JPS5822194B2 - Novel microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism belonging to the genus Stachybo.

The microorganism according to the present invention is a new bacterial species as described below,
The inventors have identified Stachybotrys sp., T-791.
Name it.

■Collection area Isolated from soil in Tokushima City, Tokushima Prefecture.

(2) Conditions on various culture media The characteristics of culturing the Deuteromycota strain 'l'-791 on various media based on macroscopic and microscopic observations (shown in Figure 1) are as follows.

A Macroscopic Observation 1) Malt Extract Agar Medium Growth is rapid and irregular, and the color of the underside is tan (
Tan, 3ie) ~ Dark Brown (DkBrown,
3pn).

The settlement is flat and sporulation is good.

Mycelium is Biscuit (Biscuit. 2ec) ~ Tan (
Tan, 3ie) and exudate droplets are observed.

No diffusible pigments are produced. 2) Growth in Vallejo glucose medium is extremely rapid, with 70% growth in 130 days at 27°C.
Reach wIL.

At the periphery, thin white mycelia are observed spreading in a tree-like manner, and droplets are also noticeable.

There is almost no sporulation. The back side is light amber (Lt
Amber, 3ie).

No diffusible dye.

3) Czapek agar medium poor growth 4) Synthetic mucor agar medium poor growth 5) Oatmeal agar medium It grows very well and covers the petri dish in two weeks.

The colony is thin and flat, and hyphae are abundantly formed from the early stage of culture, and spore formation is rapid.

The bacterial flora color ranges from white to dark brown (DkBrown).

3 pn) as the culture progresses.

A large amount of droplets are produced on the hyphae.

No diffusible pigments are produced.

■Physiological properties It is an aerobic bacterium with the following physiological properties.

■Morphological properties The following can be seen from Figure 1.

That is, the inflorescence pedicel forms simple branches and stands upright.

It swells slightly into a club shape at its tip.

However, the standard strain Stachybo 1-IJ Suechinata IF mentioned later
There is no significant enlargement as observed in O7525 and 8856.

The mycelium width is 4.0-4.5μ, which is slightly larger than the inflorescence.

From vegetative or aerial hyphae, the pedicel has podocytes and is branched upright, with 2 to 3 septa and 40 to 80 x 3.
．． It exhibits a size of 0 to 3.5μ.

No thorn-like projections are observed on the cell wall of the pedicel, which is smooth.

3 to 6 inflorescences are produced from the apical ampulla of the inflorescence stalk, and a 4.3 to 5.2×3. Globular to subglobular inchospores with a size of 0 to 4.2 μ and one cell with spiny projections are produced continuously, forming a chain of 2470 spores.

Kyoko has a sake bottle-shaped shape and measures 669 to 10.7X.
3.5-4.7 Fish stalk and stalk are colorless.

The stalks are coffee (3pn) to black in color.

Search for the taxonomic position of this bacterium with the above mycological properties using the search guide “Zadienella obhyeophomycetes from soil (
ThaGeneraofHyphomycetesFr
omSoil, TheWiIIIiams & Wilkin
sCompany Baltimore, G, L, Bar
ron, 1968) and AMANUAL OF SOIL FUNGI, Thelowa State University, Press AmesIowaU.
S.A.

J. C. Gilman, 1971) and [The Genera of Fnn.
giSporolatinginPureCulture
e.

VERAGYONJ,CREMER3301LEHRE
, J.A., VONARX, 1970.According to Saccardo's taxonomy system, this bacterium belongs to the genus Stachybotrys (genus Menmoniera) in the Hyphomycetes network Demachacarea.

In other words, this strain T-791 has the property that dark brown inchospores are continuously produced from the inflorescence, and the produced spores are long chains. The properties are
It corresponds to that of the genus Stachybotrys (genus Menmoniella).

The properties of this bacterium can be found in the search guide above and in Zutsuk R, K (1
946, Mycologia, 38:69-76) and Smith G. (1962, Trans.

Br1t, Mycal, soc, 45:387-39
4), as well as IFO preservation standards, it was identified that the property of this strain T-791 to continuously produce basophores from the sycamore is that it belongs to the Menmoniella genus Menmoniellae, which was named by Hohnel. Chinata (Me
nmoniellaechinata).

However, based on the report by Zook and Smith mentioned above, this strain T-
Since strain 791 was considered to be a bacterial species related to Stachybotrys aechinata, it was compared with two strains of Stachybotrys aechinata IFO7525 and 8856.

Morphologically, in the cell wall of this strain T-791, the spiny protrusions that are specific to both standard strains are not observed in the cell wall of the inferno pedicle, and the tip of the inflorescence pedicle is 2 to 3 times the width of the hyphae in the standard strain. Compared to this strain, no significant expansion was observed in this strain.

Furthermore, the two standard strains showed good growth on medium, especially on Vallejo glucose agar medium, forming round, slightly raised colonies, with abundant hyphae, and significant spore formation. In contrast, this strain exhibits tree-like, irregular growth as described above, and has poor hyphal formation and spore formation.

Based on the above-mentioned differences in mycological properties, this bacterial strain was recognized as a new bacterial species and named Stachybotrys sp. T-791.

The display description of the same color is [Color Harmony Manual (Co
llorHarmonyManual.

1958, Container Corporation
The notation method of nofAmerica) was followed.

The above-mentioned new strain Stachybotrys sp.

T-791 was entrusted to FERM with accession number FERM-PA3803.

The microorganism of the present invention belonging to the genus Stachybotrys produces a novel compound represented by the following formula CI) in the culture solution by culturing it.

The compound represented by the above formula CI) is 6,7-cyhydroxy-2,5,5,8a-tetramethyl-1,2,3,
4,4a,5,6,7,8,8a-decahydronaphthalene-1-spiro-27-(6',7'-diformyl-4
7-hydroxy-2'.

3'-dihydrobenzofuran).

It has an anti-complement activity and is useful as a therapeutic agent for autoimmune diseases, rheumatism nephritis, collagen diseases, etc.

Specifically, the method for obtaining the compound represented by the structural formula CI) using the microorganism of the present invention is carried out as follows.

That is, the microorganism is first cultured in a medium containing conventional nutrients and additives.

Nitrogen sources commonly used as culture media include, for example, soybean flour, soybean oil, corn steep liquor, yeast extract,
Examples include dry yeast, oatmeal, meat extract, casein hydrolyzate, ammonium salts, nitrates, etc. Carbon sources include, for example, glucose, glycerin, maltose, starch,
Examples include lactose, sucrose, and molasses.

In addition, examples of additives added to the medium include inorganic salts such as calcium carbonate, sodium chloride, magnesium sulfate, and phosphoric acid, and metals such as iron, copper, manganese, and zinc may be added to the medium as necessary. It may contain a small amount of salt.

Cultivation can be carried out in a normal aqueous medium containing the above-mentioned culture medium, either by surface culture or submerged aerated agitation culture.

It is preferable to perform deep aeration agitation culture.

The culture conditions are normal aeration conditions, and the liquid pH is 3.5 to 1.
1.5 preferably pH 4.5-9.5 and culture temperature 15
Culture can be advantageously carried out at a temperature of -35°C, preferably 20-32°C, usually for 3-7 days.

Next, the above-mentioned compound produced in the culture solution after the above-mentioned culture [
I).

The collection method is not particularly limited, and any known method that utilizes the physical and chemical properties of the produced compound can be employed.

For example, methods that utilize differences in solubility with impurities, differences in adsorption affinity for ordinary adsorbents such as activated carbon, XAD-2, silica gel, ion exchange resins, Sephadex, etc., differences in distribution ratio between two liquid phases, etc. It can be carried out by a combination of the same methods.

More specifically, the culture solution is filtered or centrifuged according to a conventional method to remove bacterial cells in advance.

Next, methanol is added to the supernatant, stirred, and left to stand for 2 to 3 hours, and the precipitate is removed by centrifugation again.

After further extraction with the same amount of ethyl acetate, the solvent was distilled off.

After pouring the extract into methanol and passing the methanol solution through an activated carbon column, the solvent of the eluate is distilled off.

The residue is gel-filtered with Sephadex LH-20, each fraction obtained is subjected to the anti-complement activity test described below, the active portion is collected, and the solvent is distilled off.

In this way, the target compound represented by the above formula (I) can be isolated and purified.

The obtained compound has the following physical and chemical properties and has the above formula (
It is identified as a compound represented by 1).

An NMR analysis diagram is shown in FIG.

In addition, the anti-complement activity of the above compound CI) is shown in "Immunochemistry", pp. 830-834 (published by Chokoku Shoten, edited by Yu Yamamura et al., 1).
973).

That is, an aqueous solution (pH ÷ 8) of the above compound CI) is placed in a test tube.
．． 5ml, 1X108 cells/ml 0 sensitized blood cells (EA)
．． 5yd, veronal buffered saline containing isotonic gelatin (
Collect 0.5 ml of complement serum (g-1) diluted 150 times with GVB++) 1d and GVB'' solution,
After incubating at ℃ for 60 minutes, 5 ml of ice-cold physiological saline was added to 2 and centrifuged, and the absorbance of the separated supernatant was measured using an absorbance meter 0D413. It is measured by determining how much hemolysis is suppressed.

The absorbance and anti-complement activity values measured according to the above method are shown in Table 1 below.

In addition, the blank in Table 1 above is a sample in which only GVB" and EA were collected without collecting compound CI), and the control* is a sample in which 5 ml of EAo and 2.0 ml of water were collected.
This is a sample showing 0% hemolysis.

And the anti-complement activity value is calculated from the sample from which compound CI) was collected.
Blank (sample 2 and 3) from 0D413 value of 16.1
] The value (OD') obtained by subtracting the 0D413 average value of the control* (Samples A6.4 and 5) is the value obtained by subtracting the blank 0D413 average value (OD
'), that is, the hemolysis ratio (y).

Therefore, if the hemolysis ratio (y) is less than 1, it can be considered to have anti-complement activity.

It can be said that the smaller the value of the hemolysis ratio (y) is, the higher the anti-complement activity is, and it can be seen from the results of this test that Compound [I] has superior anti-complement activity.

The method for pure isolation of the microorganism of the present invention from soil and the reproducibility by subculturing the microorganism will be described in detail below with examples.

Example 1 The collected soil was diluted 1000 times with sterile physiological saline,
The ITLl and 9'Inl of the following isolation agar medium (I)
The cells were mixed together, placed in a sterile petri dish, and cultured at 28°C for 5 days.

Colonies generated by the above culture are transferred to a slanted agar medium (II) having the following composition using a platinum loop, and cultured at 28°C for 3 days.

One platinum loopful of the bacteria generated on the medium by the above culture was diluted 10,000 times with physiological saline, 1 ml of it was mixed with 9 ml of isolation agar medium (1), and the mixture was placed in a sterile petri dish for 28 hours.
Culture at ℃ for 5 days, and confirm visually and microscopically that the resulting colonies are not different from each other.

10 of the above colonies were inoculated onto slanted agar medium (II) and cultured at 28°C for 3 days, and the bacteria on the 10 slanted medium (II) were macroscopically and microscopically the same bacteria. This was confirmed, and it was also confirmed that the properties and physiological properties on each medium of the 10 second-class cultured bacteria were the same.

The properties and physiological properties of each of the above-mentioned media are as described above.

As a result of the above test, it was found that all of the 610 cultured bacteria were pure monobacteria isolated from nature.

Next, a protective agent (10% skim milk and 1% sodium glutamate aqueous solution) was added to the pure cultured bacteria on the slanted agar medium (II), and the bacteria were cultured on the slanted agar medium (II).
Prepare the spore suspension above.

Approximately 1 ml of this spore suspension is dispensed into freeze-drying ampoules and freeze-dried.

The freeze-drying is carried out by rapidly freezing the ampoule containing the spore suspension in dry ice-acetone, setting the ampoule in a freeze dryer, and setting the degree of vacuum to 0.03) or less.

Then, it is vacuum sealed with a gas burner and stored at 4°C.

After storing the freeze-dried bacteria (standard sample) obtained in this way for 3 months, open the ampoule and transfer it to a sterile test tube using a sterilized mini-spartil, and add condensate solution (an aqueous solution of 1% glucose and 1% yeast extract). After adding and leaving it for more than 1 hour,
As a result of examining the properties and physiological properties on each medium under the same conditions as above, no changes were observed compared to the bacteria before freezing (standard sample).

Furthermore, the properties and physiological properties of the bacteria on each culture medium were similarly examined after the above-mentioned freezing and regeneration was repeated five times every month, and no changes were observed.

This shows that the same results can be reliably reproduced by subculturing the symptomatic organisms of the present invention.

Next, an example in which compound CI) was produced using the symptomatic organism of the present invention will be listed as a reference example.

Reference Example 1 Each of the Stachybotrys sp. T-791 pure cultured in Example 1, the regenerated bacteria after freezing, and the subcultured bacteria were placed in a 500 ml volume Sakaro flask with 100 ml of a medium having the following composition, and incubated at 25°C. , culture with reciprocal shaking for 4 days at pH=5.5.

Put 307 volumes of one seed culture obtained above into a jar fermenter and add 201 medium of the above composition, culture temperature: 25°C.
Aeration rate: 11/11 medium/min, stirring rate: 5 at 300 revolutions/min
Culture was performed for 1 day.

The obtained culture solution was centrifuged at 8000 revolutions/rotation speed to remove bacterial cells, and methanol 51 was added to this supernatant, stirred and left for 3 hours, then centrifuged to remove the precipitate, etc. of ethyl acetate.

After the solvent in the ethyl acetate layer was distilled off under reduced pressure, the residue was dissolved in methanol and passed through an activated carbon column. The eluate was concentrated to dryness under reduced pressure, and then chloroform:ethyl acetate - 1:1 (V/V) Sephadex L
Gel filtration with H-20 column, thin layer chromatography (ethyl acetate-chloroform-acetic acid = 50:50:2 (
V/V)) to Rf = 0.34, and similar thin layer chromatography (benzene-butanol-acetic acid = 60:15).
:5 (V/V) and an anti-complement active substance corresponding to Rf=0.58 is taken and the solvent is distilled off from it to obtain 6°, which is a pale yellow weakly acidic substance with anti-complement activity. 7-cyhydroxy-2,5t5,8a-tetramethyl-1,2,
3,4,4a,5,6,7,8,8a-decahydronaphthalene-1-spiro-2'(6't7'-diformyl-
47-hydroxy-27,3/-dihydrobenzofuran) was obtained.

The physicochemical properties of this compound were as described above.

From this reference example, it is clear that the ability of the organism of the present invention to produce an anti-complement active substance is sufficiently reproducible.

[Brief explanation of drawings]

FIG. 1 shows a microscopic photograph of Stachybo IJ SP T-791, which is the microorganism of the present invention. FIG. 2 shows anti-complement active substances produced by the organisms of the present invention. 6,7-cyhydroxy-2,5,5゜8a-tetramethyl-1,2,3,4,4a, 5゜6.7,8,8a-decahydronaphthalene-1-spiro-2ot (6', 7'-
1 shows a nuclear magnetic resonance spectrum analysis diagram of diformyl-47-hydroxy-2/, 3/-dihydrobenzofuran).

Claims (1)

[Claims] 1. Belongs to the genus Stachybos t-IJ, the pedicel is simply branched, stands upright, has no spiny projections on its cell wall, and the tip of the pedicel is slightly enlarged compared to the width of the hyphae. Sukibotrys sp., strain T-791, deposited as Microtechnical Research Institute No. 3803, shows irregular tree-like growth on a potato glucose agar medium and exhibits poor hyphal formation and spore formation.