JPH05163289A - New immunosuppressive substance, m1710-51f6 substance and its production - Google Patents

Abstract

PURPOSE:To provide the subject new antibiotic leaving both immunosuppressive activity and antibacterial activity. CONSTITUTION:It has been recognized that an antibiotic having high immunosuppressive activity can be produced and accumulated in a culture solution of microorganisms belonging to Amycolatopsis newly separated from soil. This substance was isolated and named M1710-51F6 substance. This substance is a new compound having both immunosuppressive activity and the high antibacterial activity on Gram-positive bacteria; however, its chemical structure has not been determined yet.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel antibiotic substance MI710-51F6 having immunosuppressive activity and antibacterial activity, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, cyclosporin A, FK506, 15-deoxyspergualin and the like are known as immunosuppressive substances produced by microorganisms.

[0003]

DISCLOSURE OF THE INVENTION An object of the present invention is to obtain a novel immunosuppressive substance useful for organ transplantation and immunodeficiency diseases.

[0004]

[Means for Solving the Problems] In order to find a useful immunosuppressive substance in a microbial product, the present inventors isolated a large number of microorganisms from natural soil and conducted various studies on the product. .. As a result, the present inventors acknowledged that antibiotics having excellent immunosuppressive activity were produced and accumulated in the culture solution of microorganisms belonging to the genus Amycolatopsis newly isolated from soil, and isolated the substance. MI710
The substance was named -51F6 substance, and its biological and physicochemical properties were examined, and it was found that this substance has immunosuppressive activity and high antibacterial activity against Gram-positive bacteria and is a novel compound.

That is, according to the first aspect of the present invention, MI710, a novel immunosuppressive substance having the following physicochemical properties:
-51F6 material is provided.

MI710-51F6 according to the first invention
The physicochemical properties of the substance are as follows.

(1) Elemental analysis: The actual measured values of elemental analysis are shown below.

C H O N 60.33% 9.52% 27.48% 2.44% (2) FAB (Fast Atom Bombardment) mass spectrum: m / z 1343 [M + H] ⁺ (3) high-resolution FAB- MS (EF-FAB [Pos.]]:
C ₇₁ H ₁₂₇ O ₂₁ N ₂ was estimated. The reference substance is P
EG1500 was used.

(4) Melting point: 137 to 139 ° C.

[0010]

(8) ¹ H-NMR spectrum (400M
Hz): As shown in FIG. 5 of the accompanying drawings. The measurement was performed using methanol (3.30 ppm) in deuterated methanol as a reference substance.

(9) ¹³ C-NMR spectrum (100M
Hz): As shown in FIG. 6 of the accompanying drawings. Methanol in deuterated methanol (49.00 ppm) was measured as a reference substance.

(10) Solubility: Soluble in methanol, water,
It is sparingly soluble or insoluble in acetone, ether, and n-hexane.

(11) Thin layer chromatography (silica gel 60F ₂₅₄ Art. 5554 manufactured by Merck & Co.): Rf values are shown in the following table.

[0015]

(12) Color reaction: Positive for vanillin sulfate and anisaldehyde sulfate.

(13) Color and properties of substance: colorless to white solid.

MI710-51F6 according to the first invention
Since the substance has a biological activity as described below, it is used for the following purposes. That is, MI710-according to the present invention
The 51F6 substance shows a strong antibacterial action against Gram-positive bacteria, an inhibitory activity against the proliferation reaction of mouse splenocytes by concanavalin A which is a mitogen selective for T cells, and a mixed lymphocyte culture reaction (Mixed Lymphoc
yte Culture Reaction) showed inhibitory activity against histocompatibility antigen-dependent T cell proliferation reaction. Furthermore, in v
In vivo , it showed inhibitory activity against delayed-type hypersensitivity in mice using sheep red blood cells as an antigen, and in a rat skin transplant rejection inhibition experiment, it strongly inhibited the rejection reaction and prolonged the graft survival time.

From these results, the MI710-51F6 substance which selectively inhibits T cells can be used as a useful drug for treating autoimmune diseases which are hyperimmune states mediated by organ transplantation and cell-mediated immune reaction. In summary, MI71
The 0-51F6 substance is useful for the treatment of Gram-positive bacterial infections, and is also useful as an immunosuppressant useful for organ transplantation and autoimmune diseases.

The physiological activity of the MI710-51F6 substance according to the present invention was evaluated in the test described below. Test Example 1 Inhibitory activity against proliferation reaction of splenocytes by mitogen CDF ₁ mouse splenocytes were cultured in RPMI1640 medium (10
%, Including fetal calf serum), and MI710-51F6 substance as a test compound was added to the mitogen (concanavalin A or LPS) at various concentrations.
Spleen cells were cultured for 72 hours at 37 ° C. and 5% carbon dioxide concentration with or without addition, and cell proliferation was determined based on the ³ H-thymidine intracellular uptake rate. The results are shown in Table 1.

[0021]

Test Example 2 Mixed Lymphocyte Culture React
ion) inhibitory activity against WKY rat spleen cells as stimulators of mixed lymphocyte culture reaction with mitomycin C 50 μg / ml at 37 ° C.
The cells were treated for 0 minutes, and non-adherent nylon wool non-adherent cells of Fischer F344 rat splenocytes were used as reaction cells.
%, Containing fetal bovine serum), MI710-51F6 substance as a test compound was added at various concentrations, and the mixture was cultured at 37 ° C. under 5% carbon dioxide concentration for 5 days. Add ³ H-thymidine 16-18 hours before the end of culture,
The rate of incorporation into the reaction cells was measured with a liquid scintillation counter. The results are shown in Table 2.

[0023]

Test Example 3 Effect on Delayed Type Hypersensitivity in Mice CDF ₁ mice were sensitized with sheep red blood cells (10 ⁵ cells / mouse) intravenously, and sheep red blood cells (10 ⁸ cells / mouse) were placed on the foot pads on the 4th day. Subcutaneous injection caused delayed hypersensitivity. Sample is 1 after sensitization
Up to the 3rd day, a solution containing the MI710-51F6 substance at each concentration was intraperitoneally administered, and on the 5th day of the sensitization, the effect was determined by measuring the thickness of the mouse footpad. The results are shown in Table 3.

[0025]

Test Example 4 Inhibitory Effect on Rat Skin Transplant Rejection The skin was collected from the tail of WKY rats (male, 13 weeks old) and transplanted to the back of F344 rats (male, 11 weeks old). MI710-51F6 substance was intraperitoneally administered from 1 to 9 days after transplantation. The evaluation was performed by setting the average number of days of graft withdrawal in the drug-non-administered group as 100. The results are shown in Table 4.

[0027]

Further, MI710-51F6 according to the present invention
The substance has antibacterial activity against certain bacteria.

Tables 5a and 5b show the measurement results of the minimum inhibitory concentration (μg / ml) of MI710-51F6 substance against various bacteria and filamentous fungi measured by the agar plate dilution method.

[0030]

[0031]

Further, according to the second aspect of the present invention, a novel immunosuppressive substance characterized by culturing a MI710-51F6 substance-producing bacterium belonging to the genus Amycolatopsis and collecting the MI710-51F6 substance from the culture. , MI710
A method of making -51F6 material is provided.

MI710-51F used in the method of the present invention
The 6-substance-producing strain is a strain of the genus Amycolatopsis, MI710.
There is no particular limitation as long as it has the ability to produce −51F6 substance. An example of the MI710-51F6 substance-producing bacterium that can be used is an actinomycete strain isolated from soil in Hayama-machi, Kanagawa prefecture in March 1988, which has the MI710-51F6 strain number.

The mycological properties of the MI710-51F6 strain will be described below.

1. Morphology Well-branched basal hyphae often have a zigzag shape,
In addition, division is recognized. The aerial hyphae are straight or wavy,
Fragmentation is observed and 10 or more cylindrical spores are formed. The spore size is approximately 0.3-0.5 x 1.4 microns and the surface is smooth. No formation of sporangia or ring branches was observed. It is non-acidic. 2. Regarding the description of colors in various media, the standard shown in [] is the Container Harmony Manual (Container Corporation of America).
Color harmony manual).

(1) Sucrose / nitrate agar medium (3
(0 ° C culture) On the development of colorless to light yellow radish [3ea, Lt Melon Yellow], aerial hyphae of light yellow radish [3ca, Shell] grow. No soluble dye is found.

(2) Glucose / asparagine agar medium (cultured at 30 ° C.) For the development of colorless to light yellow [2 gc, Bamboo], brown white [3 b
a, Pearl] to light yellow radish [3ca, Shell] on the aerial mycelium, and no soluble pigment is observed.

(3) Glycerin-asparagine agar medium (ISP-medium 5, culture at 30 ° C.) For the development of light yellow radish [3ea, Lt Melon Yellow], yellowish ash [2ca, LtIvory] to light yellow radish [3e]
a, Lt Melon Yellow] aerial hyphae are settled, and soluble pigment is not observed.

(4) Starch / inorganic salt agar medium (ISP
-Medium 4, culture at 30 ° C) Yellowish white to yellowish ash [2ca, Lt Ivory] on the development of light yellow
Of the aerial mycelium and no soluble pigment is observed.

(5) Tyrosine agar medium (ISP-medium 7, culture at 30 ° C.) light yellow [2ic, Honey Gold] to light yellow daidai [3ea,
Lt Melon Yellow ~ 3pc, Amber] develops aerial hyphae of [3ea, Shell ~ 3ea, Lt Melon Yellow] on development. No soluble dye is found.

(6) Nutrient agar medium (30 ° C. culture) White aerial hyphae are slightly grown on the growth of light yellow [2ic, Honey Gold]. No soluble dye is found.

(7) Yeast-malt agar medium (ISP-
Medium 2, culture at 30 ° C) Aerial hyphae of pink white [5ba, Shell Pink] are grown on the development of light yellow radish [3gc, Lt Tan]. No soluble dye is found.

(8) Oatmeal agar medium (ISP-medium 3, cultivated at 30 ° C.) On the development of colorless to light yellow, white aerial mycelium grows and no soluble pigment is observed.

(9) Glycerin / nitrate agar medium (30
Cultivation) Cultivation of light yellow daidai [3ea, Lt Melon Yellow] to light olive [1 1/2 ie, Lt Olive], yellowish white to light yellow daidai [3ca, Shell to 3ea, Lt Melon Yellow]
], Aerial hyphae are settled, and soluble dye is not observed.

(10) Starch agar medium (cultured at 30 ° C.) On the development of colorless to light yellow, yellowish-white aerial mycelium is grown.
No soluble dye is found.

(11) Lime malate agar medium (30 ° C. culture) light yellow [2ic, Honey Gold] to light yellow dai [3ea,
Lt Melon Yellow], and aerial hyphae of white to light yellow are colonized. No soluble dye is found.

(12) Cellulose (synthetic solution containing filter paper pieces,
Cultivation at 30 ° C) Growth is colorless, white aerial hyphae are settled, and soluble pigment is not observed.

(13) Gelatin puncture culture In a 15% simple gelatin medium (cultured at 20 ° C.), yellowish-white aerial mycelium grows on the development of colorless to light yellow. In glucose, peptone, and gelatin medium (cultured at 27 ° C), the growth is pale yellow, and white aerial hyphae slightly colonize. Soluble dyes are not found in both.

(14) Skim milk (cultured at 37 ° C.) Growth is light yellow, and white aerial mycelium slightly grows,
No soluble dye is found.

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar medium (glucose 1.
0%, asparagine 0.05%, dipotassium phosphate
0.05%, string agar 3.0%, pH 7.0), 6 ° C, 20 ° C, 24 ° C, 27 ° C, 30 ° C, 37 ° C,
As a result of testing at each temperature of 50 ° C, growth was observed at any temperature except 6 ° C and 50 ° C, but the optimum growth temperature seems to be around 27 ° C to 30 ° C.

(2) Liquefaction of gelatin (15% simple gelatin medium, 20 ° C. medium; glucose, peptone, gelatin medium, 27 ° C. culture) In both cases, liquefaction was observed from about 5 days after culturing, and the action was strong. is there.

(3) Starch hydrolysis (starch / inorganic salt agar medium and starch agar medium, 30
(° C culture) In starch-inorganic salt agar medium, hydrolyzability was observed from about 14 days after culture, but its action was extremely weak,
Not found on starch agar.

(4) Coagulation / peptonization of defatted milk (defatted milk, 37 ° C. culture) About 9 days after culturing, peptone formation started without coagulation and was completed within 2 to 3 days. Its action is moderate to strong.

(5) Production of melanin-like pigment (tryptone yeast broth, ISP-medium 1; peptone, yeast, iron agar medium, ISP-medium 6; ISP-medium 7; both cultures at 30 ° C.) Any medium But it is negative.

(6) Utilization of carbon source (Pridham, Godlieve agar medium, ISP-medium 9; cultivated at 30 ° C.) D-glucose, L-arabinose, D-xylose,
It grows using D-fructose, sucrose, inositol, rhamnose, and D-mannitol. Raffinose and lactose are also probably used.

(7) Dissolution of lime malate (lime malate agar medium, culture at 30 ° C.) Dissolution of lime malate was observed from about the 3rd day after culturing, and its action was moderate to strong.

(8) Nitrate reduction reaction (0.1% potassium nitrate-containing peptone water, ISP-medium 8, 30 ° C. culture) Positive.

(9) Decomposition of cellulose (synthesis solution with filter paper pieces added, culture at 30 ° C.) Negative.

Summarizing the above properties, MI710-5
The 1F6 strain has fragmentation in the basal hyphae and aerial hyphae, and the basal hyphae often have a zigzag shape. 10 or more cylindrical spores are formed on the aerial mycelium, and the surface is smooth. No helix formation, limbus, or sporangium is observed.
It is non-acidic. On various mediums, yellow-white to light yellow-white aerial mycelium grows on the development of light-yellow yellow-white. No soluble dye is found. The formation of melanin-like pigment is negative. The water-degradability of starch is extremely weak and the proteolytic power is moderate to strong. The nitrate reduction reaction is positive.

By the way, the cell components of the MI710-51F6 strain are meso type of 2,6-diaminopimelic acid contained in the cell wall, and the reducing sugars in all the cells are A type containing arabinose and galactose. In addition, it contains no mycolic acid, the phospholipids are of PII type (containing phosphatidylethanolamine and no phosphatidylcholine and unknown glucosamine-containing phospholipids), and the major menaquinone is MK.
A _{-9 (H 4), MK-} 9 (H 2) was also observed.

[0061] From the above properties, MI710-51F6 strain, Amycolatopsis (Amycolatop sis, literature, Intern
ational Journal of Systematic Bacteriology, 36
Vol., 29-37, 1986). Searching for known species of the genus Amycolatopsis,
Amycolatopsis orientalis ( Amycolatopsis or
ientalis , ibid.) and Amycolatopsis mediterranei , ibid.
However, it was listed as a closely related species. Currently MI710-5
The 1F6 strain and the above-mentioned two strains preserved by this institute are currently being compared and examined. Therefore, at this time, MI710-51
The F6 strain was designated as Amyco latopsis sp.
p. ).

The MI710-51F6 strain was applied for deposit at the Institute of Microbial Technology, Institute of Industrial Science, and was applied on February 2, 1991.
On the 0th day, the work was entrusted as Microbiology Research Institute No. 12021.

The MI710-51F6 substance can be produced by aerobically culturing the MI710-51F6 producing bacterium belonging to the genus Amycolatopsis in an appropriate medium and collecting the target substance from the culture.

The culture medium may contain any nutrient source that can be utilized by the MI710-51F6 substance-producing bacterium. Specifically, for example, glucose, fructose, maltose, starch and oils and the like can be used as a carbon source, soybean flour, cottonseed meal, dried yeast extract, organic substances such as polypeptone and corn steep liquor, and ammonium salts as a nitrogen source. Alternatively, minerals such as nitrates such as ammonium sulfate, sodium sulfate and ammonium chloride can be used. Inorganic salts such as sodium chloride, potassium chloride, calcium carbonate, phosphate, and heavy metal salt can be added as required. In order to suppress foaming during fermentation, an appropriate defoaming agent, for example, a silicone-based defoaming agent can be added according to a conventional method.

The culture method may be any generally used method for producing physiologically active substances and the like, and the aerobic liquid submerged culture method is particularly suitable. The culture temperature is suitably 20 to 37 ° C, preferably 27 to 30 ° C. This way M
The production amount of the I710-51F6 substance reaches the maximum in 3 to 5 days of culture in both shaking culture and aeration-agitation culture. In this way a culture of MI710-51F6 material is obtained. In the culture, the MI710-51F6 substance is present in the cells and in the culture filtrate, but more in the cells.

From such a culture, MI710-51F
Any suitable method can be used to collect the 6 substances. One of the methods is based on the principle of extraction. Specifically, for example, MI71 in the culture filtrate is used.
For the 0-51F6 substance, a method of extracting with a water-immiscible organic solvent such as butyl acetate or n-butanol,
Alternatively, for the MI710-51F6 substance in the microbial cells, the microbial cell assembly obtained by filtration or centrifugation is used for methanol,
There is a method of recovering by treating with ethanol or acetone. The culture itself may be subjected to the above extraction operation without separating the cells. Countercurrent partitioning with a suitable solvent can also be included in the extraction category. For example CPC (C
entrifugal Partition Chromatograph) (manufactured by Miki Engineering Co., Ltd.) can also be used.

One of the other methods of collecting MI710-51F6 substance from the culture is based on the principle of adsorption. For example, a suitable adsorbent, gel filtration agent, for example, silica gel, for the culture filtrate or the extract obtained by performing the extraction operation as described above,
Sephadex LH-20 (Pharmacia), Toyopearl HW-40 (Tosoh Corp.), Diaion HP-
Column chromatography using 20 (manufactured by Mitsubishi Kasei Co., Ltd.), Nucleosil 5C ₁₈ (manufactured by Nagel, West Germany)
The MI710-51F6 substance can be obtained by adsorbing and then eluting the MI710-51F6 substance by high performance liquid chromatography or the like using, for example. MI710-51F6 thus obtained
If the substance-containing solution is concentrated to dryness under reduced pressure, MI710-
A crude product of 51F6 material is obtained.

MI710-51 thus obtained
In order to further purify the crude product of F6 substance, the above-mentioned extraction method and adsorption method may be combined as necessary and performed a required number of times. For example, column chromatography using an adsorbent such as Diaion HP-20 and Sephadex LH-20 or a gel filtration agent, centrifugal liquid distribution chromatography using CPC, normal phase chromatography using silica gel and Nucleosyl 5C. High performance liquid chromatography using ₁₈ or the like can be performed in an appropriate combination.

[0069]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

In the following, "%" means "w / v%".

Example 1 (1) Preparation of Seed Mother The medium used was 1 liter of the components shown in the table below.
It was dissolved in water. The pH was not adjusted.

[0072]

110 ml of the above medium was dispensed into a 500 ml Erlenmeyer flask and sterilized, and then Amycolatopsis SP M
One platinum loop of the I710-51F6 strain (Microtechnology Research Institute, No. 12021) was ingested from the slant, and the culture obtained by rotating and culturing for 72 hours on a rotary shaker at 30 ° C. and 180 rpm was used as a seed mother.

(2) Culture The culture medium used was the same as the culture medium used for the preparation of the seed mother. This culture medium was dispensed and sterilized into a 500-ml Erlenmeyer flask by 110 ml, and 2.2 ml of the seed seed was added to the medium, and the mixture was cultivated at 28 ° C. and 180 rp using a rotary shaker.
Rotational culture was performed at m.

(3) Collection of MI710-51F6 substance After culturing for 4 days under the conditions of (2) above, about 20 l of culture solution
Were centrifuged (3,000 rpm, 15 minutes), the bacterial cells were treated with methanol, the methanol was distilled off, and then acetonitrile was added to obtain a 50% acetonitrile aqueous solution.
After standing at 0 ° C. overnight, the precipitate was separated by filtration and extracted with a small amount of methanol. Acetone was added to the methanol extract until it became cloudy, and the mixture was left at 10 ° C. overnight. When the precipitate was separated by filtration, 3.5 g of MI710-51F6 crude extract was obtained. The obtained crude extract was treated with Sephadex LH-20.
It was subjected to gel chromatography using (Pharmacia). Elution with methanol MI710-5
Fractions containing 1F6 substance were collected and concentrated to dryness under reduced pressure.
About 1.8 g of the obtained MI710-51F6 crude purified product was applied to a centrifugal liquid-liquid partition chromatograph (CPC) (manufactured by Miki Engineering Co., Ltd.). Chloroform-methanol-
The lower layer of water (2: 2: 1) was used as the stationary phase and the upper layer was used as the mobile phase, and separation and purification was performed by the ascending method under the conditions of 25 ° C, 400 rpm, and 1 ml / min. MI710-5 in the normal elution fraction
The 1F6 material was eluted and concentrated to dryness under reduced pressure.
Approximately 350 of the crudely obtained MI710-51F6 substance
mg is dissolved in methanol, and a certain amount is dissolved in a capsule pack 5C ₁₈ column (manufactured by Shiseido Co., Ltd.) (20 mmφ × 250)
mm), eluting with methanol-50 mM ammonium acetate-acetonitrile (65: 30: 5) to give MI710-51F6.
Obtain the material fraction. When this was concentrated to dryness under reduced pressure, a colorless to white solid of MI710-51F6 substance 250
mg was obtained.

[0076]

INDUSTRIAL APPLICABILITY As described in detail above, according to the present invention, MI710-51F6 substance was obtained as a novel substance having an immunosuppressive activity and an antibacterial activity against Gram-positive bacteria, and a method for producing the same was provided.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum diagram of MI710-51F6 substance in a methanol solution.

FIG. 2 MI in 0.02N HCl-methanol solution
It is an ultraviolet absorption spectrum figure of 710-51F6 substance.

FIG. 3 M in 0.02N NaOH-methanol solution
It is an ultraviolet absorption spectrum figure of I710-51F6 substance.

FIG. 4 is an infrared absorption spectrum diagram of MI710-51F6 substance by a KBr disk method.

FIG. 5 is a ¹ H-NMR spectrum diagram of MI710-51F6 substance in deuterated methanol at 400 MHz.

FIG. 6 is a ¹³ C-NMR spectrum diagram of MI710-51F6 substance in deuterated methanol at 100 MHz.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 9, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

(1) Elemental analysis: Measured values of elemental analysis are shown below. CHON 60.33% 9.52% 27.48% 2.44%

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

(2) FAB (Fast Atom Bombardment) mass spectrum: m / z 1343 [M + H] ⁺ (3) High resolution FAB-MS (EF-FAB [Pos.] ) :
C ₇₁ H ₁₂₆ O ₂₁ N ₂ was estimated. The reference substance is P
EG1500 was used.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction content]

1. Morphology Well-branched basal hyphae often have a zigzag shape,
In addition, division is recognized. The aerial hyphae are straight or wavy,
Fragmentation is observed and 10 or more cylindrical spores are formed. The spore size is approximately 0.3-0.5 x 1.4 microns and the surface is smooth. No formation of sporangia or ring branches was observed. It is a non-acid-resistant. 2. Regarding the description of colors in various media, the standard shown in [] is the Container Harmony Manual (Container Corporation of America).
Color harmony manual).

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0048

[Correction method] Change

[Correction content]

(13) Gelatin puncture culture In a 15% simple gelatin medium (cultured at 20 ° C.), yellowish-white aerial mycelium grows on the development of colorless to light yellow. Oite glucose, peptone, gelatin medium (27 ℃ culture), slightly epiphytic growth house yellow orange, the aerial mycelium of white. Soluble color element is not observed even with both.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction content]

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar medium (glucose 1.
0%, asparagine 0.05%, dipotassium phosphate
0.05%, string agar 3.0%, pH 7.0 )
6 ° C, 20 ° C, 24 ° C, 27 ° C, 30 ° C, 37 ° C, 50
As a result of test at each temperature of ° C., 6 ° C., with the exception of 50 ° C., was growth in any of its temperature, optimum growth temperature is 27 ° C.
It seems to be around -30 ° C.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0054

[Correction method] Change

[Correction content]

[0054] (5) production of melanin-like pigment (tryptone East broth, ISP- medium 1; peptone, yeast, iron agar medium, ISP- medium 6; ISP- medium 7;
Nor any is 30 ° C. culture) negative in either medium.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0059

[Correction method] Change

[Correction content]

Summarizing the above properties, MI710-5
The 1F6 strain has fragmentation in the basal hyphae and aerial hyphae, and the basal hyphae often have a zigzag shape. 10 or more cylindrical spores are formed on the aerial mycelium, and the surface is smooth. No helix formation, limbus, or sporangium is observed.
It is a non-acid-resistant. On various mediums, yellow-white to light yellow-white aerial mycelium grows on the development of light-yellow yellow-white. No soluble dye is found. The formation of melanin-like pigment is negative. The water-degradability of starch is extremely weak and the proteolytic power is moderate to strong. The nitrate reduction reaction is positive.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number in the agency FI technical display location C12R 1:01) (72) Inventor Masa Hamada 26-1 Naitocho, Shinjuku-ku, Tokyo Hidewa Residence 405 (72) Inventor Kenji Maeda 2-46-11 Gotongi, Meguro-ku, Tokyo (72) Inventor Tomio Takeuchi 5-11-11 Higashi-Gotanda, Shinagawa-ku, Tokyo New Mansion 701

Claims (3)

[Claims] 1. An immunosuppressive substance having the following physicochemical properties, MI710-51F6 substance. (1) Elemental analysis: The measured values of elemental analysis are shown below. C H O N 60.33% 9.52% 27.48% 2.44% (2) FAB mass spectrum: m / z 1343 [M + H] ⁺ (3) Melting point: 137 to 139 ° C. (7) ¹ H-NMR spectrum (400 MHz): As shown in FIG. 5 of the accompanying drawings. The measurement was performed using methanol (3.30 ppm) in deuterated methanol as a reference substance. (8) ¹³ C-NMR spectrum (100 MHz): As shown in FIG. 6 of the accompanying drawings. Methanol in deuterated methanol (49.00 ppm) was measured as a reference substance. (9) Solubility: Soluble in methanol, hardly soluble or insoluble in water, acetone, ether, and n-hexane. (10) Color and properties of substance: colorless to white solid. 2. The immunosuppressive substance according to claim 1, which belongs to the genus Amycolatopsis, MI710-51F6 substance-producing bacteria are cultured, and the immunosuppressive substance, MI710-5, is cultured from the culture.
A method for producing a novel immunosuppressive substance, MI710-51F6 substance, which comprises collecting 1F6 substance. 3. The MI710-51F6 substance producing bacterium is Amycolatopsis sp. MI71.
The method according to claim 2, which is strain 0-51F6.