JP5020839B2 - Novel microorganism and method for producing compound using novel microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism producing a new compound which has high binding ability to iron ions and helps stable existence of iron ions in an aqueous solution, and to provide a method for producing the microorganism. <P>SOLUTION: Disclosed are a microorganism strain YM5-799 (NITE AP-480) having production ability of compounds represented by formula (I) and (II), and a method for producing compounds represented by formula (I) and (II). <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a novel microorganism having the ability to produce a novel compound, which is one of compounds having a high binding ability to trivalent iron ions collectively called siderophore, and the novel compound using the microorganism It relates to a method of manufacturing.

  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 trivalent iron ions called siderophores and relatively low molecular organic compounds that have a high affinity in order to take up iron ions that are slightly present in the environment. It binds to trivalent iron ions present in a minute amount in the environment to form 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 is extremely low. Therefore, it is known that the growth of marine organisms such as phytoplankton, which mainly absorb carbon dioxide in the ocean, is suppressed (see Non-Patent Document 4), and the iron ion concentration in the surface area of the ocean is increased. If possible, the photosynthetic ability of phytoplankton in the marine environment can be enhanced, and as a result, the ability to fix the 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, few compounds have been known so far that can increase the iron ion concentration in the marine environment, and furthermore, no microorganism has been known so far that can produce such compounds. The present invention relates to a novel microorganism that produces a compound having a high binding ability with iron and capable of increasing the iron ion concentration in the marine environment, and the iron ion concentration in the marine environment using the microorganism. It is an object to provide a method for producing a compound that can be enhanced.

  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, and the compound obtained from the culture solution of the microorganism strain had high binding properties with iron ions, and trivalent iron The present inventors have found that the compound is a novel compound having the characteristic of assisting the stable presence of ions in an aqueous solution, and has led 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.

および下記の式（II）：

で表される化合物並びに／またはそれらの塩を生産する能力を有する微生物ストレプトマイセス・スピーシーズYM5-799株（NITE AP-480）に関する発明である。 The gist of the present invention is as follows.
(1) The following formula (I):

And the following formula (II):

And / or a microorganism Streptomyces sp. YM5-799 strain (NITE AP-480) having the ability to produce a salt thereof.

(2) Moreover, the present invention is characterized by culturing the microorganism in a medium, producing and accumulating the compound in the culture, and then collecting the compound from the culture. And a process for the preparation of compounds of formula (II) and / or their salts.

(3) Furthermore, the present invention separates the thus-prepared compounds of formula (I) and formula (II) and / or their salts using column chromatography and / or high performance liquid chromatography. The present invention relates to a method for producing a compound of formula (I) and / or a salt thereof, characterized by obtaining a compound of formula (I) and / or a salt thereof.

(4) Furthermore, the present invention provides the compounds of formula (I) and formula (II) and / or their salts produced as described above using column chromatography and / or high performance liquid chromatography. The present invention relates to a method for producing a compound of formula (II) and / or a salt thereof, characterized by separating to obtain a compound of formula (II) and / or a salt thereof.

  The present invention can provide a novel microorganism that produces a novel compound having a high binding ability with iron ions. In addition, a novel compound having a high ability to bind to iron ions can be produced using the novel microorganism. Furthermore, the produced novel compound is useful as a marine environment modifier such as an algae growth promoter that improves the iron ion concentration in the marine environment, an antibacterial agent, and an iron discharge agent.

  In addition, the microorganism itself can be used as a microorganism preparation.

The present invention is described in detail below.
(1) Microorganism of the present invention The microorganism of the present invention is Streptomyces sp. YM5-799 strain belonging to the genus Streptomyces. 2-5-8), deposited on January 24, 2008 as accession number NITE AP-480.

  The strain is composed of about 300 strains of bacteria obtained by crushing algae and sea cucumber collected from the coastal area of Hokkaido in sterile seawater, applying them to a marine broth agar medium, and culturing them for 7 days. The strain obtained by evaluating the production ability of the siderophore by the CAS (Chrome azurol S) assay described in the above and confirming that it has the production ability of the compounds of formula (I) and formula (II).

  The mycological properties of the Streptomyces species YM5-799 strain of the present invention 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) was the most popular. Although it showed high homology (99%), a completely identical sequence was not confirmed. Therefore, it was a novel microorganism not reported so far. There is no report that Streptomyces sp. AR17 strain showing the highest homology produces siderophore.

(2) Microbial culture For the culture of the microorganism according to the present invention, a normal microorganism culture method is used. 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 25-37 ° C., the pH of the medium during the culture is preferably maintained at 7-9, and the culture is preferably performed by rotating or reciprocating shaking culture at a shaking speed of 30-120 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) Method for Producing Compound The method for producing (isolating / purifying) the compound of the present invention from the culture can be carried out according to a method commonly used 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, the culture filtrate or the extract of the culture filtrate 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. The compound of the present invention can be obtained. By examining whether or not the obtained compound 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 technique 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) are different in molecular weight and polarity, 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 to collect fractions containing siderophore, and CAS assay is performed to measure siderophore activity. The fraction having a peak in the siderophore (I) and the 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 a pharmaceutical The compound of the present invention or a salt thereof can be used as an antibacterial agent as described in Non-Patent Document 2 or an iron excretion drug as described in Non-Patent Document 3.

  The compound of this invention can be made into the form of a salt according to a use. Examples of salt forms include hydrochloride, hydrobromide, sulfate, nitrate, acetate, methanesulfonate, toluenesulfonate, citrate, etc. Thus, solubility in solvents such as water solubility and fat solubility can be improved.

(5) Use as a microbial preparation As described above, a microorganism that produces siderophore can be cultured in large quantities by a culture method, and a compound having siderophore activity can be purified from the culture. Can be used at the application site by applying the microorganism itself or a carrier mixed with the microorganism without 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 it can be used as a solution because it can be mixed more homogeneously with the carrier described below. Is preferable.

  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.

  Hereinafter, the present invention will be described specifically by way of examples. However, the technical scope of the present invention is not limited by these examples.

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 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 PCR primers, universal primers 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. For 16S rDNA fragment partial sequence, PCR is performed using universal primer for 16 S rDNA, 341F (5'-CTC CTA CGG GAG GCA GCA G -3 '), and base sequence is determined using capillary sequencer PE3730 did. 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 completely new and named Streptomyces species YM5-799.

(Example 2)
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 (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 is based on the CAS (Chrome azurol S) assay [Schwyn, B. and Neilands, JB: Analytical Biochemistry, Vol. 160, 47-56 (1987)] used to evaluate the binding ability between siderophore and trivalent iron ions. went. 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 of the compound of formula (I) and 20 mg of the compound of formula (II) by dry weight, respectively.

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.

(Example 3)
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 an 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 the compound of formula (I). In this case, D represents the amount of increase in the chlorophyll fluorescence amount 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 uptake of a very 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 4
A carrier mixed with siderophore-producing microorganisms was buried along the coastline, and the effect on the seaweed bed construction 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 (total of 10 flasks, 10 L), 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, aerated and stirred for 3 days, 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. The seaweeds overgrowth was obtained by collecting seaweeds that grew in a 1m square section on the seabed at the inspection site and measuring their mass. The iron concentration in sea water was analyzed by ICP emission spectrometry in accordance with JIS K0102. The survey results are shown in Table 2. 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 1 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.

  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. When about 100 colonies were confirmed by the CAS (Chrome azurol S) assay described in Non-Patent Document 5, the presence or absence of siderophore production ability, 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.

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 (4)

Microbial Streptomyces sp. YM5-799 strain (NITE AP-480) having the ability to produce the following compound (I) and compound (II) and / or their salts
Compound (I)

Compound (II)

  A microorganism according to claim 1 is cultured in a medium, and after the compounds of formulas (I) and (II) and / or their salts defined in claim 1 are produced and accumulated in the culture, A method for producing a compound of formula (I) and (II) and / or a salt thereof, which comprises collecting a compound of formula (I) and (II) and / or a salt thereof.   The compounds of formula (I) and formula (II) and / or their salts produced by the production method according to claim 2 are separated using column chromatography and / or high performance liquid chromatography, and the compounds of formula (I) A process for producing a compound of formula (I), characterized in that a compound of formula (I) and / or a salt thereof is obtained.   The compounds of formula (I) and formula (II) and / or their salts produced by the production method according to claim 2 are separated using column chromatography and / or high performance liquid chromatography, and the compounds of formula (II) A process for producing a compound of formula (II), characterized in that a compound of formula (II) and / or a salt thereof is obtained.

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