JP2000247996A - New fo-7314 substance and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound inhibiting chitinase activity, having low toxicity and useful as a insecticide for controlling or exterminating insect pests. SOLUTION: This FO-7314 substance is represented by the formula. The compound is obtained by culturing a microorganism [preferably Clonostachys sp. FO-7314 (FERM P-17200) fungal strains] having ability to produce the compound of the formula in a culture medium, accumulating the compound of the formula in culture products and collecting the compound of the formula from the culture products. The compound of the formula has the following physicochemical properties. Appearance: white powder; melting point: 270 deg.C; molecular weight: 675.3214 (M+H, by high-resolution, high-speed shock mass spectrometry); molecular formula: C29H42N10O9; specific optical rotation [α]D25: +52.1 deg. (c=0.1 in an aqueous solution); ultraviolet absorption spectrum has maximum absorption at 200 nm (ε=31,400) (in an aqueous solution).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel FO-7314 which can be used as an insecticide for controlling or controlling pests.
It relates to substances and their production.

[0002]

2. Description of the Related Art It is certain that insecticides have greatly contributed to the increase and stable supply of food resources, but it is well known that pesticides have also caused significant problems such as residual toxicity and destruction of ecosystems. It is on the street. Therefore, the development of highly safe insecticides has been promoted, and today, those which have been sufficiently considered in terms of toxicity and the like have been used.

[0003]

However, there is still a strong need for a more safe, less toxic, highly selective and easily degradable insecticide for the purpose of pursuing more safety. Furthermore, in tropical and subtropical regions, several insect-borne infections account for a large proportion of the causes of death, and there is a strong need for better insecticides to control the vehicle, a sanitary pest. Requested. The present invention provides a FO-7314 substance which can be used as an insecticide which can sufficiently satisfy such a demand, and a method for producing the same.

[0004] In general, chitinase, a chitin-degrading enzyme, is known to be an essential enzyme in the growth and molting of arthropods such as insects, but is hardly used in mammals. Therefore, it is thought that chitinase inhibitors can be extremely low-toxic insecticides, but there is no report that they have been put to practical use to date.

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
Focusing on chitinase, a chitin-degrading enzyme, we continued to search for metabolites produced by microorganisms for inhibitors of this enzyme. As a result, FO-
It was found that a substance having chitinase inhibitory activity was produced in the culture of strain 7314. Next, as a result of separating and purifying a substance that inhibits chitinase activity from the culture, a substance having a chemical structure represented by the following [I] was found. 7314 substance.

The present invention has been completed on the basis of this finding, and has the following formula [I]

[0007]

Embedded image The present invention relates to a novel FO-7314 substance represented by

The present invention further relates to the following formula [I]

[0009]

Embedded image A microorganism having an ability to produce a FO-7314 substance belonging to the filamentous fungus represented by
O-7314 substance is accumulated and FO-7 is removed from the microorganism.
The present invention relates to a method for producing a new FO-7314 substance for collecting 314 substances.

According to a preferred embodiment of the present invention, the microorganism belonging to the filamentous fungus and capable of producing the FO-7314 substance is Clonostakis sp .
achys sp. ) FO-73 which is FO-7314
This is a method for producing 14 substances. The present invention further relates to a microorganism belonging to a filamentous fungus and having an ability to produce a FO-7314 substance. The microorganism is, according to a preferred embodiment of the present invention, Clonostakis sp .
stachys sp. ) FO-7314. The present invention further relates to Clonostakis sp.
chys sp. ) FO-7314 (FERM P-1)
7200).

The FO- of the present invention represented by the above formula [I]
Microorganisms capable of producing 7314 substances (hereinafter referred to as
“FO-7314 substance-producing bacterium” belongs to the genus Clonostakis . For example, the present inventors have newly isolated Clonostakis sp. FO- from soil collected in Oshima-cho, Tokyo. 7
The 314 strain is an example of a strain most effectively used in the present invention. Hon Chronostakis SP ( Clo
nostachys sp. ) The bacteriological properties of the FO-7314 strain are as follows.

1. Morphological Properties Clonostakis sp.
s sp. ) FO-7314 strain grows relatively well on potato / glucose agar medium, corn / meal agar medium, Tzapek agar medium, Miura agar medium, wort agar medium, etc. Glucose agar, Miura agar, wort agar, corn
Good on meal agar medium and slightly inhibitory on Tzapek agar medium.

When a colony grown on a corn / meal agar medium is observed with a microscope, the hyphae are transparent and have partition walls, and the conidium (45 to 100 μm × 2.5 to 3.5 μm)
m) grows directly from the basal hypha and branches off two to several times.
The branched conidiophores are brush-like (penicillat)
e), and a conidium-forming cell is formed at the tip.
Conidium-forming cells (10-14 μm × 1.5-3.0 μm)
m) is colorless, endogenous sprouting type, phyaro type, and is a club-like shape with a slightly swollen base. Most are formed in groups of two to four (at the tips of the conidiophores). Conidia (5.
(7.5 to 7.5 μm × 1.5 to 2.0 μm) is colorless, elliptical to wide elliptical, viscous, and linked in a rice ear shape (10
0 μm or more).

2. Cultural properties on various agar media The results of macroscopic observation of the strain when cultured on various agar media at 25 ° C. for 14 days are shown in Table 1 below.

[0015]

[Table 1]

3. Physiological properties (1) Optimal growth conditions The optimal growth conditions for this strain are pH 5-7, temperature 13-28.
° C. (2) Growth range The growth range of this strain is pH 3-10, temperature 9-37 ° C. (3) Aerobic and anaerobic distinction

The strain FO-731 described in detail above.
Based on the morphological characteristics, culture characteristics and physiological characteristics of the four strains, an attempt was made to compare the strain with known bacterial strains. As a result, this strain was identified as a strain belonging to the genus Clonostakis, and Clonostakis sp. FO- 7314
It was named. In addition, this strain is Clonostakis sp. FO-7314.
As a FERM P-17200 on February 4, 1999 at the Institute of Biotechnology and Industrial Technology, located at 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture.

[0018] The FO-7314 substance producing microorganism used in the present invention, said Kuronosutakisu sp (Cl
onostachys sp. ) The FO-7314 strain is mentioned as the most preferred example, but as a general property of the fungus, the mycological properties are extremely susceptible to variation, are not constant, and are naturally or normally carried out by ultraviolet irradiation or mutated derivatives, For example, N-methyl-N'-nitro-N-
It is a well-known fact that mutations are made by artificial mutation means using nitrosoguanidine, 2-aminopurine, etc., and such artificial mutants, as well as cell fusions and genetically engineered strains, belong to filamentous fungi, The FO-7314 substance of the present invention represented by the formula [I] (hereinafter referred to as "FO-7314 unless otherwise specified")
All strains capable of producing -7314 substance ") can be used in the present invention.

In practicing the present invention, first, a FO-7314 substance-producing bacterium belonging to a filamentous fungus is cultured in a medium.
It is performed by separating and purifying from the culture. As a nutrient source suitable for the above FO-7314 substance production,
A natural medium or a synthetic medium containing a carbon source suitable for cultivation of ordinary filamentous fungi, an assimilable nitrogen source and inorganic substances, and if necessary, other nutrients can be used. As the nutrient source used for the medium, any type can be used as long as it can be used as a nutrient source for filamentous fungi.

As a nutrient source, for example, commercially available peptone,
Meat extract, corn steep liquor, cottonseed flour, peanut flour, soy flour, yeast extract, NZ-amine, casein hydrate, nitrogen sources such as sodium nitrate, ammonium nitrate, ammonium sulfate, glycerin, starch, glucose, galactose , Mannose and other carbohydrates or fats and other carbon sources, and salts, phosphates, calcium carbonate, magnesium sulfate and other inorganic salts can be used alone or in combination.

In addition, if necessary, trace amounts of metal salts such as magnesium salts, calcium salts, and sodium salts, and animal, plant, and mineral oils as antifoaming agents can be added. Any of these may be used as long as they are required by the FO-7314 substance-producing bacterium to contribute to the production of the FO-7314 substance of the present invention, and any known filamentous fungal culture material can be used. Liquid culture is preferable for mass culture of the FO-7314 substance of the present invention.
It can be applied to the extent that O-7314 can be produced. Culture conditions can be appropriately selected according to the properties of the FO-7314 substance producing bacterium to be used.

[0022] The FO-7314 substance of the present invention is contained in the culture supernatant and the cultured cells, and contains methanol,
It can be extracted with a water-miscible organic solvent such as acetone. In addition to the above-described extraction methods, known methods used for collecting water-soluble substances, such as ion-exchange chromatography, adsorption chromatography, distribution chromatography, gel filtration chromatography, and high-performance liquid chromatography, may be used. By combining or repeating, the FO of the present invention can be purified from the culture supernatant and the cultured cells.
-7314 material can be collected.

The physicochemical properties of the FO-7314 substance of the present invention are as follows. (1) Property: white powder (2) Melting point: 270 ° C. (decomposition) (3) Molecular weight: 675.3214 (M + H, by high-resolution high-speed atom bombardment mass spectrometry) (4) Molecular formula: C ₂₉ H ₄₂ N ₁₀ O ₉ (5) Specific rotation: [α] _D ²⁵ = + 52.1 ° (c = 0.
1, aqueous solution)

(6) Ultraviolet absorption spectrum (aqueous solution):
As shown in FIG. 1, it has a maximum absorption at 200 nm (ε = 31,400). (7) Infrared absorption spectrum (KBr tablet): 3310, 3220, 1710, 1650, 1 as shown in FIG.
575, 1530, 1435, 1390, 1245cm
^It has a maximum absorption around ^-1 .

(8) Proton nuclear magnetic resonance spectrum: 1
% Chemical shift (ppm) and spin coupling constant (Hz) in heavy dimethyl sulfoxide containing trifluoroacetic acid are shown in Table 2.
As shown in FIG. (9) ¹³ C nuclear magnetic resonance spectrum: chemical shift in heavy dimethyl sulfoxide containing 1% trifluoroacetic acid (pp
m) is as shown in Table 2. (10) Solubility in solvent: easily soluble in acidic water. Soluble in water and dimethyl sulfoxide, poorly soluble in methanol and ethyl acetate.

[0026]

[Table 2]

As described in detail above, the FO- of the present invention
As a result of detailed examination of various physicochemical properties and spectrum data of the 7314 substance, it was determined that the FO-7314 substance of the present invention had the following chemical structure.

[0028]

Embedded image

As mentioned above, the FO-7314 of the present invention
Although various physicochemical properties of the substance have been described in detail, no compound corresponding to such properties has been reported so far, and therefore, the FO-7314 substance according to the present invention was determined to be a novel substance.

Next, the chitinase inhibitory activity of the FO-7314 substance of the present invention will be described in detail. 1 fly pupa
After homogenization in a 00 mM sodium phosphate solution (pH 7.0), the mixture was centrifuged at 100,000 × g for 60 minutes to collect the supernatant, which was used as a crude chitinase enzyme in the following.

After the sample solution was added to the 96-well microplate, the solvent was distilled off under reduced pressure. To this, 20 μl of a 100 mM sodium phosphate solution (pH 7.0) was added, and after dissolving the sample, 30 μl of chitinase crude enzyme was added.
Preincubation was performed at 5 ° C. for 5 minutes. Then, 50 μl of 0.8 mM 4-methylumbelliferyl triacetylchitotriose (manufactured by Sigma) was added thereto,
37 with a fluoroplate reader (Dainippon Pharmaceutical)
The mixture was incubated at 10 ° C. for 10 minutes, and umbelliferone released by the reaction was excited at 360 nm.
It was measured every 90 seconds at a fluorescence intensity of 60 nm.

As a result of quantifying the slope of the increase in the fluorescence intensity as the chitinase enzyme activity, the FO-7314 substance of the present invention inhibited the chitinase activity by 50% at a concentration of 0.15 μM. The FO of the present invention thus inhibiting chitinase
It is expected that the -7314 substance can be used as a composition for protecting or controlling pests.

Next, the antibacterial activity of the FO-7314 substance of the present invention will be described in detail below. 10 μl of a 1 mg / ml aqueous solution of the FO-7314 substance of the present invention was immersed in a filter paper disc (Advantech, 6 mm in diameter),
After air-drying for a certain period of time to remove the solvent, the resultant was stuck on a bacteria-containing agar plate, cultured at 27 ° C. or 37 ° C. for 24 hours, and the diameter of the growth inhibition circle formed around the filter paper disk is shown in Table 3. . In Table 3, *: cultivation was performed at 27 ° C., and the others were cultivated at 37 ° C.

[0034]

[Table 3]

[0035]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited thereto. Clonostachys sp. FO-731 cultured on an agar slant medium
From 4 strains (FERM P-17200), glucose 2.0%, polypeptone (Nippon Pharmaceutical) 0.5%, agar 0.1%, yeast extract (Oriental Yeast Co., Ltd.)
0.2%, magnesium sulfate heptahydrate 0.05%, potassium dihydrogen phosphate 0.1% liquid medium (pH 6.
One platinum loop was inoculated into one 500 ml Erlenmeyer flask into which 100 ml of each of (0) was dispensed, and cultured with shaking at 27 ° C. for 4 days.

Using this as a seed culture, maltose 5.0
%, Dried yeast 3.0%, potassium bromide 1.0%, magnesium sulfate heptahydrate 0.05%, and potassium dihydrogen phosphate 0.05% in a liquid medium (pH 6.0) of 100 m.
1m into 80 500ml Erlenmeyer flasks dispensed by l
Each l was inoculated and cultured with shaking at 27 ° C. for 140 hours. The culture was centrifuged, and the obtained cells were extracted with 5 liters of methanol, concentrated under reduced pressure, and the methanol was distilled off. This was adsorbed on an NH ₄ ⁺ type cation exchange resin Diaion SK-1B column (manufactured by Mitsubishi Chemical Corporation), washed with purified water, and eluted with 0.5 to 2.0 N aqueous ammonia.

Among them, chitinase inhibitory activity was observed.
Neutralize fractions and use adsorbent Diaion HP20 column
(Manufactured by Mitsubishi Chemical Corporation) and washed with purified water for 10-8
Elution was performed with 0% methanol water. Meta of this active fraction solution
After the methanol was distilled off under reduced pressure, NH _Four ⁺Type SP Sephadex
Adsorbed on a Cousin C-25 column (Pharmacia)
And eluted with 0.1 M ammonium acetate. This active image
Adsorbed to an ODS column (manufactured by Senshu Chemical Co., Ltd.)
After washing with purified water, elution was carried out with 40% methanol water.

Next, the solvent of the active fraction was distilled off under reduced pressure, dissolved in a small amount of purified water, applied to a Sephadex LH-20 column (manufactured by Pharmacia), and eluted with 65% aqueous methanol. After evaporating the solvent of this active fraction under reduced pressure, the residue was dissolved in a small amount of purified water, applied to a Bio-Gel P-2 column (manufactured by Bio-Rad), and eluted with 0.05% aqueous acetic acid. The active fraction was evaporated under reduced pressure to remove the solvent, and FO-731 as a white powder was removed.
25.0 mg of four substances were obtained.

[0039]

As described above, the novel F of the present invention is used.
O-7314 substance is expected to be a satisfactory drug as an insecticide for protecting or controlling pests because it inhibits chitinase activity.

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum (aqueous solution, 40 μg / ml) of a FO-7314 substance of the present invention.

FIG. 2 shows an infrared absorption spectrum (KBr method) of the FO-7314 substance of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) C12R 1: 645) (C12P 21/04 C12R 1: 645) (72) Inventor Ruro Masuma Port of Tokyo 5-9-1, Shirokane-ku, Tokyo, Japan Kitasato Research Institute (72) Inventor Jou Iwai 5-9-1, Shirokane, Minato-ku, Tokyo F-term (reference) 4B064 AG21 AG37 BA07 BC05 BE01 BE19 BG01 BH01 BH02 BH04 BH05 BH06 CA05 CE08 CE10 CE11 DA12 4B065 AA58X AC12 AC14 BD16 CA24 CA48 4H011 AC01 BB09 BB21 DG05 4H045 AA10 AA20 BA13 BA35 CA15 DA55 EA06 FA73 GA01 GA22 GA23 GA25 HA02 HA31 HA31

Claims (6)

[Claims] [Claim 1] The following formula [I] A novel FO-7314 substance which is a compound represented by the formula: 2. The following formula [I] belonging to a filamentous fungus: A novel microorganism characterized in that a microorganism capable of producing the FO-7314 substance represented by the formula is cultured in a medium, the FO-7314 substance is accumulated in the culture, and the FO-7314 substance is collected from the culture. Method for producing FO-7314 substance. 3. A microorganism belonging to a filamentous fungus and having an ability to produce a FO-7314 substance, is Clonostasis sp. FO-731.
3. The method according to claim 2, wherein the number is 4. 4. A microorganism belonging to a filamentous fungus and capable of producing a FO-7314 substance. 5. The microorganism according to claim 4, wherein the microorganism is Clonostchys sp. 6. Clonostakis sp ( Clono)
stachys sp. ) FO-7314 (FERM
P-17200) strain.