JPH10298007A - Agent for preventing adhesion of aquatic organism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an aquatic organism adhesion-preventing agent low in toxicity and capable of exhibiting an aquatic organism-repelling action without accompanying an environmental disruption, and to provide a coating containing the aquatic organism adhesion-preventing agent. SOLUTION: This aquatic organism adhesion-preventing agent and the coating containing the aquatic organism adhesion-preventing agent contain a carbonic anhydrase as an active ingredient, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an aquatic organism adhesion inhibitor,
In particular, the present invention relates to an aquatic organism adhesion inhibitor comprising, as an active ingredient, carbonic anhydrase having an action of repelling organisms such as marine fouling organisms.

[0002]

2. Description of the Related Art In the bottom of ships and fishing nets, water intake channels for condenser cooling water in thermal power plants or heat exchanger cooling water in the petrochemical industry, buoys and other submarine equipment, harbors and dams, etc. It has been reported that aquatic organisms such as barnacles, mussels, and algae attach and breed, and obstacles such as closing of intakes are observed. Conventionally, in order to prevent the attachment of these aquatic organisms, an aquatic organism adhesion inhibitor containing an organotin compound as an active ingredient has been used, but this use pollutes the river or marine environment or uses fish as a medium. There has been a social problem that the human body may be damaged, and the use and production of the organotin compound are subject to legal regulations, and there has been a demand for the development of an aquatic organism adhesion inhibitor instead.

[0003] For this reason, recently, safe inhibitors containing various organic compounds such as physiologically active substances extracted from marine organisms and the like have been developed. However, the technique of applying or immobilizing these substances on a base (iron plate, concrete, or the like) of an artificial facility or the like and actually repelling the above organisms has not yet been developed. Therefore, development of an effective biological repellent substance that does not affect the environment and can solve these problems has become an urgent issue.

[0004]

DISCLOSURE OF THE INVENTION The present invention relates to an enzyme protein which does not adversely affect the environment and has an effective repellent action against marine fouling organisms, and an aquatic organism adhesion inhibitor containing this enzyme protein as an active ingredient. The purpose is to provide.

[0005]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that a protein having carbonic anhydrase activity exhibits an excellent aquatic organism adhesion inhibitory effect, The present invention has been completed. That is, the present invention provides an aquatic organism adhesion inhibitor containing carbonic anhydrase as an active ingredient.

Examples of the carbonic anhydrase include those derived from cnidarians. The present invention also provides a paint containing the aquatic organism adhesion inhibitor.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The aquatic organism antifouling agent of the present invention is obtained by collecting carbonic anhydrase or its contents from cnidarians such as sea anemones, mammals such as cattle and horses, plants such as spinach, and microorganisms such as Escherichia coli. , Prepared using this.

For example, a method for preparing a biofouling inhibitor containing carbonic anhydrase as an active ingredient from a sea anemone belonging to the family Anemonidae will be described below.

[0009] 300 g of a sea anemone, which is obtained by removing fine sand and seaweed attached to its epidermis,
The (wet weight) is placed in 1.2 l of a buffer (pH 7.5) containing 1 mM EDTA, 1 mM DTT and 0.5 M NaCl, and crushed using a blender (manufactured by Trio Science) to obtain a crude extract.

Next, the extract is centrifuged to remove solid substances, and a supernatant is obtained. An appropriate amount of ammonium sulfate is added to the supernatant to carry out salting out to recover only the protein.
The collected precipitate is dissolved in a solution used in the subsequent steps, and dialysis is performed. The dialysis can be performed according to a normal dialysis method.

For example, a dialysis membrane (cellulose tube: VIS
A sample is placed in a cellulose tube made by KASE Co., Ltd.) and placed in a sufficient amount of a solution having conditions necessary for removing low-molecular substances such as inorganic salts contained in the sample, for example, 0.1 M NH ₄ HCO ₃ 5 l. Dialyze at 4 ° C for 24 hours. The inner dialysis solution (protein) thus obtained is purified by a method used for general enzyme purification.

For example, affinity chromatography [ANALYTICAL BIOCHEMISTRY 57, 467-476 (1974), THE
Journal of Biological Chemistry 270,5985-5993 (19
95), ARCHIVES OF BIOCHEMISTORY AND BIOPHYSICS 312,
429-435 (1994), etc.] or by performing ion exchange, gel filtration, chromatography on a hydrophobic resin or the like. Thus, a purified protein having carbonic anhydrase activity having the following enzymatic properties is obtained.

As the instruments, reagents and the like used in each chromatography, commercially available ones can be used. Examples of those used for affinity chromatography include p-aminomethylbenzenesulfonamide agarose gel (manufactured by SIGMA).

[0014] as eluent in this case, like 0.1 M NH ₄ HCO ₃ containing 0.4 M NaN ₃ is used. The carbonic anhydrase used in the present invention is an enzyme having the following action and substrate specificity.

(1) Action: Acts on carbon dioxide and water to catalyze the reaction that produces hydrogen ions and hydrogen carbonate ions, and also catalyzes the reverse reaction. (2) Substrate specificity: It acts on carbon dioxide, acetaldehyde and other carbonyl compounds, and also acts on esters such as p-nitrophenyl acetate.

The aquatic organism adhesion inhibitor containing carbonic anhydrase having the above-mentioned properties may be used as it is, but may be used by mixing it with a paint or the like. As the above-mentioned paint, a water-soluble emulsion paint, for example, a water-soluble acrylic copolymer paint containing a hydrazine group crosslinking agent is preferable.

The paint containing such an aquatic organism adhesion inhibitor is applied to pottery, a plastic plate, concrete or the like and dried before use. In this case, even if immersed in seawater or freshwater for a long time (3 to 6 months), it does not peel or deteriorate,
It has an aquatic organism adhesion preventing effect.

[0018]

The present invention will be described below more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1] (1) Purification of carbonic anhydrase From a rock anemone (Anthopleura japonica VERRILL) obtained from a rock reef at Jogashima, Kanagawa, fine sand and seaweed attached to the epidermis were removed. 300g
(Wet weight) 25 mM containing 1 mM EDTA, 1 mM DTT, 0.5 M NaCl
Put in 1.2 L of HEPES buffer (pH 7.5) and blend it.
(Trio science).

The obtained homogenized suspension was cooled on ice to obtain a crude extract. Next, the extract was centrifuged at 10,000 × g for 20 minutes (5 ° C.), and 1.6 l of the supernatant was collected and 30%
Ammonium sulfate was added so as to be saturated, and ammonium sulfate precipitation was performed.

The precipitate is removed by centrifugation at 10,000 × g for 30 minutes,
Ammonium sulfate was added to 1.6 liters of the supernatant so as to be 80% saturated, and ammonium sulfate was precipitated. The precipitate was recovered by centrifugation at 10,000 × g for 30 minutes. The collected precipitate was dissolved in 0.1 M NH ₄ HCO ₃ , transferred to a dialysis membrane of a cellulose tube (manufactured by VISKASE), and dialyzed against 5 l of 0.1 M NH ₄ HCO ₃ at 4 ° C. for 24 hours.

To 1 liter of the dialyzed solution, 30 ml of p-aminomethylbenzenesulfonamide agarose gel (manufactured by SIGMA) equilibrated with 0.1 M NH ₄ HCO ₃ was added, and the enzyme was adsorbed with occasional gentle stirring. . After washing with the same buffer to remove unnecessary proteins, the gel was columnized (diameter 3 cm).
0.1M NH ₄ HCO ₃ 100ml containing the filled 0.2 M NaI in × 20 cm)
After washing with, elution was performed with 100 ml of 0.1 M NH ₄ HCO ₃ containing 0.4 M NaN ₃ .

The eluate was transferred to a dialysis membrane, and 1 mM EDTA, 1 mM DTT,
4 ° C for 3 L of 25 mM HEPES buffer (pH 7.5) containing 0.5 M NaCl
For 24 hours. Furthermore, the buffer was exchanged and dialysis was performed three times.

Ultrafiltration membrane device manufactured by Amicon (molecular weight cut off 1)
0000) to 2 ml. This fraction showed a uniform and single band by SDS-polyacrylamide gel electrophoresis and had a molecular weight of about 28,000. FIG.
Is a view showing the result.

Next, to determine the internal partial amino acid sequence, Achromobacter protease I (manufactured by Takara) was added to the denatured fraction containing 2 nmol of protein, and treated at 37 ° C. overnight. A number of peptide fragments were obtained. Then this is CA
0.1% TFA using PCELL PAK C18 SG120 (Shiseido) column
Chromatography was performed by applying a gradient of 0 to 80% of an aqueous solution and acetonitrile containing 0.1% TFA, and peak portions were respectively collected.

These peaks were subjected to a 473A protein sequencer (manufactured by Applied Biosystems) to analyze their sequences. As a result, the amino acid sequences shown in SEQ ID NOS: 1 and 2 were obtained.

The enzymatic chemical properties of the purified product obtained above are shown below. 1) Action: Acts on carbon dioxide and water to catalyze the reaction that produces hydrogen ions and bicarbonate ions, and also catalyzes the reverse reaction. 2) Substrate specificity: It acts on carbon dioxide, acetaldehyde and other carbonyl compounds, and also acts on esters such as p-nitrophenyl acetate.

3) Optimum pH: Optimum pH is 8 to 10 when esterase activity is used as an index. 4) Molecular weight: about 28000 (SDS-polyacrylamide electrophoresis) 5) Partial amino acid sequence: as shown in SEQ ID NOS: 1 and 2.

(2) Measurement of enzyme activity 1) A method of measuring the rate of pH change (change of hydrogen ion concentration caused by enzyme reaction).

3 ml of veronal buffer (0.022 M veronal and 0.022 M sodium salt, pH 7.95), a few drops of bromothymol blue, 2.3 ml of water or enzyme solution are placed in a 15 ml sealed container, and placed on ice water for 15 minutes. Cool for minutes. To this, 5 ml of ice-cooled saturated carbonated water is added to start the reaction. At 0 ° C, the pH of the reaction solution is measured based on the change in color of bromothymol blue.
Measure the time to reduce to 6.3. When the time in the presence of the enzyme t, the time in the enzyme absence was _{t 0, (t 0 -t)} /
The value of t is defined as the activity unit (U) of the enzyme.

According to this method, the activities of carbonic anhydrase purified from a sea anemone and a commercially available bovine erythrocyte-derived carbonic anhydrase (Funakoshi) were 4000 U / mg and 1900 U / mg, respectively. .

2) Method for measuring esterase activity: Transfer 0.6 ml of 15 mM Tris-sulfate buffer (pH 7.6) into a microtube, add 15 μl of an appropriately diluted enzyme solution, and reach a temperature equilibrium of 37 ° C. Add 0.3 ml of 1.5 mM acetic acid p-nitrophenyl ester solution to start the reaction. 0
The reaction is allowed to proceed for 4040 minutes, and the reaction is stopped by adding 0.15 ml of 1N HCl.

The change in absorbance at 348 nm of the reaction solution is examined using an absorbance meter. One unit is the amount of the enzyme that produces 1 μmol of p-nitrophenol per minute at 37 ° C.

In an experiment for examining the inhibitory effect of sulfanilamide, a specific inhibitor of carbonic anhydrase, 0.6 ml of 15 mM Tris-sulphate (Tris-sul
fate) Sulfanylamide is dissolved in a buffer solution (pH 7.6) to a predetermined concentration in advance, and the reaction is performed.

The specific activity of the carbonic anhydrase purified from the sea anemone by the above method was measured and found to be 1590 U / mg. The activity almost disappeared in the presence of 100 μM sulfanilamide.

Example 2 Repellent action against barnacles and mussels against carbonic anhydrase purified as in Example 1 and commercially available bovine erythrocyte-derived carbonic anhydrase (Funakoshi) A bioassay was performed using as an index. The method is disclosed in JP-A-8-1930
The method described in No. 97 was followed.

A multiwell (17 culture holes, 24 culture holes; manufactured by Falcon) having a diameter of 17 mm, a depth of 15 mm, and a capacity of 2 ml was filled with the fractionated solution prepared at each concentration, and barnacle nauplii larvae were introduced. Activity and life and death were observed and counted.

The swimming behavior of barnacle nauplii larvae is
As shown in FIG. 2, it is divided into the following five stages. I: Swim vigorously and do not stick to the bottom of the container. II: Swim or move around again, even if it hits the bottom of the container.

III: While still at the bottom of the container,
It moves around when other individuals come in contact. IV: appendages occasionally move while at the bottom of the container
(It does not move around even if other individuals touch it). V: Completely stop moving (protozoa develop in dead individuals).

The effect was judged by the state of the above III-V (Immobilized) in which the swimming ability was lost and stopped at the bottom of the multiwell.
Condition: swimming behavior inhibition / stop state) is calculated over time under an inverted stereomicroscope, and a percentage (index) with respect to the total number of input individuals is calculated. The index is converted to IMI (Immobilized In
dex), which was used as an index for determining the effect. The results are shown in FIG.

For carbonic anhydrase (hereinafter, referred to as CA) purified from sea anemones, 0.6 m
The IMI value was 90% at g / ml, and the commercially available bovine erythrocyte-derived carbonic anhydrase had an IMI value of 50% at 1.0 mg / ml, indicating a repellent effect. As a control, bovine serum albumin was measured in the same manner, and the results are shown in FIG.

Example 3 Method for Preparing Enzyme-Containing Paint and Immobilizing Method Thereof An equal amount of water-soluble paint to protein solution (0.5-2.5 mg / ml)
(Water-soluble emulsion paint: manufactured by Mitsubishi Chemical Corporation) was added and mixed. This was applied in an amount of 2 ml on an unglazed plate 35 cm ² , allowed to stand at room temperature for 24 hours, and dried.

[Example 4] Field immersion test of paint According to Example 3, a paint (repellent substance) of the present invention was applied to an unglazed plate
(35 cm ² ) was applied at 0.5 to 2.5 mg, and immersed for 14 days in the tidal zone of Isogo Ward, Yokohama City, Kanagawa Prefecture. In the test, as a control group, a case not coated (control) and a case coated with a conventional paint not containing the protein of the present invention (emulsion paint) were provided. The state of attachment was determined by measuring the numbers of adult barnacles, barnacles, cypris larvae, and mussels attached to the unglazed plate. FIG. 4 shows the results. From FIG. 4, the number of barnacles adhered was reduced in the group where the protein of the present invention was mixed with the paint and applied compared to the control group.

[0044]

According to the present invention, it is possible to provide a paint containing carbonic anhydrase having an excellent biological repellent action as an active ingredient and a method for producing the same. The paint of the present invention has a low toxicity since the enzyme is an active ingredient,
The repellent action of aquatic organisms can be exhibited without causing environmental destruction.

[0045]

[Sequence list]

SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Sequence type: Partial sequence of royal sea anemone carbonic anhydrase Sequence: Thr Asn Glu Gly Thr Asn Leu Ser Gly Gly Pro Leu Gly His Asn Tyr Arg Phe Glu Gln Phe

SEQ ID NO: 2 Sequence length: 15 Sequence type: amino acid Sequence type: partial sequence of royal anemone carbonic anhydrase Sequence: Thr Val Pro Ala Glu Leu His Leu Met Gln Trp Asn Thr Phe Leu

[Brief description of the drawings]

FIG. 1 is a view showing the results of SDS polyacrylamide electrophoresis.

FIG. 2 is a view showing swimming behavior of barnacle novius larvae.

FIG. 3 is a diagram showing the results of a bioassay using barnacle nauplii larvae.

FIG. 4 is a view showing the results of a biological repellent effect in a field immersion test of the paint of the present invention.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Tatsuo Horiuchi 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation (72) Inventor Kanae Matsuzaki 23-6-204 Denen Chofu Minami, Ota-ku, Tokyo Genes Denon Chofu (72 Inventor Ikuko Aoyama 9-15 Beniyacho, Hiratsuka-shi, Kanagawa 402

Claims (3)

[Claims] 1. An aquatic organism adhesion inhibitor comprising carbonic anhydrase as an active ingredient. 2. The aquatic organism adhesion inhibitor according to claim 1, wherein the carbonic anhydrase is derived from a cnidarian. 3. A paint comprising the aquatic organism adhesion inhibitor according to claim 1 or 2.