JP5073518B2 - Siderophore, marine environment modifier containing the siderophore, and marine environment reforming method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a siderophore having high ability to bind to iron ion and thus assisting the stable existence of iron ion in its aqueous solution, and to provide a marine environment modifier containing the siderophore. <P>SOLUTION: The siderophore is a compound represented by chemical structural formula (I) or (II) or a salt thereof. The marine environment modifier containing the siderophore is also provided. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a siderophore composed of a novel compound having a high binding ability with iron ions, and a technique for preserving the marine environment using the compound.

  Iron is an essential element for most organisms. Although iron is the most abundant element on earth, it is easily oxidized from divalent ions to trivalent ions in the presence of oxygen, and these trivalent iron ions are used by organisms in neutrality or alkalinity. Produces sparingly soluble salts that cannot be made. This nature of iron ions makes it difficult for organisms to take up iron ions.

  Therefore, many microorganisms secrete low molecular weight organic compounds having high affinity with trivalent iron ions called siderophores in order to take up iron ions that are slightly present in the environment. It binds to trivalent iron ions that are present in a trace amount, thereby forming an iron-siderophore complex. This complex is taken into the cell via an outer membrane receptor specific to the siderophore, and the complex is formed from the incorporated complex by hydrolysis of the siderophore or an enzyme that reduces iron bivalently. It is off the body and used by microorganisms. Thus, certain microorganisms can effectively take in trace amounts of iron ions in water by secreting siderophores.

  In addition, since the production of microorganisms, particularly siderophores of pathogenic bacteria, is deeply involved in host infection and pathogenic expression, the production of siderophores of pathogenic bacteria has been well studied and many siderophores have been isolated and structurally determined ( Non-patent document 1). For certain microorganisms, only specific siderophores can be used, and growth may be suppressed by other siderophores, and there is an idea of using siderophores as antibacterial agents (see Non-Patent Document 2). ). Some siderophores have been put to practical use as iron excretion drugs such as deferoxamine B produced by several actinomycetes strains including Streptomyces (see Non-patent Document 3).

  Furthermore, in the marine environment, especially in the surface layer where sunlight necessary for photosynthesis reaches, iron ions are rapidly oxidized to trivalent by dissolved oxygen, and the iron ions settle as iron hydroxide, so the concentration becomes extremely low. For this reason, it is known that the growth of marine organisms such as phytoplankton, which mainly absorbs carbon dioxide in the ocean, is suppressed (see Non-Patent Document 4), and it is possible to increase the iron ion concentration in the surface area of the ocean. If possible, the photosynthesis ability of phytoplankton in the marine environment can be increased, and as a result, the ability to fix carbon dioxide in the ocean can be enhanced, which helps to counter global warming.

Shigeo Yamamoto: Pharmacia, Vol. 31, No. 6, 588 (1995) Ward, C. Gillon, The Journal of Trauma: Injury, Infection, and Cinical Care, Vol. 41, No. 2, pp 356-361 (1996) Bullen, J. J., European Journal of Clinical Microbial Infection Dis., 10, 613 (1991) Price, NL, Morel, FMM, Biological cycling of iron in the ocean.In Metal Ions in Biological Systems, Shigel, A., Shigel, H., Eds .; Marcel Dekker Inc .: New York, 1998; Vol. 35, pp 1-36. Schwyn, B. and Neilands, J.B., Analytical Biochemistry, 160 (1), 47 (1987)

  However, almost no siderophore that can increase the iron ion concentration in the marine environment has been known so far, and no method for modifying the marine environment using the siderophore has been known.

  The present invention relates to a siderophore having high binding ability with iron ions and capable of increasing the iron ion concentration in the marine environment, a marine environment modifier containing the siderophore, and a marine environment using the siderophore. An object is to provide a reforming method.

  The present inventor was able to obtain one strain of siderophore-producing microorganism by conducting CAS (Chrome azurol S) assay from a large number of microorganisms obtained from seaweed and sea cucumber. As a result of the examination, it was found that the microorganism was a novel microorganism belonging to the genus Streptomyces, but when the microorganism strain was cultured, the compound obtained from the culture had high binding properties with iron ions. And it discovered that it was a novel siderophore which has the characteristic which assists that a trivalent iron ion exists stably in aqueous solution. Furthermore, this siderophore has been found to have a growth promoting effect on algae cultured in a poor iron environment, leading to the present invention.

  The CAS assay is a method for determining the presence of a siderophore and is described in Non-Patent Document 5, for example. CAS is a pigment compound that develops a characteristic color when bound to iron ions. When a compound with a high iron-binding ability such as siderophore is added to a solution containing CAS, siderophore absorbs iron ions from the CAS-iron complex. As a result, the coloration characteristic of the CAS-iron complex is eliminated, so that the determination can be made. In other words, this evaluation method is considered to reflect the binding ability between siderophore and trivalent iron ions in seawater.

で表される化合物もしくはその塩、または下記の化学構造式（II)：

で表される化合物もしくはその塩であるシデロフォアに関する発明。
（２）また、本発明は、（１）記載のシデロフォアを含有し、海水中の鉄イオン濃度を向上させる機能を有することを特徴とする海洋環境改質剤に関する。
（３）また、本発明は、（１）記載のシデロフォアを生産するストレプトマイセス属に属する微生物であるストレプトマイセス・スピーシーズYM5-799株（NITE AP-480）を土に添加し、当該微生物が添加された土を、海底に投入または埋設することにより海水中の鉄イオン濃度を向上させることを特徴とする、海洋環境改質方法に関する。 The gist of the present invention is as follows.
(1) The following chemical structural formula (I):

Or a salt thereof, or the following chemical structural formula (II):

The invention regarding the siderophore which is a compound represented by these, or its salt.
(2) Moreover, this invention relates to the marine environment modifier which contains the siderophore of (1) description, and has the function to improve the iron ion density | concentration in seawater.
(3) Moreover, this invention adds the Streptomyces species YM5-799 strain | stump | stock (NITE AP-480) which is microorganisms which belong to the Streptomyces genus which produces the siderophore of (1 ) to soil, The said microorganisms soil but which has been added, characterized in that to improve the concentration of iron ions in seawater by on or embedded in the seabed, related to the marine environment modification method.

  The siderophore according to the present invention has a high binding ability with iron ions, increases the bioavailable iron ion concentration in the marine environment, and is useful as a marine environment conservation or algal growth promoter. Thereby, it is considered possible to improve the fixing ability of carbon dioxide in the ocean.

で表される化合物もしくはその塩、または下記の化学構造式（II)：

で表される化合物もしくはその塩である。
塩としては、塩酸塩、リン酸塩、硫酸塩、フマル酸塩、マレイン酸塩等の酸付加塩、ナトリウム塩等のアルカリ金属塩等本化合物の鉄結合能および生物利用性に影響を与えない範囲で適宜選択できる。 The present invention is described in detail below.
(1) Structure and properties of the compound of the present invention The siderophore of the present invention has the following chemical structural formula (I),

Or a salt thereof, or the following chemical structural formula (II):

Or a salt thereof.
As salts, acid addition salts such as hydrochloride, phosphate, sulfate, fumarate and maleate, and alkali metal salts such as sodium salt do not affect the iron binding ability and bioavailability of this compound. It can be selected appropriately within a range.

  When the compound of the present invention is used as a medicine, the salt is desirably pharmaceutically acceptable.

  As its use, it can be used as a therapeutic agent for iron overload caused by blood transfusion or as a therapeutic agent for wounds, like deferoxamine mesylate, which is a siderophore currently in practical use as a pharmaceutical. Moreover, the compound of this invention can be utilized as an antibiotic with respect to the microorganisms which cannot be utilized as a siderophore.

  Examples of the pharmaceutically acceptable salt include conventional non-toxic acid addition salts. More specifically, inorganic acid addition salts (for example, hydrochloride, sulfate, phosphate, etc.), organic carboxylic acid addition salts or organic sulfonic acid addition salts (for example, formate, acetate, trifluoroate, maleate) Acid salts, tartrate salts, methanesulfonate salts, benzenesulfonate salts, paratoluenesulfonate salts, etc.), salts with basic amino acids or acidic amino acids (for example, arginine, aspartic acid, glutamic acid, etc.).

  Properties of the compound of the chemical structural formula (I) and the compound of the chemical structural formula (II) are as follows.

<Properties of compound of formula (I)>
(1) Color of the substance: colorless (2) Molecular weight: 804.80
(3) Molecular formula: C ₃₄ H ₄₈ N ₁₀ O ₁₃
(4) Mass spectrometry: High resolution FABMS measured value 805.3454 (M + H) ⁺
Calculated value 805.33481 (C ₃₄ H ₄₉ N ₁₀ O ₁₃ )
(5) Infrared absorption spectrum (KBr): ν _max 3423, 1638, 1543, 1386, 1262, 1205, 1132 cm ^-1
(6) UV absorption spectrum (H ₂ O, pH 4.0): λ _max (log ε): 247 (3.99), 317 (3.65), 357 (3.44) nm

(7) ¹ H-NMR (measured in deuterated dimethyl sulfoxide, 750 MHz)
δ ppm 11.80 (brs, 1H), 11.67 (brs, 1H), 9.36 (brs, 1H), 9.34 (brs, 1H), 8.81 (d, 1H), 8.79 (d, 1H), 8.42 (brs, 1H) , 8.08 (brd, 1H), 7.52 (brt, 1H), 7.50 (brs, 1H), 7.40 (m, 2H), 6.94 (m. 2H), 6.72 (m, 2H), 5.32 (m, 1H), 4.93 (brd, 1H), 4.70 (m, 2H), 4.64 (brd, 1H), 4.36 (dd, 1H), 4.29 (brm, 1H), 3.14 (m, 4H), 1.86 (m, 2H), 1.75 (m, 2H), 1.58 (m, 4H), 1.15 (t, 3H), 1.08 (t, 3H)

(8) ¹³ C-NMR (measured in deuterated dimethyl sulfoxide, 125 MHz)
δ ppm 171.89, 171.66, 170.59, 169.32, 168.56, 168.34, 156.66, 148.39, 148.15, 146.03, 146.00, 118.78, 118.71, 118.56, 118.35, 118.14, 116.09, 115.87, 70.52, 66.21, 57.19, 54.77, 52.53 40.54, 40.41, 29.14, 29.01, 25.18, 20.12, 16.24

(9) Solubility: Easily soluble in water, soluble in DMSO and methanol, hardly soluble in acetone, ethyl acetate and chloroform.

(10) CAS assay: The iron binding ability of this compound was evaluated by the CAS assay described in Non-Patent Document 5. CAS (Chrome azurol S) is a pigment compound that develops a characteristic color when combined with iron ions. When a compound with a high iron-binding ability such as siderophore is added to a solution containing CAS, siderophore becomes a CAS-iron complex. The iron color is taken away from the body, and as a result, the coloration characteristic of the CAS-iron complex is eliminated, so that the presence of the siderophore can be determined. In other words, this evaluation method is considered to reflect the binding ability between siderophore and trivalent iron ions in seawater. This evaluation method showed three times the activity (ED ₅₀ 0.1 mM) of the control deferoxamine B (ED ₅₀ 0.3 mM).

<Properties of compound of formula (II)>
(1) Color of the substance: colorless (2) Molecular weight: 1180.18
(3) Molecular formula: C ₅₁ H ₆₉ N ₁₅ O ₁₈
(4) Mass spectrometry: High resolution FABMS measured value 1180.5004 (M + H) ⁺
Calculated 1180.5023 (C ₅₁ H ₇₀ N ₁₅ O ₁₈ )
(5) Infrared absorption spectrum (KBr): ν _max 3422, 1751, 1675, 1543, 1265, 1203, 1137 cm ^-1
(6) UV absorption spectrum: λ _max (MeOH) (log ε) 246 (3.08), 320 (2.62), 357 (1.98) nm

(7) ¹ H-NMR (measured in deuterated dimethyl sulfoxide, 750 MHz)
δ ppm 11.55 (s, 3H), 9.41 (brs, 3H), 8.79 (brs, 6H), 7.47 (brt, 3H), 7.40 (d, 3H), 6.93 (d, 3H), 6.70 (t, 3H) , 5.43 (s, 3H), 4.88 (d, 3H), 4.78 (brs, 3H), 3.20 (brs, 3H), 3.10 (brs, 3H), 1.90 (brs, 3H), 1.80 (brs, 3H), 1.63-1.58 (brd, 6H), 1.15 (d, 9H)

(8) ¹³ C-NMR (measured in deuterated dimethyl sulfoxide, 125 MHz)
δ ppm 172.28, 168.10, 168.07, 156.68, 147.78, 145.94, 118.75, 118.61, 118.24, 116.46, 70.80, 55.36, 52.61, 40.61, 28.89, 24.94, 16.15

(9) Solubility: Easily soluble in water, soluble in DMSO and methanol, hardly soluble in acetone, ethyl acetate and chloroform.
(10) CAS assay: The iron binding ability of this compound was evaluated by the CAS assay described in Non-Patent Document 5 described above. The CAS assay showed 1.5 times the activity (ED ₅₀ 0.2 mM) of the control deferoxamine B (ED ₅₀ 0.3 mM).

Next, an example of a method for producing a siderophore according to the present invention will be described.
The compounds of the formulas (I) and (II) of the present invention can be produced by culturing a microorganism in a medium, producing and accumulating the compound in the culture, and collecting the compound from the culture.

(1) Microorganism As a microorganism that can be used in the production method of the present invention, a compound belonging to the genus Streptomyces and represented by the above formulas (I) and (II) or a salt thereof is produced. There is no particular limitation as long as it is a microorganism having ability.

  Here, “microorganisms having the ability to produce compounds represented by formulas (I) and (II) or salts thereof” typically means that the strain is 1 L containing 200 mL of marine broth medium. Incubate in an Erlenmeyer flask with baffle at 30 ° C for 5 days by rotating and shaking (100 rpm) to make an inoculum, and inoculate the inoculum with 1 L of medium for production of the compound (ASG medium) 10 Inoculate 10 mL each of this (total 10 L), and culture (10 x L, 30 minutes) 10 L of the culture solution obtained by rotating and shaking (100 rpm) for 7 to 14 days at 30 ° C. The bacterial cells and the supernatant are separated, the supernatant is treated with HP20 resin, the compound is adsorbed on the resin, the resin is washed with distilled water, and the compound is eluted with a mixed solvent of methanol-distilled water, When the eluted fraction is purified by high performance liquid chromatography, 1 mg or more per 10 L of culture solution is more preferable. Ku is 5 mg or more, the formula most preferably in an amount of more than 10 mg (I), it means a microorganism capable of producing compounds or salts thereof represented by (II).

  Examples of such microorganisms include Streptomyces sp. YM5-799 strain. These strains are the following non-patent documents from about 300 bacteria obtained by crushing algae and sea cucumber collected from the coastal area of Hokkaido in sterilized seawater, applying them to a marine broth agar medium, and culturing them for 7 days. 5. A siderophore production capacity was evaluated by the CAS (Chrome azurol S) assay described in 5, and confirmed to have the capacity.

  Streptomyces sp. YM5-799 is registered as an independent administrative agency, Product Evaluation Technology Foundation, Patent Microorganisms Deposit Center (2-5-8, Kazusa Kamashika, Kisarazu City, Chiba Prefecture) with a deposit number of NITE as of January 24, 2008. Deposited as AP-480.

The mycological properties of Streptomyces sp. YM5-799 are as follows.
a. Morphological and cultural properties 1) Presence / absence of spores: Yes 2) Shape of aerial hyphae With branching. Width 0.8-1.0mm.
3) Presence of motility: None 4) ISP medium No.2 Plate culture: Colony size Diameter 1-2 mm. Colony surface shape Striped pattern. Color tone Front (aerial mycelium) brown, back side (basic mycelia) brown. Brown water-soluble pigment production.
5) ISP medium No.3 Plate culture: Grows. Front: Gray, Back: Gray
6) ISP medium No.4 Plate culture: Grows. Front: Gray, Back: Gray
7) ISP medium No.5 Plate culture: Grows. Front: Gray, Back: Gray

b. Physiological and biochemical properties (+: positive,-: negative)
1) Liquefaction of gelatin:-
2) Starch hydrolysis: +
3) Nitric acid reduction reaction: +
4) Peptonization and coagulation of skim milk powder:-
5) Growth temperature range: 20-30 ° C. 37 ° C Does not grow.
6) Salt tolerance: 1 to 4% grows. 5% does not grow.
7) Availability of carbon sources
ISP medium No. 9 (negative control):-
Glucose (positive control): +
L-rhamnose:-
D-mannitol: +
D-fructose: +
L-arabinose ： −
Raffinose:-
Sucrose ： −
D-xylose:-
Inositol:-
8) Formation of melanin-like pigment
ISP medium No.6: +
ISP medium No.7:-
9) 16S rDNA sequence Shown in the sequence listing (SEQ ID NO: 1).

  Based on this sequence, a homology search (BLAST search) against the nucleotide sequence database of DDBJ (Japan DNA Data Bank) revealed that Streptomyces sp. AR17 (EF672649.1) had the highest homology (99%) However, since a completely identical sequence was not confirmed, it was a novel microorganism not reported so far. There is no report that Streptomyces sp. AR17 strain showing the highest homology produces siderophore.

  Furthermore, mutant strains derived from Streptomyces sp. YM5-799 and having the ability to produce the compounds represented by the above formulas (I) and (II) can also be used. The “mutant strain” here is obtained by mutagenesis treatment using any appropriate mutagen, and the term “mutagen” in the broad sense includes not only a drug having a mutagenic effect, for example. It should be understood to include treatments with mutagenic effects such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, N-methyl-N′-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines, but any other Effective mutagens can also be used.

(2) Microbial culture In the present invention, a normal microorganism culture method is used for the culture of the microorganism. As the medium, any of a synthetic medium or a natural medium can be used as long as it contains an assimilated carbon source, nitrogen source, inorganic substance, and necessary growth / production promoting substances as appropriate. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses and the like can be used alone or in combination. Furthermore, hydrocarbons, alcohols, organic acids, amino acids (such as tryptophan) and the like can be used as necessary. As the nitrogen source, use ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean flour, cottonseed meal, casamino acid, etc. alone or in combination. Can do. In addition, inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, and zinc sulfate can be added as necessary. . Furthermore, trace components that promote the growth of the microorganisms to be used and the production of the compounds of the present invention can be added appropriately, and those skilled in the art can select appropriate components as such components.

  As a culture method, liquid culture is suitable, but is not limited thereto. The culture temperature is suitably 25 to 37 ° C., the pH of the medium during the culture is preferably maintained at 7 to 9, and the culture is preferably performed by rotating or reciprocating shaking culture at a shaking speed of 30 to 120 rpm. When culturing is usually performed for 5 to 14 days in liquid culture, the target compound is produced and accumulated in the culture solution. Usually, the culture is stopped when the production amount in the culture reaches the maximum.

(3) Production of Siderophore Production of the siderophore of the present invention from the culture can be carried out according to a commonly used method for isolating and purifying microbial metabolic products from the culture. Here, “culture” means any of culture supernatant, cultured cells, or disrupted cells. For example, the culture is separated into a culture filtrate and cells by filtration or centrifugation, and the cells are extracted with an appropriate solvent. The culture filtrate can be extracted with ethyl acetate, chloroform or the like. A compound can also be extracted using a synthetic adsorbent or the like. Next, the bacterial cell extract, culture filtrate or culture filtrate extract is concentrated alone or in combination of two or more thereof, and purified by column chromatography, preparative thin layer chromatography, high performance liquid chromatography, etc. And the siderophore of the present invention can be obtained. By examining whether or not the obtained siderophore exhibits the properties described in the above-mentioned “properties of the compound of the formula (I) and the compound of the formula (II)” by an ordinary chemical method such as NMR analysis, It can be confirmed that it is a compound of the present invention.

  In addition, since the compound of formula (I) and the compound of formula (II) have different molecular weights and polarities, they can be separated by high performance liquid chromatography (HPLC) using a reverse phase column according to a conventional method.

  For example, the above extract containing the compound of formula (I) and the compound of formula (II) is purified by HPLC, fractionated and fractions containing siderophore are collected, and CAS assay is performed to measure siderophore activity. A fraction having a peak in the siderophore (I) and a fraction having a peak in the siderophore (II) may be collected. In order to obtain a compound with higher purity, the fraction collected once may be further purified by HPLC, and after fractionation, the fraction may be collected repeatedly.

(4) Use as an antibacterial agent or iron excretion agent (medicine) The compound of the present invention or a salt thereof is an antibacterial agent as described in Non-Patent Document 2 or an iron excretion drug as described in Non-Patent Document 3. Can be used as

Moreover, the compound of this invention can be made into the form of a salt according to a use. For example, hydrochloride, hydrobromide, sulfate, nitrate, acetate, methanesulfonate, toluenesulfonate, citrate, etc., and water solubility and fat solubility by changing the salt form Thus, the solubility in a solvent can be improved.
Furthermore, the compound of the present invention and a salt thereof can be used as a mixture.

(5) Use as a microbial preparation As described above, a microorganism that produces siderophore can be cultured in a large amount by a culture method, and a compound having siderophore activity can be purified from the culture. In the case of use, the microorganism itself or a carrier mixed with the microorganism can be applied and produced at the application site without performing high-level purification.

  For example, a microorganism product can be prepared by obtaining a culture obtained by culturing a microorganism that produces siderophore by the above-described culture method and mixing it with a carrier such as soil or humus. The culture and the carrier are mixed, kneaded or allowed to stand for several hours to several days, and the microorganisms producing the siderophore are adapted and cultured. The culture temperature is suitably 25 to 37 ° C. in order to activate the growth, and is kept moist so as not to be dried.

  By applying a microbial preparation containing siderophore-producing bacteria prepared as described above to agricultural land, it is possible to promote the iron uptake of plants growing there, and the microbial preparation is buried or laid on the bottom of the coastal water By doing so, the supply of bioavailable iron in the surrounding sea area is promoted, and the growth of seaweeds can be accelerated.

(6) Utilization as a marine environment modifier A compound of the present invention or a salt thereof, or a mixture of a crude product containing the compound of the present invention and a carrier containing iron is used as a marine environment modifier. be able to.

  The roughly purified product containing the compound of the present invention is obtained by culturing a microorganism strain producing the compound of the present invention in an appropriate culture medium, culture supernatant, culture filtrate, cultured cells, or disruption of cells. A culture product such as a product, or an extract obtained by extracting a fraction containing a siderophore from the culture solution by an appropriate method can be used alone or in combination of two or more. The compound of the present invention or a salt thereof, or a roughly purified product containing the compound of the present invention can be used as a dry powder, but since it can be mixed more homogeneously with the carrier described below, It is preferable to use it.

  As the carrier containing iron, iron powder, iron pieces, steel slag, humus and humus can be used alone or in combination of two or more.

  The mixture of the compound of the present invention or a salt thereof, or a roughly purified product containing the compound of the present invention and a carrier containing iron is filled in an appropriate container to be submerged or buried in a sea area where the marine environment is to be improved. By doing so, iron and siderophores are released from the mixture into the seawater, and the iron concentration in the surrounding sea area, particularly the bioavailable iron concentration, is increased, so that the growth of seaweeds can be accelerated. The container filled with the mixture is not particularly limited as long as it can hold the mixture and has a function of releasing the iron content and the compound of the present invention from the mixture into seawater. Cloth bags, net bags, steel rivets, steel or concrete boxes with small holes on the wall.

Example 1
Algae and sea cucumber collected from the coastal area of Hokkaido were crushed in sterile seawater, and the crushed material was applied to a marine broth agar medium and cultured for 7 days to obtain about 300 bacteria. With respect to these bacterial strains, the aforementioned CAS assay was performed to obtain one bacterial strain having the ability to produce siderophore.

DNA of the strain (YM5-799 strain) was extracted and analyzed for 16S rDNA base sequence. That is, genomic DNA of the strain was extracted using an instagene DNA purification matrix, and a 16S rDNA fragment was amplified by PCR. As a PCR primer, universal primer for 16S rDNA amplification, 27F (5'-GGC TAC CTT GTT ACG ACT T -3 ') (SEQ ID NO: 2) and 1492R (5'-AGA GTT TGA TCC TGG CTC AG -3') (SEQ ID NO: 3) was used. The reaction volume per sample was 25 μl, and LA Taq polymerase (TaKaRa Biochem) was used as the PCR enzyme. The composition of the reaction mixture was DNA 1 μl, 10 x LA buffer 2.5 μl, 25 mM MgCl ₂ 2.5 μl, dNTP Mixture 2.0 μl, 25 μM primer 0.25 μl, enzyme 0.1 μl, PCR amplification was performed at 95 ° C. for 1 min. A cycle of 58 ° C., 1 minute, 72 ° C., 1 minute 30 seconds was repeated 30 times. A 96-sample thermal cycler (Techne, Genius) was used for the PCR reaction. The amplified product was purified using Montage PCR96 (Millipore) and the nucleotide sequence was determined. The partial sequence of the 16S rDNA fragment was subjected to PCR using the universal primer for 16S rDNA, 341F (5'-CTC CTA CGG GAG GCA GCA G -3 ') (SEQ ID NO: 4), and the capillary sequencer PE3730 was used. The base sequence was determined. The obtained base sequence data was visually confirmed on the base sequence editing software SeqED. The obtained sequence was subjected to a BLAST search in the DDBJ database. As a result, the strain was estimated to be of the genus Streptomyces.

  YM5-799 strain was cultured as follows. This strain was cultured in a 1 L baffled Erlenmeyer flask containing 200 mL of marine broth medium at 30 ° C. for 5 days by rotating and shaking (100 rpm) to obtain an inoculum. Production medium (ASG medium: casamino acid 5 g / L, glycerin 3 mL / L, glycerophosphoric acid 0.1 g / L, sodium chloride 15.5 g / L, potassium chloride 0.8 g / L per 2 L baffled Erlenmeyer flask , Magnesium sulfate heptahydrate 12.4 g / L, Calcium chloride dihydrate 2.9 g / L, Ammonium chloride 1.0 g / L, HEPES sodium salt 2.6 g / L, Sodium bicarbonate 0.17 g / L, Iron chloride 0.01 μM (pH 6.8 before sterilization) 1 L was added, and 10 mL of the precultured inoculum was inoculated into each autoclave-sterilized flask (total of 10 flasks, 10 L), and cultured at 30 ° C for 10 days with shaking. (100 rpm) was performed. During the cultivation, pH was not particularly controlled.

  The siderophore was purified from the culture as follows. Purification was performed using the CAS assay as described above. The cells were separated from the supernatant by centrifugation (6000 × g, 4 ° C., 20 min). The pH of the separated supernatant was adjusted to pH 3 with hydrochloric acid, 2 L of aromatic synthetic adsorption resin HP20 resin (Mitsubishi Chemical Corporation) was added, and the mixture was allowed to stand at 4 ° C. for 24 hours. The resin was filtered using a glass filter (3G), and the separated resin was washed with 10 L of ultrapure water adjusted to pH 3 with hydrochloric acid. The washed HP20 resin was transferred to a beaker and immersed in 5 L of methanol for 24 hours. Next, methanol and the resin were separated by filtration using a glass filter, and the obtained methanol solution was concentrated under reduced pressure using an evaporator to obtain 12 g of a brown solid.

  Next, column chromatography using LH-20 resin was performed. A glass column with an inner diameter of 3 cm and a length of 80 cm is filled with 150 g of LH-20 resin (manufactured by Amersham Pharmacia) previously swollen in 50% methanol-ultra pure water. A solution of g dissolved in 5 ml of 50% methanol-ultra pure water was loaded and eluted with 50% methanol-ultra pure water. Fractionation was performed 20 mL each, and the fraction containing iron chelator was detected by CAS assay. Fractions containing an iron chelator were collected, concentrated under reduced pressure using an evaporator, and lyophilized to obtain 680 mg of a light brown solid.

  Next, purification was performed by high performance liquid chromatography (HPLC) using a reverse phase column. Cosmo seal 5C18-AR-II (inner diameter 20 mm, length 250 mm) was used for the column, 45% methanol-ultra pure water was used as the mobile phase, and fractionation was performed at a flow rate of 10 mL / min. Fractionation was performed every 2 minutes (20 mL), CAS assay was performed, fractions containing siderophore were collected, concentrated under reduced pressure with an evaporator, and lyophilized to obtain 130 mg of a pale yellow solid. Next, TSK GEL ODS 80Ts (inner diameter 7.8 mm, length 300 mm) was used for the column, and two kinds of solvents (solvent A: 0.1% trifluoroacetic acid-ultra pure water, solvent B: 0.1% trifluoroacetic acid- A gradient elution using acetonitrile was performed (the initial concentration of solvent B was 10%, and the concentration was increased to 40% after 30 minutes, flow rate 2 mL / min). A chart of ultraviolet absorption at a wavelength of 220 nm is illustrated in FIG. The fraction corresponding to each peak was separated and recovered, and the activity of the siderophore was measured by the CAS assay described in Non-Patent Document 5 above, and it was confirmed that peak A and peak B in the figure had the activity. . Purification was repeated under the same separation conditions, and the fractions of peak A and peak B were collected to obtain 10 mg and 20 mg, respectively, by dry weight.

The composition formula of each compound was determined by high-resolution FAB-MS, and the structure was further determined by detailed analysis of NMR data such as ¹ H NMR, ¹³ C NMR, HSQC, and HMBC. From the peak B, the compound of formula (II) was obtained.

  The two kinds of compounds obtained here exhibited the above-mentioned physicochemical properties (the properties of the compound of formula (I) and the compound of formula (II)).

(Example 2)
The marine green alga Chlorococcum littorale (Chlorococcum littorale) NBRC102761 was used to examine whether the compound of formula (I) and the compound of formula (II) have a growth promoting effect on the unicellular algae.

In general, marine phytoplankton is known to be difficult to proliferate at low iron concentration levels (several nM) in seawater, and it is known that when iron supply to cells is promoted, proliferation is also promoted. First, basic growth medium (A5 medium: sodium nitrate 1.5 g / L, magnesium sulfate heptahydrate 0.1 g / L, monopotassium phosphate 35 mg / L, dipotassium phosphate 45 mg / L, iron-EDTA 12 mg / L , Calcium chloride dihydrate 9 mg / L, boric acid 70 μg / L, manganese sulfate heptahydrate 150 μg / L, zinc sulfate heptahydrate 300 μg / L, copper sulfate pentahydrate 300 μg / L L, sodium molybdate 3μg / L, cobalt chloride 70μg / L, seawater 1L), medium lacking iron-EDTA from basic growth medium (however, the trace amount of iron derived from seawater used for medium preparation Present; hereinafter referred to as iron-deficient medium) and subcultured twice every 3 days to produce iron-deficient cells (hereinafter referred to as iron-deficient cells). Next, an iron-deficient medium, a medium obtained by adding 0.1 mM deferoxamine B to the iron-deficient medium, a medium obtained by adding 0.1 mM of the compound of formula (I) to the iron-deficient medium, and 0.1 mM of the compound of formula (II) to the iron-deficient medium. Each of the added media was prepared, inoculated with the iron-deficient cells, and statically cultured in an incubator at a temperature of 25 ° C. and a light intensity of 50 μmoL m ⁻² s ⁻¹ to examine proliferation.

  The results are shown in FIG. The vertical axis indicates the average value (n = 12) of the difference in chlorophyll fluorescence intensity at the start of culture and 44 hours after culture, and means the amount of microalgae grown during the period. Bars indicate standard error (n = 12). Chlorophyll fluorescence was measured using a fluorescence spectrophotometer to measure the intensity at a fluorescence wavelength of 645 nm when the excitation wavelength was 485 nm.

  In FIG. 2, A is an iron-deficient medium, B is an iron-deficient medium supplemented with deferoxamine B, and C is an iron-deficient medium supplemented with a compound of formula (I). In this case, D represents the amount of increase in the amount of chlorophyll fluorescence of chlorococcum litorale in the case of using a medium in which the compound of formula (II) is added to the iron-deficient medium.

  In the medium supplemented with deferoxamine B, the amount of fluorescence increase observed in the iron-deficient medium was lower. That is, when deferoxamine B was added, the growth of the microalgae was suppressed. From this result, it is considered that when deferoxamine B binds to a small amount of iron in the medium, the microalgae cannot take it, and the growth is suppressed. In contrast, the addition of the compound of formula (I) or the compound of formula (II) promoted growth more than in the case of an iron-deficient medium. That is, it is considered that the compound of the formula (I) and the compound of the formula (II) promoted the uptake of an extremely small amount of iron from seawater by chlorococcum literale. That is, it was confirmed that the compound according to the present invention has an effect of promoting the growth of algae.

(Example 3)
A carrier mixed with microorganisms that produce siderophores was buried along the coastline in the shoreline portion at a width of 2 m and a length of 1 to 2 m over a length of 20 m, and the effect on seaweed formation in the sea area was confirmed.

  Streptomyces sp. YM5-799 was cultured as follows. This strain was cultured in a 1 L baffled Erlenmeyer flask containing 200 mL of marine broth medium at 30 ° C. for 5 days by rotating and shaking (100 rpm) to obtain an inoculum. Production medium (ASG medium: casamino acid 5 g / L, glycerin 3 mL / L, glycerophosphoric acid 0.1 g / L, sodium chloride 15.5 g / L, potassium chloride 0.8 g / L per 2 L baffled Erlenmeyer flask , Magnesium sulfate heptahydrate 12.4 g / L, Calcium chloride dihydrate 2.9 g / L, Ammonium chloride 1.0 g / L, HEPES sodium salt 2.6 g / L, Sodium bicarbonate 0.17 g / L, Iron chloride 0.01 μM (pH 6.8 before sterilization) 1 L was added, and 10 mL of the precultured inoculum was inoculated into each autoclave sterilized flask (10 tubes, 10 L in total), and cultured at 30 ° C for 10 days with shaking. (100 rpm) was performed.

  10 L of the above culture solution was added per 100 kg of commercially available humus soil, aerated and stirred for 3 days, packed in a cloth bag, and embedded in the shoreline of the test sea area. In the examples, about 1 t of humus was used. As a comparative example, 10 L of marine broth medium per 100 kg of commercially available humus soil was added, and after aeration and stirring for 3 days, it was packed in a cloth bag and embedded at a point 100 m away from the embedding point of the example with a shoreline in the test sea area. In the comparative example, about 1 t of humus soil was used.

After embedding the materials of Examples and Comparative Examples, one year and two years later, the state of overgrowth of seaweeds and the concentration of iron in seawater in the vicinity of the respective embedding points were investigated. As for the state of overgrowth of seaweeds (for example, kombu, hirakotoji, pine tree), seaweeds proliferated in a 1 m square section on the sea bottom of the inspection point were collected and their mass and weight were measured. The iron concentration in seawater was analyzed by ICP emission spectrometry in accordance with JIS K0102. The survey results are shown in Table 1. Symbol seaweed overgrowth situation, less than 100 g / m ² "-", less than 100 g / m ² or more 1 kg / m @ 2 "+", and expressed as 1 kg / m ² or more "++".

  Before application, both the examples and comparative examples were sea areas where the iron concentration in seawater was low and seaweed was hardly grown, but after one year of application, the growth of seaweed was confirmed in the examples, and two years after application Remarkably thrived. The iron concentration in seawater was about 1.9 μg / L before application, but increased to 10 μg / L or more after application, and the iron concentration level was maintained even after 2 years of application. On the other hand, in the comparative example, growth of seaweed could not be confirmed 1 year after application, but some growth of seaweed was observed 2 years after application. This is because the sea area where seaweeds have prospered has expanded its effect area centering on the application point of the example, and the effect area of the example expanded to the application point of the comparative example at the point of 2 years after application. It is. The iron concentration in the seawater in the comparative example was 1.8 μg / L, which was about the same as that in the example before application, and was about 2.1 μg / L almost unchanged from that before application even after one year of application. Although it slightly increased two years after application, this is considered to be an expansion of the effect area of the examples as described above.

また、施用２年後の観察を行った後に実施例および比較例の埋設担体を採取し、滅菌海水中で分散させ、その上清をマリンブロス寒天培地に塗布し、７日間培養して得たコロニー約100個について、非特許文献５に記載のCAS（Chrome azurol S）アッセイによってシデロフォアの生産能力の有無を確認したところ、23個の分離株についてシデロフォア活性が観察された。シデロフォア活性が確認された23株についてＤＮＡを抽出し、16S rDNAの塩基配列を調べたところ、すべてがYM5-799株と同一であった。 As described above, a clear correlation was observed between the iron concentration in seawater and the state of overgrowth of seaweed. Iron ions are dissolved as ions in an acidic liquid, but form a hydroxide in a neutral or alkaline liquid to cause precipitation and cannot be dissolved in water. Compared with the comparative example, in the examples, the iron concentration in the seawater was kept high, and the overgrowth of seaweed was remarkable. This is because iron and / or iron ions in seawater were supplied from the carrier by applying a carrier containing humus containing iron and microorganisms producing siderophores to the sea area as in the examples. It is thought that the high bioavailability of iron content, which exists as a dissolved state in seawater due to chelation by siderophore, increased, and as a result, the growth of seaweed was promoted.

Further, after observing two years after application, the embedded carriers of Examples and Comparative Examples were collected, dispersed in sterilized seawater, and the supernatant was applied to a marine broth agar medium and cultured for 7 days. About 100 colonies were examined for the presence or absence of siderophore production ability by CAS (Chrome azurol S) assay described in Non-Patent Document 5, siderophore activity was observed for 23 isolates. When DNA was extracted from 23 strains in which siderophore activity was confirmed and the base sequence of 16S rDNA was examined, all were the same as those of the YM5-799 strain.

Example 4
A steel unit filled with a carrier containing an extract containing siderophore was submerged on the bottom of the sea at 80m offshore from the shoreline of the burnt sea area, and the effect on seaweed bed development in the sea area was confirmed.

  Siderophore was prepared from a culture of Streptomyces sp. YM5-799 strain. That is, this strain was cultured in a conical flask with 1 L baffle containing 200 mL of marine broth medium, and cultured at 30 ° C. for 5 days by rotary shaking (100 rpm) to obtain an inoculum. Production medium (ASG medium: casamino acid 5g / L, glycerin 3mL / L, glycerophosphoric acid 0.1g / L, sodium chloride 15.5g / L, potassium chloride 0.8g / L, magnesium sulfate per 1L Erlenmeyer flask with baffle・ Heptahydrate 12.4g / L, Calcium chloride ・ Dihydrate 2.9g / L, Ammonium chloride 1.0g / L, HEPES sodium salt 2.6g / L, Sodium bicarbonate 0.17g / L, Iron chloride 0.01μM （ Pre-sterilization (pH 6.8)) 1L was placed, and each flask (total 100, 100L) sterilized by autoclaving was inoculated with 10mL of the pre-cultured inoculum, followed by shaking culture (100rpm) for 10 days at 30 ° C. It was.

  The cells were separated from the supernatant by centrifugation (6000 × g, 4 ° C., 20 minutes). The pH of the separated supernatant was adjusted to pH 3 with hydrochloric acid, 20 L of aromatic synthetic adsorption resin HP20 resin (manufactured by Mitsubishi Chemical Corporation) was added, and the mixture was allowed to stand at 4 ° C. for 24 hours. The resin was filtered using a glass filter (3G), and the separated resin was washed with 50 L of ultrapure water adjusted to pH 3 with hydrochloric acid. The washed HP20 resin was transferred to a beaker and immersed in 50 L of methanol for 24 hours. Next, methanol and the resin were separated by filtration using a glass filter, and the resulting methanol solution was concentrated under reduced pressure using an evaporator to obtain about 100 g of a brown solid. It was confirmed by CAS assay that the solid had siderophore activity. In addition, by dissolving a part of the solid in pure water, high performance liquid chromatography (HPLC) using a reverse phase column (Cosmo Seal 5C18-AR-II (inner diameter 20 mm, length 250 mm)) When analyzed by passing 45% methanol-ultra pure water as the mobile phase at a flow rate of 10 mL / min, a peak was confirmed at the same residence time position as that of the compounds (I) and (II) of the present invention. Therefore, it contained the compound (I) and compound (II) of the present invention in the solid.

  100 g of the solid matter was dissolved in 50 L of pure water, sprayed on steel slag 3t, kneaded well, and then filled into a steel box (W2000 × D1000 × L500) with small holes on the sides. A steel unit was used. Moreover, what filled the steel box of the same type as an Example with steel slag 3t which is not spraying the solution containing siderophore was created as a comparative example.

  The steel unit of the example was sunk on the bottom of the water at a point 80 m offshore from the coastline of the sea where the steel was burnt. A survey was conducted about half a year after installation, and no seaweeds were observed in the vicinity of the steel unit of the comparative example, as in the case before the construction. It was thriving.

  In addition, when seawater was collected immediately above the steel units of the examples and comparative examples, and the iron concentration was measured, it was 2.3 μg / L in the comparative example, whereas it was markedly 15.8 μg / L in the example. High concentration. The iron concentration in the sea area 100 m away from the place where the steel unit was installed was 1.6 μg / L, indicating that iron was released from the steel unit of the example. Iron was also released from the steel unit of the comparative example, but it was rapidly oxidized in seawater and precipitated as iron hydroxide, which had no effect on the growth of seaweeds. On the other hand, the siderophore is released from the steel unit of the embodiment together with the iron, and they can be combined to stably exist in the seawater, that is, the available iron for seaweed increases. As a result, seaweeds are thought to have flourished. That is, according to the present invention, the burnt marine environment could be improved.

It is an example figure of the HPLC chart in the siderophore refinement | purification process of this invention. It is an example of the result of examining the effect of siderophore on the growth of marine green algae.

Claims (3)

The following chemical structural formula (I),

Or a salt thereof, or the following chemical structural formula (II),

A siderophore, which is a compound represented by the formula:   A marine environment modifier containing at least one kind of siderophore according to claim 1 and having a function of improving iron ion concentration in seawater. Streptomyces sp. YM5-799 strain (NITE AP-480) , which is a microorganism belonging to the genus Streptomyces that produces at least one kind of siderophore according to claim 1 , is added to the soil, A marine environment reforming method characterized by improving the iron ion concentration in seawater by introducing or burying added soil into the seabed.

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