JPS586473B2 - Method for producing 3-methoxytropolone - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 3-methoxytropolone by fermentation.

More specifically, it is characterized by culturing 3-methoxytropolone-producing bacteria belonging to the genus Strebto Verticillium in a nutrient medium, accumulating 3-methoxytropolone in the culture, and collecting 3-methoxytropolone from the culture. The present invention relates to a method for producing 3-methoxytropolone using a fermentation method.

3-Methoxytropolone is a substance useful as an antibiotic.

It is known that 3-methoxytropolone, 2-hydroxy-3-methoxy-2,4,6-cyclohexatrienone, has the following structural formula and can be produced by conventional organic chemical synthesis methods (Y. Kitah et al., Sci,
Repts. Tohoku University Firstse
r. 39, 265-274 (1956), Chemic
alAbstract 51, 128746 (1957
)).

Structural formula of 3-methoxytropolone.

However, there is no knowledge that 3-methoxytropolone can be obtained by fermentation.

The present inventors obtained a large number of microorganisms from the natural world and three
-The productivity of methoxytropolone was studied.

As a result, when a strain (referred to as strain MK-100) isolated from the soil of a field outside Kanagawa Prefecture's Kanadeno City was cultured in a medium,
It was discovered that 3-methoxytropolone was produced in the culture.

The method for producing 3-methoxytropolone will be described in detail below.

The preferred strain used in the present invention is MK-100, and its mycological properties are as follows.

■ Morphological characteristics This strain shows good growth on starch inorganic salt agar, oatmeal agar, and yeast malt agar, and the color of the basal hyphae is light brown to dark brown.

On the other hand, the attachment of aerial hyphae is generally not good, but the color is white to grayish white.

When aerial hyphae are observed under an optical microscope, they are found to have axle-like branches, with ten or more spores attached to their tips in most cases, and the sporophyte shape is straight or curved.

The shape of the spore is cylindrical and its size is 0.5 x 1μ, and the surface of the spore is smooth when observed with an electron microscope.
It has no flagella.

Also, no sporangial morphology was observed.

■ Growth status on various media 1. Growth on Czapek agar medium: Poor Substrate mycelium color: Brick red (6 ng) to deep brown mahogany (6 pl) Poor aerial mycelia growth, white (a) Soluble pigment: None 2. Glucose-asparagine agar medium Growth: Ordinary basal mycelium Color: Mustard (21e) to camel (3i)
e) Aerial hyphal bicoccysis poor Soluble pigment: light purple 3. Glycerol-asparagine agar growth:
Color of normal basal hyphae: Gold (21c) ~ Amber (31c)
) Poor aerial mycelial growth, white (a) to eggshell (
2ca) Soluble pigment: None 4. Starch inorganic salt agar medium Growth: Good Substrate mycelium color: Mustard gold (2ne) to amber (3nc) Aerial mycelium biphytic normal, white (a) to light gray (
c) Soluble pigment: light purple5. Tyrosine agar medium Growth: Ordinary basal mycelial color: Mustard (21e) to amber (3n)
c) Aerial mycelium, poor soluble pigment: None 6. Nutrient agar medium Growth: Ordinary basal mycelial color: Mustard (21e) ~ Dull gold (
2ng) Aerial hyphae: Non-settled Soluble pigment: None 7. Yeast malt agar medium Growth: Good Substrate color: Bamboo (2gc) to cinnamon (31e)
) Aerial mycelium: Poor settlement, white (a) Soluble pigment: None 8. Oatmeal agar medium Growth: Good Substrate color: Light wheat (2ea) ~ camel (
3ie) Aerial mycelium: Poor settlement, white (a) to oyster white (b) Soluble pigment: Light purple brown9. Bennett agar medium Growth: Normal color of basal hyphae: Mustard gold (2ne) Poor aerial mycelium, white (a) Soluble pigment: Light brown 10. Growth on Emerson agar medium: Normal color of basal hyphae: Colorless to bright gold (2 nc) Aerial hyphae inability to attach to hyphae Soluble pigment: Light brown 11. Pebton yeast on iron agar medium Growth: Ordinary Substrate Color of hyphae: Sepia brown (3pn) to chocolate brown (4pn) Aerial hyphae: Non-settleable Soluble pigment: Dark brown Purple or higher after 2 weeks at 30°C This is an observation result.

Also, the color display is based on Color Harmony Man
ual (Container Corporation
of America).

■ Physiological properties 1. Assimilation of carbon sources Assimilates D-glucose and i-inositol.

Does not utilize D-raffinose, D-arapinose, D-mannitol, L-rhamnose, and D-xylose.

It also utilizes D-fructose and sutucarose very poorly.

2. Liquefaction effect of gelatin: Yes 3. Starch hydrolysis effect: Yes 4. Pebtonization of skim milk: Very weak.

5. Coagulation of skim milk: Yes6. Production of melanin-like pigment: Yes (Tyrosine agar medium and Pebton yeast iron agar medium) 7. Optimal growth temperature: 27°C to 37°C or higher.
These are the observation results after 2 weeks at 30°C.

However, the optimum growth temperature for No. 7 was obtained after 5 days, and the results for No. 4 and No. 5 on skim milk were obtained after three weeks.

■ Identification As seen above, this strain MK-100 forms true aerial hyphae on an agar medium, and its branching method is axle branching, which is a typical strain belonging to the genus Strebto verticillium.

Thus, the present MK-100 strain was designated as Streptoverticillium sp.MK-100.

Regarding the classification of the genus Streptoverticillium, see R.
THE GENUS STREP by Locci et al.
TOVERTICILLIUM (Giorn.Micr
obiol. 17 (1969)) and Bergy'
s Manual of Determinative
Bacteriology 8th Ed. p. 82
However, according to the latter classification, based on the color of the basal hyphae and aerial hyphae, the closely related strains are Strepto verticillium viverticillatum, Strept verticillium salmonis, and Strepto verticillium.
Examples include strains of the Lilacinum series.

However, when these strains and the MK-100 strain are cultured simultaneously and compared, the following is found.

Strebto verticillium (hereinafter abbreviated as Stv.).

) There are six known strains of the Viverticillatum series, but Stv. In Viverticillatum, the color of basal mycelia on glucose-asparagine agar medium and tyrosine agar medium is burnt red (6 pg), respectively.
MK-1 because it exhibits rose wine (8nc)
Unlike the 00 strain, Stv. Aureoversales and Stv. Pentateium differs from the MK-100 strain in that the basal hyphae exhibit a reddish color on starch inorganic salt agar medium and glucose-asparagine agar medium.

Also Stv. Roseoverticillatum also Stv. In addition to the same reasons as in the case of Viverticillatum, M
It is a different strain from the K-100 strain.

Furthermore, Stv. Roseoverticillatum, subspecies albosborum is known for its ability to produce red to purplish basal mycelium on glycerol-asparagine agar and starch inorganic salt agar, and Stv. Rubuloverticillatum differs from strain MK-100 in that it produces soluble pigments on nutrient agar, and also produces colorless to beige (3ge) basal hyphae on peptone yeast iron agar. This is also different from the MK-100 strain.

Next, Stv. As a strain of the Salmonis series, S.
tv. There is one strain of Salmonicida, but the color of the basal hyphae on yeast malt agar medium is prickly (6 ng).
It differs from the MK-100 strain in that it produces a soluble pigment on a nutrient agar medium.

Also, Stv. As a strain of Lilacinum series,
Stv. Lilacinum and Stv. There are two strains of Cashmirens, Stv. In Lilacinum, the color of the basal hyphae on glycerol-asparagine agar medium is prickly (6n).
g), which is significantly different from the MK-100 strain, and i
- Also in terms of assimilating inositol, Stv. 'J Lacinum assimilates only poorly, whereas MK-100 assimilates as well as D-glucose.
MK-100 strain is Stv. Different from lilacinum.

Also Stv. The color of aerial mycelia on yeast malt agar medium is rose mist (7ec), and MK-100 produces soluble pigments on this medium and does not assimilate D-glucose and i-inositol at all. Different from stocks.

As described above, the present strain MK-100 was not found to have any characteristics consistent with these 3-series strains.

Therefore, the MK-100 strain was considered a new species, and was named after the place where it was collected.
onense) MK-100.

The strain has been deposited with the Microtech Institute, and its deposit number is No. 4025.

As seen in the case of other actinomycetes, this strain can be mutated by artificial mutagenic means using, for example, ultraviolet rays, Co60 irradiation, X-rays, various mutagenic drugs, etc., and such mutant strains Any strain capable of producing 3-methoxytropolone can be used in the present invention.

In the cultivation of the present invention, ordinary methods for culturing actinomycetes are generally used.

Various sources of nutrients can be used for culture.

As the carbon source, glucose, starch, glycerin, mannose, fructose, inositol, sucrose, molasses, etc. are used alone or in combination.

Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used.

Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, and sodium nitrate; natural nitrogen sources include pebtone, meat extract, yeast extract, dried yeast, corn stew liquor, soy flour, and casamino acids. , soluble vegetable protein, etc. may be used alone or in combination.

In addition to adding inorganic salts such as table salt, potassium chloride, calcium carbonate, and phosphates as necessary,
- Organic or inorganic substances that promote the production of methoxytropolone can be appropriately added.

As a culture method, a liquid culture method, especially a deep agitation culture method, is suitable.

It is desirable that the culture be carried out at a culture temperature of 20 to 40°C and a pH of around neutrality.

When culture is carried out in liquid culture for usually 1 to 5 days, 3-methoxytropolone is produced and accumulated in the culture solution.

Stop the culture when the production amount in the culture medium reaches the maximum,
The target product is purified and isolated from the culture solution obtained by separating the bacterial cells.

For purification and isolation of 3-methoxytropolone from culture filtrate,
Separation and purification methods commonly used to isolate microbial metabolic products from their culture fluids are utilized.

Since 3-methoxytroborone is soluble in organic solvents such as alcohols and ethyl acetate, it can be collected by methods that take advantage of its properties.

In other words, methods for purifying 3-methoxytropolone include extraction using water-insoluble organic solvents such as butanol and ethyl acetate, or extraction using Amberlite Adsorption/desorption method using resins such as silica gel, Sephadex LH-20, Sephadex G-15
(manufactured by Pharmacia Fine Chemicals, Inc., USA) and the like can be mentioned as a purification method in which methods such as column chromatography are appropriately combined.

For example, culture solution f is adjusted to pH around 7 and passed through a column filled with Diaion UP-10.

Since 3-methoxytropolone is adsorbed on Diaion HP-10, it is washed with water and then eluted with an 80% methanol aqueous solution.

After concentrating the eluted 3-methoxytropolone fraction and removing methanol, the concentrated solution was adjusted to pH 2 with mineral acid,
Extract with ethyl acetate.

The ethyl acetate layer is concentrated to dryness to obtain a crude powder of 3-methoxytropolone.

This crude powder was placed on a silica gel column, eluted with ethyl acetate, and the eluted 3-methoxytroborone fraction was concentrated under reduced pressure and left in a cold room (0°C).
Pale yellow crystals of methoxytropolone can be obtained.

The physicochemical properties of the obtained 3-methoxytropolone were as follows, and were in good agreement with the standard product.

(2) Pale yellow needle-shaped crystals with a melting point of 113-114°C.

■ Methanol, ethanol, acetone, ethyl acetate,
Soluble in chloroform, carbon tetrachloride, and water.

■ Color reaction The ferric chloride reaction is positive and the ninhydrin reaction is negative.

■ The ultraviolet absorption curve is as shown in Figure 1.

The methanol solution shows extremely thick absorption at 246 nm and 325 nm, and a shoulder at 269 nm.

A 0.1N hydrochloric acid methanol solution (3-methoxytropolone dissolved in methanol and made acidic with 0.1N hydrochloric acid) shows absorption maxima at 248 nm and 324 nm.

A 0.1N caustic potash methanol solution (3-methoxytropolone dissolved in methanol and made alkaline with 0.1N caustic potash) shows an absorption maximum at 249 nm and 338 nm, and a shoulder at 269 nm.

■ Molecular weight according to high-resolution mass spectrum is 152.0
470 (theoretical amount: 152.0473).

Molecular formula: C8H8O3 ■ Infrared spectrum is second
As shown in the figure.

Absorption is 3260, 1598, 1553, 1428, 1260, 1080, 845 and 792c
It is located at m-1.

■ Nuclear magnetic resonance spectrum (solvent: deuterochloroform) Singlet of 3 protons at δ4.03 Multiplet of 4 protons at δ7.8-6.7 δ8.
Broad singlet of 1 in 50 protons ■ R of paver chromatography using various developing agents
The f value is as shown in Table 1.

Table 1: Rising Paver Chromatography Next, Table 2 shows the antibacterial spectrum of 3-methoxytropolone against various microorganisms at pH 7.0.

Table 2 Acute toxicity value in mice of minimum inhibitory concentration of 3-methoxytropolone determined by agar dilution method (
LD50) is 137.5 mg/kg (i.v.).

Examples of the present invention will be shown below.

Example 1 (A): Using Strebto verticillium Hadanonense MK-100 (FEI Deposit Accession No. 4025) as the inoculum, 1 g/dl of glucose/1 g/dl of soluble starch, and 1 g/dl of yeast as the first type medium. A medium containing 0.1 g/dl of extract, 0.5 g/dl of peptone, and 0.1 g/dl of CaCO3 (pH 7.2 before sterilization) was used.

Inoculate 1 platinum loopful of inoculum into 30 ml of the above first type medium in a 250 ml Erlen-Meyer flask, and incubate at 30°C for 48 hours.
Cultured with shaking for hours.

30 ml of the first type culture is inoculated into 300 ml of the second type medium contained in a 2-liter Erlen-Meyer flask.

As the second type medium, a medium having the same composition as the first type medium is used.

The second type culture is also cultured with shaking at 30°C for 48 hours.

1.54 ml of this second type culture solution is inoculated into 15 liters of the main medium in a 30 liter stainless steel jar fermenter.

The medium composition of this medium is glucose 2g/dl, P. biralex (animal oil: manufactured by Adeka Fine Chemicals) 2g/dl, soybean meal 2g/dl, and meat extract 0.5g.
/dl, K2HPO40.05g/d4MgSO4・7
H20 0.05g/dl, KCl0.03g/dl,
CaCO30.3g/dl (pH 8.0 before sterilization).

This jar fermenter culture was carried out at 30°C for 60 hours using an aeration agitation method (rotation speed 300 rpm, aeration rate 15 l/mi).
n).

As a result, about 100 γ/ml of 3-methoxytropolone accumulates in the culture solution.

(B): The culture solution obtained in (A) was adjusted to pH 7.0 with hydrochloric acid, and 800 g of P super-aiding agent Radiolite No. 600
(manufactured by Showa Kagaku Kogyo Co., Ltd.), filtered under reduced pressure, and 15L
Obtain the filtrate.

This filtrate is passed through a column filled with 1 liter of Diaion HP-10 (manufactured by Mitsubishi Chemical Corporation).

3-Methoxytropolone is adsorbed on this resin, and after washing with water, it is eluted with an 8.0% aqueous methanol solution.

In this way 3 l of 3-methoxytropolone active fraction was obtained, containing 3-methoxytropolone at a rate of 300 gamma/ml.

(C): The active section obtained in (B) was heated for 300 m under reduced pressure.
The pH of the concentrated solution was adjusted to 2.0 with hydrochloric acid, and the mixture was extracted three times with 100 ml of ethyl acetate.

3-Methoxytropolone is transferred to the ethyl acetate layer.

This ethyl acetate layer was concentrated to dryness under reduced pressure, and the purity was approximately 30%.
About 1.8 g of coarse powder was obtained.

(D): The crude powder obtained in (C) was placed on a 500 ml silica gel column, eluted with ethyl acetate, the obtained active fraction was concentrated to about 1 ml, and kept in a cold room (0'C) overnight. When left to stand, pale yellow crystals are obtained.

The crystals were filtered and re-crystallized in carbon tetrachloride to give approximately 150%
- pale yellow crystals of 3-methoxytropolone can be obtained.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of 3-methoxytropolone. The solid line is a methanol solution, the dotted line is a 0.1N HCl methanol solution (3-methoxytropolone dissolved in methanol and acidified with 0.1N HCl), and the dashed line is a 0.1N HCl methanol solution. IN
The case is shown in which a KOH methanol solution (3-methoxytropolone dissolved in methanol and made alkaline with o.INKOH) is used. FIG. 2 shows the infrared absorption spectrum of 3-methoxytroborone.

Claims (1)

[Claims] 1. A method for producing 3-methoxytropolone by a fermentation method, which comprises culturing 3-methoxytropolone-producing bacteria belonging to the genus Streptoverticillium in a nutrient medium, and collecting 3-methoxytropolone produced and accumulated from the culture.