JPS5822193B2 - Novel microorganism - Google Patents

Description

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

The microorganism according to the present invention is a new bacterial species as described below,
The inventors have identified Stachybotrys sp., T-78.
Name it 9.

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

■Characteristics on various cultures Stachypotrys sp. T-789 The characteristics of culturing Stachypotrys sp. T-789 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 extremely slow and cultured at 27℃ for 30 days. 17-20mm in length.

The area around the village grows in waves and is localized.

The center of the village shows a convex bulge, and the periphery shows radial sliding.

Aerial hyphae are thin and epiphytic over the entire surface, and spore formation gradually occurs from white at the initial stage of culture, and in the center, spore formation occurs.
, 1), and grows densely, but sporulation can be seen in a ring around the periphery.

The back side is Cork Tan (4ie)
.

Diffusible pigments are abundant and amber (Amber, 31e
).

2) Czapek agar medium Growth was good and after culturing at 27℃ for 30 days. Grows into 45 dragons.

The colony has a convex center and a radial curve, and the periphery shows wavy growth.

Thin, white aerial mycelium grows concentrically outward from the central convex part, and spore formation occurs concentrically only at the periphery, and the convex part is extremely moist with dissolved hyphae.

The back side is mustard brown. Brown, 2pi).

No diffusible pigments are produced. ■ Physiological properties It is an aerobic bacterium with the following physiological properties.

pH Temperature Conditions for growth 3.5-11.5 15-38°C Optimum growth conditions 4.5-10.5 20-32°C Morphological properties The following is clear from Figure 1.

That is, no sclerotia or other sexual reproductive organs were observed on the various media, and asexual sporulation of the ascospore type was observed.

Hyphae are formed on a variety of media, are intricately branched, and have a diameter of 2 to 4 μm.
The hyphae extend horizontally and vertically with a width of .

The pedicel has simple branches from the hyphae, and there is no branching from one pedicle to other pedicles) It has 3 to 4 septa from the podocyte at the base, and is either erect or gently curved. and expand.

The hyphae width of the pedicel is 4.3-4.7μ at the base and 3μ at the center.
．． 6-4.5μ.

The tip of the pedicel is slightly swollen and the size is 7.5mm from the top.
Three to seven elliptical to bottle-shaped stalks, measuring 9 to 9.3 x 3.6 to 4.7 microns, are formed upright.

The stalks are smooth and colorless to pale yellowish brown.

The mycospores are produced by basophilically connecting from the tips of the sycamores and do not form straight chains, but instead form semi-moon-shaped clusters of 7 to 26 pieces.

The pedicel is 4.9-8. It is subglobular to ovoid with an OX of 3.3 to 4.7μ, and its surface is rough to warty.

Also, the color tone is dark eye buoy (D, k Ivy,
24po) to exhibit a gray-black color.

No coating of viscous material was observed on the mycospore mass.

The taxonomic position of this bacterium, which has the above mycological properties, was determined in the handbook ``The Genera of Hyphomycetes from Soil''.
cetes From 5oil, TheWilliam
ms & Wilkins CompanyVoltim
ore, G.L., Barron, 1968) and [A MANUAL OF SO'IL F.
UNGI,.

The Iowa University
Press Ames Iowa USA, J
, C, Gi 1man.

1971)” and “The Genera of Hungjai Sporating in Pure Culture (T.
he Genera of Fungi Sporola
ting -in Pure Culture, VER
AG VON J.

CREMER3301LEHRE, J, A, VONAR
X, 1970), etc., this bacterium is classified as Hyphomycetes network Demachacarea, according to the classification system of Satucardo (5accardo).

Belongs to the genus Squirrel.

That is, the characteristics of this strain have the property that it does not have asci or other sexual reproductive organs, produces dark brown inchospores from the inflorescence, and that the produced inchospores gather in a half-moon shape at the apex of the inflorescence. The characteristics match those of the genus Stachybotrys.

The properties of this bacterium can be found in the above search guide and Bisbee G, R (1
943): Trans.

Br1t Mycol, soc, 26: 133
-143, Zutsuku RK (1946): Myco
Logia, 38:69-76, Baron G, L (19
61), (::an.

J. Bot, 39:153-157, and I
FO preservation preservation standard comparison Identified Stachybotrys
Lobrata (S tac, hypot-r, ys・Iob
ulata IFO5369) was determined to be the most similar.

However, Stachybotrys lobulata differs in that some of the inflorescences reach up to 1 mTIL, and unlike the present strain, which forms simple branches, the inflorescences of Stachybotrys lobulata have the property of branching and elongating from one inflorescence to another.

In addition, this strain is found to form good inchospores on Czapek's agar medium, whereas Stachybotrythrobrata has a culture difference in that almost no inchospore formation is observed on Czapek's agar medium.

This strain also differs from the above-mentioned strains in that its optimal growth range is broadly from pH 4.5 to 10.5.

Therefore, for the above reasons, this bacterial strain was recognized as a new bacterial species and named Stachybotrys sp. T-789.

Display descriptions of the same color can be found in [Color Harmony Manual (
Col for Harmony Manual.

1958, Container Corporation
on of America).

The above-mentioned new strain Stachybotrys sp.

T-789 was entrusted to FERM-P3802 with accession number FERM-P 3802.

When the microorganism of the present invention belonging to the genus Stachybotrys is cultured, it produces a novel compound represented by the symbol below in the culture solution.

The compound represented by the above formula [■] is 6,7-dihydroxy-2,5,5,8a-tetramethyl-12,3,4
,4a,5,6,7,8,8a-decahydronaphthalene-1-spiro-27(6/,7/-diformyl-47-
Hydroxy-27,3/-dihydrobenzofuran).

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

Specifically, the method for obtaining the compound represented by the structural formula CI, 1 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, and 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 by surface culture or deep aeration agitation culture, but deep aeration agitation culture is preferably carried out.

What are the culture conditions? Under normal aeration conditions, the liquid has a pH of 8.5~
11.5 Preferably pH 4.5 to 9.5 and culture temperature 1
Culture can be advantageously carried out at 5 to 35°C, preferably 20 to 32°C, usually for 3 to 7 days.

Next, the above compound C produced in the culture solution after the above culture
I) is collected.

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, differences in solubility with impurities, differences in adsorption affinity for common adsorbents such as activated carbon, XAD-2, silica gel, ion exchange resins, Sephadex, etc., two liquids.

This can be carried out by a combination of a method that utilizes the difference in distribution ratio between phases, and a secondary method.

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 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 [D] can be isolated and purified.

The obtained compound has the following physicochemical properties and is identified as the compound represented by Inuyama above.

1) Optical rotation 0 (b) D = 48° (C = 2.5. methanol) 2)
Elemental analysis value C23H3006 Calculated value (ring C68,64,H7,51 Actual measurement @ C68,58,H7,553) Ultraviolet absorption spectrum (UV) analysis Ethanol λmax = 246 nm (ε = 16474) 307n
m (ε=6659) 350 nm (ε=5427) 4) Nuclear magnetic resonance spectrum (NMR) analysis Pyridine-d
An NMR analysis diagram using 5 (DSS) as a solvent is shown in FIG.

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

That is, in a test tube, put an aqueous solution (pH: 8) of the compound marked above.
．． 5ml, I x 10” cell/1111! (7)
Sensitized hemocytes (EA) 0.5 r/l, complement sensitization (g-p-c) diluted 150 times with 1 r ILl of veronal buffered saline (GVB++) containing isotonic gelatin and GVB''.
After collecting 0 and 5 rnl and incubating at 37°C 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 determined by determining how much the dissolution of sensitized blood cells is inhibited by the supplied compound mark.

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

The blank in Table 1 above is a sample in which only the compound [GVB'' and EA was collected without collecting D, and the control* is a sample in which 5 ml of E A O and 2.0 rnl of water were collected. This is a sample that shows

And the anti-complement activity value is calculated from the sample from which compound CI) was collected.
The value (OD') obtained by subtracting the OD4□3 average value of the blank (sample/462 and 3) from the 0D413 value of 161 is
Value COD obtained by subtracting the 0D413 average value of the blank from the 0D413 average value of the control* (Samples 44 and 5)
'], that is, the molten metal ratio (yl).

Therefore, if the solubility 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 above-mentioned solubility surface ratio (y), the higher the anti-complement activity, and it can be seen from the results of this test that Compound [D] has superior anti-complement activity.

Hereinafter, a method for pure isolation of non-inventive microorganisms from soil and reproducibility by subculturing the microorganisms will be described in detail with examples.

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

Agar medium for separation (I) Composition Potato infusion 200g Glucose 21 Agar 15g Water 1000rrLlpH
G5.6 Transfer the colonies generated by the above culture to a slanted agar medium (ml) having the following composition using a platinum loop, and culture at 28°C for 3 days.

Slanted agar medium (II) Composition Adult malt extract 205' Yeast extract 2g Peptone 1g Glucose 4g Agar 20.9 Water 1000mg One platinum loop of bacteria generated on the medium by the above culture was diluted 100 times with physiological saline, Mix 1rnl of it with 9M of isolation agar medium (I) and place it 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 (n) and cultured at 28°C for 3 days, and the bacteria on the 10 slanted medium (I) 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 10 cultured bacteria were 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 (n), and a spore suspension was prepared on the slanted agar medium (IIJ). do.

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

The freeze-drying is carried out by quickly freezing the ampoule containing the spore suspension in dry ice-acetone, setting it in a freeze dryer, and setting the degree of vacuum to 0.03 torr 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, transfer it to a sterile test tube using a sterile mini-spertil, and add condensate (an aqueous solution of 1% glucose and 1% yeast extract) to this. ) was added and allowed to stand for over 1 hour, and the properties and physiological properties were examined on each medium under the same conditions as above, and no changes were observed from 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 Stachybotrys sp. T-789 pure cultured in Example 1 above, the regenerated bacteria after freezing, and the subcultured bacteria were each placed in a 500 d Sakaro flask in medium 1 with the following composition.
00rnl and cultured at 32°C and pH=7.5 for 4 days with reciprocal shaking.

Glycerin 0.5% Glucose 1.2% Corn Stable - 0.5% Dried yeast 0.1 malt extract 0.2% Mg50
．． 0.05% NaC1O, 3% Add 301 volumes of one bottle of the seed culture obtained above, and culture 201 volumes of the medium with the above composition in a jar fermenter.

Cultivation temperature: 32°C, aeration rate: 11/l medium/min, number of stirring: 300
Culture was performed for 5 days at rotation/min.

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) The solution was dissolved in ethyl acetate, gel filtrated with a column of Sephadex LH-20, and subjected to thin layer chromatography (ethyl acetate-chloroform-acetic acid-50:50:
2 (V/V)) to Rf-〇, 34, and similar thin layer chromatography (benzene-butanol-acetic acid-60:
By taking the anti-complement activity fraction corresponding to Rf=0.58 (15:5 (V/■)) and distilling off the solvent, a pale yellow weakly acidic substance with anti-complement activity is obtained. There is 6゜7
-cyhydroxy-2,5,5,8a-tetramethyl-1
,2,3,4,4a,5,6,7,8゜8a-decahydronaphthalene-1-spiro-2'(6',7'-diformyl-47-hydroxy-2/,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 micrograph of Stachybotrys sp. T-789, which is the microorganism of the present invention. Figure 2 shows the anti-complement active substance 6,7-hydroxy-2,5,5,8a-tetramethyl-1,2,3,4,4a,5,6,7° produced by the organism of the present invention. 8.8a-decahydronaphthalene-1-spiro-2/ (6/, 7
FIG. 2 shows a nuclear magnetic resonance spectrum analysis diagram of 2/-diformyl-47-hydroxy 2/, 3/-dihydrobenzofuran).

Claims (1)

[Claims] 1 Belonging to the genus Stachypotrys, the pedicel is simply branched, good spore formation is observed on Czapek agar medium, and the optimum growth pH is in the range of 4.5 to 10.5. Stachybotrys sp. strain T-789 deposited as No.