one of a biochemical mounting method and a physicochemical mounting method, or by mixing two methods. Harmful algae control method using.
10. The method of claim 8, wherein the biochemical loading method is coexpression using gene recombination, or self assembly using dissociation or re-association buffer. Harmful algae control method using a nano-capsid, characterized in that carried out by.

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