JPH08193097A - Organism-repellent protein and its immobilizing method - Google Patents

Abstract

PURPOSE: To obtain a protein for coating components, having organism-repelling action and antimicrobial action by homogenizing a marine organism belonging to sea anemones and centrifuging the marine organism, dialyzing the supernatant, treating the dialyzed inner solution with chromatography. CONSTITUTION: This organism repellent protein is obtained by homogenizing a marine organism (e.g. Anthopleura japonica VERRILL) belonging to the genus sea anemones) in a buffer solution, centrifuging the resultant crude extracted solution, putting a supernatant after centrifugation into a dialyzed membrane of a cellulose tube, dialyzing the supernatant to buffer solution at 5 deg.C for 24hr and subjecting the resultant dialyzed inner solution to ion exchange chromatography and then, get chromatography. The organism-repellent protein has about 40000 molecular weight and has organism repelling action and antimicrobial action. Furthermore, the protein is reacted with a copolymer compound expressed by the formula (R is a 2-6C alkylene) and having carbonyl group (e.g. water-soluble acrylic copolymer). Thereby, an organism-repellent coating can be obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein having an action of repelling organisms such as marine fouling organisms and an antibacterial action, a method for immobilizing the protein, a paint containing the protein as an active ingredient, and a method for producing the paint. .

[0002]

2. Description of the Related Art Barnacles, mussels, etc. attach to thermal power, cooling water channel systems of nuclear power plants, marine artificial facilities, ship bottoms, fishing nets, etc., resulting in water intake obstacles, deterioration of facilities, and navigation. It is a marine fouling organism that causes various obstacles such as reduced speed or obstruction of fishing.

For this reason, many control measures against fouling organisms have been attempted to control these organisms (Marine attached organisms, 1974, Marine Science, Symposium-54, Ocean Publishing Co .; Takeshi Kajiwara, Ed. 1986, Marine periphyton and aquaculture, Seiseisha Koseikaku). Recently, research and development have been conducted on physiologically active substances extracted from marine products (Marine Biotechnology Research Group, 1992, 1st Marine Biotechnology Research Presentation Abstract).

However, with respect to these physiologically active substances, a technique for actually repelling the above-mentioned organisms by coating or immobilizing these physiologically active substances on the base (iron plate, concrete, etc.) of cooling water channels, artificial facilities, etc. has not yet been developed. It has not been. In addition, fouling organism control substances containing heavy metals and organic poisons (cuprous oxide, triphenyltin, etc.) have been developed (environmental standards), but their use is not preferable because it causes environmental damage.

Therefore, there is an urgent need to develop an effective bio-repellent substance that does not affect the environment and can solve these problems.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a protein which does not affect the environment, has an effective repellent action against marine fouling organisms, and the action is long-lasting, and immobilization of the protein. An object of the present invention is to provide a method, a paint containing the protein as an active ingredient, and a method for producing the paint.

[0007]

[Means for Solving the Problems] As a result of intensive studies based on the above problems, the present inventors have found that physiologically active substances extracted from marine products belonging to sea anemones are effectively repelled against marine fouling organisms. It has been found that it has an action and an antibacterial action, and has completed the present invention. That is,
The present invention is to homogenize marine organisms belonging to the sea anemone, centrifuge the crude extract obtained, dialyzing the supernatant after centrifugation, the resulting dialyzed liquid is ion exchange chromatography, then gel. It is a biorepellent protein having a molecular weight of about 40,000 and having biorepellent and antibacterial activities, which is obtained by chromatography.

[0008] Examples of marine organisms belonging to the sea anemones include organisms belonging to the family Anemones, such as the sea anemone. Further, the invention provides the biorepellent protein, Formula I:

[0009]

Embedded image

In the method for immobilizing the protein on the copolymer compound, the compound represented by (R represents an alkylene group having 2 to 6 carbon atoms) and a copolymer compound having a carbonyl group are reacted. is there. Furthermore, the present invention is a paint containing the above-mentioned bio-repellent protein as an active ingredient.

The organisms targeted in the above-mentioned biological repellent action include marine fouling organisms such as barnacles and mussels. Further, the present invention provides formula I:

[0012]

[Chemical 4]

A method for producing the above-mentioned paint, characterized in that a compound represented by the formula (R is the same as above), a copolymer compound having a carbonyl group and the biorepellent protein are reacted. As the copolymer compound having a carbonyl group, a water-soluble acrylic copolymer paint containing a hydrazine group crosslinking agent (for example, Acronal: Mitsubishi Kasei Co., Ltd.)
Manufactured) and the like.

The present invention will be described in detail below. (1) Production of protein of the present invention The protein of the present invention is produced by the following method. That is, a marine organism belonging to the sea anemone is homogenized to obtain a crude extract, which is centrifuged, and the resulting supernatant is dialyzed using a cellulose tube or the like. Then, the obtained dialyzed liquid is subjected to ion exchange chromatography and then gel chromatography to obtain a fraction in which activity is recognized as the protein of the present invention.

The marine organism used is not particularly limited as long as it belongs to the sea anemone, and examples thereof include the sea anemone of the family Anemoneaceae. The homogenization of the sample (Euphoria anemones) is performed, for example, by the following method.

0.15 M per 30 g of anemone sample from which impurities (sand, seaweed, etc.) adhering to the body wall have been removed
Of NaCl (pH 7.0) containing 5 mM NaCl, 5 mM EDTA and 0.1 mM PMSF (phenylmethanesulfonyl fluoride)
Is added, and the tissue is destroyed using a homogenizer (manufactured by Tokyo Rika) to obtain a brown crude extract. Then, the crude extract is left for 30 minutes under ice cooling and used for the following purification.

Centrifugation is carried out under conditions such that solid substances (cell crushed substances) in the sample solution can be removed, and usually centrifugation is performed at 10,000 × g for 20 minutes. Dialysis can be performed according to a usual dialysis method. For example, put a sample in a dialysis membrane (cellulose tube: cellulose tube made by VISKASE, etc.), and a sufficient amount of buffer solution with the conditions necessary to remove low molecular weight substances such as inorganic salts contained in the sample, for example, It is put in a buffer solution (pH 6.9; 3 L) containing 0.15 M NaCl and 0.5 mM EDTA, and dialyzed at 5 ° C. for about 24 hours. This is repeated 3 to 4 times as needed, and 10 mM Tris (pH
Stabilize with 8.0).

The dialysis inner solution thus obtained is purified by ion exchange chromatography and then gel chromatography. Equipment used for each chromatography,
As the reagents and the like, those commercially available can be used.
Examples of the ion exchanger used in the ion exchange chromatography include DEAE Toyopearl (manufactured by Tosoh Corporation) and Q Sepharose (manufactured by Pharmacia Biotech Co., Ltd.). As an instrument used in gel chromatography, the emission limit molecular weight is 3,000 to 70,000. Prepack column (Pharmacia Biotech Co., Ltd.) and the like, and 10 mM Tris and NaCl (0 to 0.1 M) are used as the extraction solution (pH 8.
0).

The protein fractionated as described above is subjected to a bioassay using the repellent action against barnacles and mussels as an index to obtain the fraction having activity as the purified protein of the present invention. The bioassay method is as follows. A multi-well (diameter: 17 mm, depth: 15 mm, volume: 2 ml) (24 culture holes; manufactured by Falcon) was filled with the fractionation solutions prepared to each concentration, and after the nauplii larvae were introduced, the activity, life and death state were observed over time. Observe and count.

Nauplius larvae swimming behavior is divided into the following 5 stages (see FIG. 1). I: Actively swim and never reach the bottom of the container. II: Will you reach the bottom of the container, but will you swim again?
Move around. III: It remains attached to the bottom of the container, but moves around when contacted by another individual. IV: The attached limb occasionally moves while still attached to the bottom of the container (it does not move around even when another individual comes into contact with it).

V: The movement stops completely (protozoa occur in dead animals). To judge the effect, an individual who lost the swimming ability and stopped at the bottom of the multiwell in the above-mentioned state of III to V (Immobilized Condition: swimming behavior inhibition / stopped state) was calculated with the use of an inverted stereomicroscope, and input. Percentage of total population (index)
To calculate. The index is defined as IMI (Immobilized Index) and is used as an index for effect determination.

Among the physicochemical properties of the protein of the present invention, the molecular weight is measured by a calibration curve of gel chromatography using a marker of known molecular weight or electrophoresis. (2) Protein Immobilization Method Next, the protein immobilization method of the present invention will be described.

The protein of the present invention can be used by mixing it with paint or the like. However, the protein of the present invention is not preferable because the protein is not uniformly mixed by simply mixing it, the amount of the substance that does not exhibit the repellent effect is not reached, and the protein flows out. Then, as a result of earnest research on a method of immobilizing the above-mentioned protein on a carrier, the above-mentioned protein and the copolymer compound which is the main component of the paint are represented by Formula I:

[0024]

Embedded image

By binding via a compound (crosslinking agent) represented by (R represents an alkylene group having 2 to 6 carbon atoms), the above protein is immobilized on the copolymer, and the activity of the above protein is Was found to be retained. The structure of the above protein thus immobilized on the copolymer compound is represented by the formula I:

[0026]

[Chemical 6]

Of the two hydrazide groups of the compound represented by (R is the same as the above), one of the hydrazide groups is bound to the above protein and the other hydrazide group is bound to the copolymer compound. It is presumed that the hydrazide group forms a crosslinked structure at room temperature with the carbonyl group of the protein and the copolymer compound. In FIG. 2, R1 and R2 represent amino acid side chains in the peptide bond. (3) Next, a coating material containing the protein of the present invention as an active ingredient and a method for producing the coating material will be described.

The above protein, the copolymer compound having a carbonyl group and the hydrazine compound represented by the formula I are mixed so as to be uniform at room temperature to obtain a paint containing the above protein. As the paint, an aqueous paint containing a copolymer compound and a hydrazine compound is particularly preferable. Examples of the copolymer compound of the paint component include acrylic acid and the like.

The mixing ratio of the protein of the present invention to the water-soluble paint is 1 ml or more to 1 ml of the aqueous protein solution, but the ratio of protein to paint is 3 mg / ml or more as long as the protein is not eluted from the paint. Is preferred. The amount of the compound of the formula I used is 6 to 34 μg of protein and 1.5 ml of the paint per 1 cm ² of the substrate. It is desirable that the reaction conditions for immobilization are such that they are mixed at room temperature so as to be uniform, and after application, the binding reaction is allowed to proceed by volatilizing water and dried for at least 24 hours.

The paint of the present invention is used for pottery, plastic plates,
When applied to concrete, etc., and dried at room temperature for about 24 hours, it is fixed to the base (wall surface), so even if it is immersed in seawater or fresh water, it may peel or deteriorate for a long time (3 to 6 months). There is no such thing. In addition, the paint of the present invention can be directly applied to the above-mentioned plastic plate, concrete or other base,
It is also possible to apply the coating material of the present invention to the front surface of the film having the adhesive agent applied to the back surface, and to use (separate) according to the size and material of the target wall surface.

[0031]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. [Example 1] Purification of protein Fine sand, seaweed, etc. adhering to the epidermis of the sea anemone (Anthopleura japonica VERRILL) obtained from the reef of Jogashima Island, Kanagawa Prefecture were removed to remove 30 g of wet weight of 0.15 M NaC.
l 300 ml buffer containing 5 mM EDTA and 0.1 mM PMSF (pH
7.0) and homogenized using a homogenizer (manufactured by Tokyo Rika). The resulting homogenized suspension was left under ice cooling for 30 minutes to obtain a crude extract. Next, add 1
Centrifugation was carried out at 0,000 xg for 20 minutes (5 ° C). After centrifugation, the supernatant was collected and dialyzed as follows. Add the supernatant to the dialysis membrane of a cellulose tube (made by VISKASE) and
It was dialyzed at 5 ° C. for 24 hours against 3 L of a buffer solution (pH 6.9) containing 15M NaCl and 5 mM EDTA. This was repeated 3 to 4 times until the solution was no longer colored, and stabilized with 10 mM Tris (pH 8.0) to obtain a sample for the chromatographic treatment described below.

The sample was applied to an anion exchanger (DEAE Toyopearl; manufactured by Tosoh Corporation) infiltrated with 10 mM Tris, 10 mM Tris and NaCl (0 to 0.1 M) were used as an extract (pH 8.0), and a concentration gradient was applied. Method (change NaCl from 0 to 0.1M)
By ion exchange chromatography (column diameter 2 cm, height 60 cm). As a result, one peak was obtained (Fig. 3), and this fraction was collected from Pharmacia Biotech Hi-L.
Using oad 16/60 Superdex75 (molecular weight 3,000-70,000), elution was performed with 150 mM ammonium acetate at a flow rate of 2.0 ml / min, and a peak (peak A) and a molecular weight of 40,000 were obtained.
A peak (peak B) of 000 or less was obtained (Fig. 4). As a result of performing a bioassay using the obtained fractions of the Fujitsubonopurius larva, a repellent effect was observed in each fraction (FIG. 5, “●” and “◯” marks). [Example 2] Method of immobilizing protein Since the repellent substance obtained in this study is a water-soluble protein, a method of retaining it on the surface without destroying the three-dimensional structure is as follows.
A chemical bond may be considered. Therefore, a water-soluble emulsion paint was selected from the three types of paint, and mixing, coating, and elution experiments were conducted. As a result, it has been found that the protein and the water-soluble emulsion paint can be uniformly mixed and can be applied to a base such as a unglazed plate, plastic, or vinyl resin.

Example 3 A coating containing protein, a method for producing the same, and a biorepellent effect thereof. For 1 mg of the protein prepared in Example 1, a water-soluble coating (water-soluble emulsion coating: manufactured by Mitsubishi Yuka Co., Ltd.) was used. Mixtures having different mixing ratios were prepared so as to be less than or equal to ml (coating 0.5 ml to 4 ml per 1 ml of protein). The protein of the present invention is water-soluble, and the protein is composed of peptide bonds. The coating material selected was a water-soluble emulsion, which was prepared by adding a hydrazine derivative which forms a cross-linked structure by a carbonyl-hydrazide group to the dispersed copolymer with evaporation of water at room temperature. Therefore, when this paint and protein are mixed and applied to a substrate, the carbonyl group of the peptide bond of the protein and the hydrazide group of the paint are chemically bonded together with the evaporation of water to form a film.

A coating material (protein of the present invention / water-soluble coating material = 0.5-4, see FIG. 6) obtained by mixing the above-mentioned proteins of the present invention having different mixing ratios was applied to a unglazed plate (35 cm ² 0.2 to 1.23 mg was applied to the solution) and left at room temperature for 24 hours. Next, each unglazed plate was immersed in the coast of Tokyo Bay for about 1 week, and the adhesion state of barnacles was tested. Regarding the adhesion state of barnacles, a group of 2 to 3 biscuit plates was used per mixing ratio. As a control, the protein-free paint of the present invention and the unglazed plate itself were used.

The results are shown in FIG. From Fig. 6, the control section (con
Compared to t.), the number of barnacles adhered decreased in the group to which the coating material of the present invention was applied. [Example 4] Field immersion test of the coating material of the present invention The coating material (repellent substance) of the present invention was applied to a biscuit board (35 cm ² ) at 0.2
Apply ~ 1.23mg, and apply to the intertidal zone of Isogo and Hakkeijima Koji,
Soaked for 51-96 days. In the test, as a control section, a case where no paint was applied (control) and a case where a conventional paint containing no protein of the present invention was applied (emulsion paint) were provided. The adhesion condition was determined by the ratio of the algae covering the substrate (coverage%).

The results are shown in FIG. From FIG. 7, the algae coverage in the case where the coating material (repellent substance 1, 2, 3) of the present invention was applied was 20% or less, which was smaller than that in the control group (uncoated: 100%). The effect of suppressing the adhesion of algae by the paint was confirmed. [Reference Example 1] Protein of the present invention (repellent substance; 0.1 to 10)
(mg / l) was used to perform a TLm test (Median Tolerance Limit; a test for measuring 50% lethal concentration with one standard value indicating the toxic effect of pollutants) against medaka, tiger shrimp, mussel, etc.

As a result, the protein concentration of the present invention was 0.1.
No deceased individuals were observed in the range of -10 mg / l, and it was found that the protein of the present invention is environmentally friendly and has no toxicity to living organisms (Table 1).

[0038]

[Table 1]

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a protein having an excellent biorepellent action, a method for immobilizing the protein, a paint containing the protein as an active ingredient, and a method for producing the paint. Since the coating material of the present invention has little toxicity, it has the excellent marine fouling organism repellent action and antibacterial action described above without causing environmental damage.

[Brief description of drawings]

FIG. 1 is a diagram showing swimming behavior of Nauplius larvae.

FIG. 2 is a view showing a binding state of a protein, a copolymer compound and a hydrazine compound.

FIG. 3 shows the results of ion exchange chromatography.

FIG. 4 is a diagram showing the results of gel chromatography.

FIG. 5 is a view showing the results of bioassay using barnacle bonito larvae.

FIG. 6 is a view showing the results of the biorepellent action in the field immersion test of the coating material of the present invention.

FIG. 7 is a photograph showing the morphology of organisms in the biorepellency of the field immersion test.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kanae Matsuzaki, Kanae Matsuzaki 5-38-3 Kamata, Ota-ku, Tokyo Asahi Building 7F Chiyoda Dame's & Moore Co., Ltd. (72) Ikuko Aoyama 5 Kamata, Ota-ku, Tokyo Chome 38-3 Asahi Building 7F Chiyoda Dames & Moore Co., Ltd. (72) Inventor Kazuya Ogawa 2-24-23 Koenji Minami, Suginami-ku, Tokyo Nakai Lab.

Claims (6)

[Claims] A marine organism belonging to the sea anemone is homogenized, the resulting crude extract is centrifuged, the supernatant after the centrifugation is dialyzed, and the resulting dialyzed liquid is subjected to ion exchange chromatography, A bio-repellent protein having a molecular weight of about 40,000 and having biorepellent and antibacterial actions, which is obtained by gel chromatography treatment. 2. The biorepellent protein according to claim 1, wherein the marine organisms belonging to the sea anemone are organisms belonging to the family Anemoneidae. 3. The organism-repellent protein according to claim 2, wherein the organism belonging to the family Anemoneaceae is the sea anemone. 4. The biorepellent protein according to any one of claims 1 to 3, a compound of formula I: (R represents an alkylene group having 2 to 6 carbon atoms) and a copolymer compound having a carbonyl group are reacted with each other to immobilize the biorepellent protein on the copolymer compound. . 5. A paint containing the biorepellent protein according to any one of claims 1 to 3 as an active ingredient. 6. Formula I: embedded image The compound represented by (R is the same as the above), a copolymer compound having a carbonyl group, and the biorepellent protein according to any one of claims 1 to 3 are reacted. Paint manufacturing method.