JPS62186787A - Novel microorganism - Google Patents

Abstract

NEW MATERIAL:A microbial strain (FERM P-8613) belonging to Actinomadura genus, capable of producing novel antibiotic substance SS50732A, B and C and having the following properties. Optimum growth temperature (cultured for 14 days in yeast-starch-agar medium), 30-40 deg.C; liquefaction of gelatin, positive; hydrolysis of starch, negative; coagulation of defatted cow's milk, positive; peptonization of defatted cow's milk, positive; formation of melanine- like pigment, negative; reduction of nitrate, positive; decomposition of cellulose, negative; cell wall composition, type III; sugar composition of whole microbial cell, type B; having adherend spore chain having about 10 links on a sporophore; free from sporangium and planosphore; etc. USE:A microorganism capable of producing antibiotic substance SS50732A, B and C. PREPARATION:The objective strain can be obtained e.g. by extracting soil collected in Isumi District, Chiba Prefecture, Japan and culturing the extracted liquid in a proper medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel microorganism belonging to the genus Actinomadilla, and more particularly to novel antibiotics SS50732A and B.
and a novel microorganism from which C can be collected.

[Conventional technology and its problems]

Several strains belonging to the genus Actinomadilla have been known, but their number is extremely small compared to strains belonging to the genus Streptomyces.

Moreover, the antibiotics produced by the genus Actinomadilla are l"R-900405 and FR-, c+oo4o6C
Journal of Antibiotyphoid Fever (Tu JOU
llAL.

IJF ANTIBIOTIC8), Volume 38, 83
Pages 5-839.

840-848 (1985)] and 5F-2140C
Journal of Antibiotics (T)LF:
JOURNAL OF ANTIBIOTIC8), 3
Volume 7, pp. 931-934, pp. 1144-1148 (1
984)], and their number was extremely limited.

[Means to solve the problem]

The present inventors isolated a large number of microorganisms from natural soil and conducted various studies on their products in order to obtain new microorganisms that produce antibiotics and physiologically active substances useful as medicines or veterinary medicines.

As a result, the present invention was completed by discovering that microorganisms belonging to the genus Actinomagilla isolated and collected from soil in Isumi District, Chiba Prefecture produce novel antibiotics SS50732A, B, and C useful as medicines.

Therefore, the present invention provides bacteria producing novel antibiotics SS50732A, B, and C belonging to the genus Actinomadilla.

An example of the method for separating the microorganisms of the present invention from soil is as follows. That is, first, the upper layer of the suspension of the collected soil was smeared onto an agar plate with a normal composition.
After culturing at 7 to 30°C for 20 to 30 days, the microbial colonies that emerged were separated into spheres with a normal composition, and 27 to 3
Cultivate at 0°C for 7 to 14 days to obtain a large number of strains. Then, each strain was incubated at 27-30°C in a medium with a normal composition.
The culture is cultured with shaking for 3 to 5 days, and the antibacterial activity of the culture solution is tested using various test bacteria, and strains having antibacterial activity are selected from among them. Furthermore, these strains were tested in more detail, and SS50732A and B described later were added to the culture solution.
and selectively isolate those that produce an antibiotic named C.

8, which is one of the microorganisms of the present invention obtained in this way.
Strain 50732 has the following mycological properties.

(I) Morphological properties Vegetative hyphae can be grown on glycerin/asparagine agar, starch agar, yeast/malt agar, oatmeal agar, tyrosine agar, nutrient agar, malic acid/
Moderately developed on lime agar, irregularly branched but usually without septa.

Aerial mycelium grows moderately on starch agar, oatmeal agar, malic acid/lime agar, and poorly on glycerin/asparagine agar, glycerin/nitrate agar, tyrosine agar, and nutrient agar; The tongs are yellowish white to pale yellowish pink. It does not grow on sucrose/nitrate agar, glucose/asparagine agar, or yeast/malt agar. When observed under a microscope, spore chains with around 10 chains are attached to the sporophyte. Spore size is 0.6-0.9 x 1.0
~1.3μ and almost oval in shape. The surface of the spore is amorphous. Single nuclei, sporangia and zoospores are not observed.

(n) Properties on various media The growth state of strain 850732 on various media is as shown in the following table. Observations were made after culturing at 28°C for 14 days. The colors are described using the systematic color names from the "Small Dictionary of Color Names" published by Nippon Shikiken Business ■ (1982).

In addition, the number in parentheses in the table indicates the color standard number.

(1) Physiological properties + 1) Growth temperature range (yeast starch agar medium, 1
4-day culture) Optimum growth temperature: 30-40°C Growth temperature: 18-43°C (2) Liquefaction of gelatin
Positive (3) Hydrolysis of starch
Negative (4) Coagulation of skim milk Positive peptonization Positive (5) Production of melanin-like pigments Negative (6) Reduction of nitrates Positive (decomposition of cellulose Negative (IV) Assimilation of carbon sources (28℃,
14-day culture) When Pridham-Godliep agar medium is used as the basal medium, growth is poor in the medium containing glucose, and no difference in growth is observed between the medium and the medium without sugar.

Therefore, 14 L on Pridham-Godliep agar
- Use asparagine as the basal medium.

L-arabinose, D-glucose, D-xylose,
D-mannitol, D-fructose, and L-rhamnose are used, but sucrose, inositol, raffinose, galactose, mannose, and inulin are not used.

(v) Chemical analysis of cells (1) Cell wall composition Diaminopimelic acid is of men type, has no glycine, and no arabinose is observed.

(2) Sugar composition of the whole bacterial cell Contains arabinose, xylose, madulose, galactose, ribose, and crucose.

Therefore, Licipalier (Inter, J, Sys
tem.

Bact, vol. 20, 435i ~ 443 Sada, 1970
The cell wall composition of this bacterium according to the classification of
walltype) is the summer type, and the sugar composition of the whole bacterial cell (Wh
ole cell agar pattern) is B
Becomes a mold.

As mentioned above, since this bacterium has an in-type cell wall composition and a sugar composition of the entire bacterial body, it has been shown that Microbispora, Streptosborangium, Svirirospora, Planomonospora, Dermadophilus, and Actinomagilla It is thought to belong to one of the genus. However, even in this form, spore chains with around 10 chains are attached to the sporophyte, and no sclerotia, sporangia, or zoospores have been found, indicating that this fungus belongs to the genus Actinomagilla. A
ctino-madura). By the way, currently, 30 species belonging to the genus Actenomadula have been approved and officially published (see references (1) to (3) below), but the species that are thought to have the above-mentioned mycological properties are Therefore, this bacterial strain was judged to be a new strain belonging to the genus Actinomagiula, and was classified as Actinomagiula sp. 850732 (Actinomagiula sp.
durasp, 850732) and was deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under accession number FERMP-8613.

Literature: +11 Skerman, V.B.D.
V, Me Gowan, andP, ratio A, 5ne
ath (ed.), 1980. Approved
1istsof bacterial names,
Int, J, 5yst, Bacte-riol,
33: 225-420 (2) W.J., C., Moor.
e, Elizabeth P., Cato, and
Lillian V, H, Moore, 1985. In
dex of the Bacterial and
Yeast NomenclaturalChang
es Published in the Inter
National Journal of Systema
tic Bacteriology Since the
1980 Approved Li5ts ofBa
cterial Names (l January
1980t.

I January 1985), Int, J.
5yst.

Bacteriol, 35: 382-407(3)
D, P, Labeda, R, T, Te5ta, M
, P. Lechevalier.

and H, A, Lechevalier, 1985.
Actinoma-dura yumaensis a
p, nov, Int, J, 5yat.

Bacteriol, 35: 333-336 Novel antibiotic 8850732A using the microorganism of the present invention,
B and C are produced, for example, as follows.

That is, the usual method for culturing actinomycetes is used to culture the strain of the present invention. As the nutrient medium, any synthetic medium, semi-synthetic medium, or natural medium can be used as long as it contains appropriate amounts of assimilable carbon sources, nitrogen sources, inorganic substances, and the like. As the carbon source, for example, glucose starch, fructose, etc. may be used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, and organic acids may also be used. As nitrogen sources, inorganic or organic nitrogen compounds, such as ammonium chloride, ammonium sulfate, ammonium nitrate, urea, sodium nitrate, monosodium glutamate, etc.; or natural products, such as soy flour, yeast extract, NZ amine, peptone, meat extract, dried yeast,
Cottonseed meal, proteose beptone, casamino acids, corn
Stape liquor and the like may be used alone or in combination. Examples of inorganic substances include calcium carbonate, sodium chloride, copper sulfate, manganese sulfate, zinc sulfate, iron sulfate, etc., which may be used alone or in combination. Others 85073
Substances that help the growth of the two strains and promote the production of SS50732A%B and C, or silicone oil or Adekanol (
A general antifoaming agent such as (trade name) can also be added to the medium as appropriate.

As the culture method, the method generally used for culturing the Ifs bacteria as described above is employed, but the liquid culture method, particularly the deep agitation culture method, is most suitable. Cultivation is carried out under aerobic conditions, and a suitable temperature for culturing is 20-35°C.

SS50732A, B, and C reach their maximum production after 3 to 5 days of culture in both shaking culture and deep agitation culture. SS50732A, B and C in culture
When the accumulated amount of f@ reaches the maximum, the f@ culture is stopped, and the target substance is isolated and purified from the culture solution.

Isolation of SS50732A, B, and C from the culture medium is carried out by using various methods alone or in appropriate combinations, taking into account the physical and chemical properties of the compounds, as shown in the Reference Examples below. That is, SS50732A,B
and C are normally present in the culture P solution and the bacterial cells, so the culture solution is separated into the culture P solution and the bacterial cells by centrifugation or filtration, and these are separated using conventional separation methods such as solvent extraction and ion exchange. Separation and purification are carried out by resin method, gel filtration method, adsorption or distribution column chromatography method, high performance liquid chromatography method, dialysis method, precipitation method, etc. alone or in appropriate combinations. In addition, the culture solution is made strongly acidic with an acidic substance such as hydrochloric acid or sulfuric acid, and after being left for a while, a residue is obtained by centrifugation or filtration, and from this, fiSS50732A, B, and C are separated by the above-mentioned normal separation means. It can also be purified.

Examples of preferable separation and purification include the following methods.

First, after the fermentation has been completed, the culture is converted to p)I2 with hydrochloric acid, and after being left for a while, centrifugation is performed to obtain a residue containing p)I2. This is extracted with a chloroform-methanol mixture (2:1.v/v) in a salt rII acid solution. After distilling off the solvent of the extract and washing with n-hexane or petroleum ether, the residue is subjected to silica gel column chromatography. Next, a suitable l@#i! When eluting with a solution, such as a chloroform/methanol mixture, first SS5
The 0732A fraction was eluted and then the SS50732Bti
ui fraction and further 58507320 fractions are eluted. Furthermore, each portion was treated with Sephadex LH20 (manufactured by Pharmacia).
When subjected to column chromatography and eluted with a suitable eluent, such as a chloroform/methanol mixture, all components can be eluted with high purity. Each component thus obtained is again subjected to silica gel column chromatography and eluted with a suitable eluent such as a chloroform/methanol mixture to obtain SS50732A, B and C in crystalline form. In addition, in the above separation and purification, an acidic substance or a basic substance such as acetic acid or ammonia may be added to the eluent.

SS50732A, B and C obtained as described above have the following physical and chemical properties.

■Ultraviolet absorption spectrum The solid line is a chart measured in ethanol. The broken line is a chart obtained by adding a trace amount of sodium hydroxide solution.

This is a chart of measurements taken. Figure 1 is for 5850732, Figure 2 is for 5850732B, Figure 3 is for SS507.
32C ultraviolet and line absorption spectra are shown.

■It is a figure measured by infrared rays absorption spectrum KJJ rden. Figure 4 is SS5
0732 A 1. Figure 5 is SS50732B, No. 6
The figure shows the infrared absorption spectrum of SS50732C.

■Molecular weightResults of measuring polyethylene glycol with different molecular weights using high performance liquid gel chromatography as a reference materialS
The molecular weight of S50732A is 1000, SS50732
B was 950, and SS50732C was 900.

■Color and properties of substances SS50732A, SS50732C from acetic acid solution
is obtained as orange crystals, and SS50732B is obtained as yellow-green crystals.

■Color reaction Both were positive with vanillin sulfate test solution and iodine.

■Solubility Soluble in chloroform/methanol mixture. n-hexane,
Insoluble in water.

■ Thin layer chromatography carrier: Silica gel play 1” TS< (manufactured by Merck & Co., Ltd.) S
S50732A, B, and C have the following biological properties.

■Antibacterial activity The minimum inhibitory concentrations (MIC) of SS50732A, B, and C against various microorganisms are shown in Table 1.

■ Tumor cell growth inhibition effect SS50732A, B, and C were tested for growth inhibition effect on L5178Y cells by the following method. 50 results
It is shown in Table 2 as % inhibitory concentration (ICso).

experimental method? L517BY cells were cultured 2X in RPMI 1640 medium (GIBcOg) supplemented with 10% tallow serum.
10' l [i/-, and SS50732A,
B, C 0.001°0.01, 0.1, 1.0μ
It was added to a final concentration of -. After culturing in a 5% carbon dioxide incubator at 37°C for 48 hours, the number of viable cells was counted. The 6.5ots inhibitory concentration (ICffi) was determined from the cell proliferation inhibition rate at each concentration using the provit-Gui 7g method.
This compound is not only useful as an antibacterial agent, but also has a growth-inhibiting effect on tumor cells, and is therefore a promising compound as an anticancer agent. Therefore, the microorganisms of the present application are excellent for producing useful antibiotics.

In addition, since the number of antibiotics that have been discovered so far in the genus Actinomadilla is extremely limited, Actinomadziula sp. expected to be produced.

Furthermore, in recent years, genetic engineering methods have been applied to the discovery of new antibiotics;
Since strain 32 can be cultured for almost the same number of days as Streptomyces, it is expected that it will serve well as a host.

〔Example〕

Next, the present invention will be explained with reference to Examples and Reference Examples.

Example ±ts1t collected in Isumi District, Chiba Prefecture and air-dried was suspended in sterilized physiological saline 9-, stirred with a mixer for 1 minute, and then serially diluted 10 times (10° to io
-'). 3 ml of each diluted solution Q is dispensed in advance into an actinomycete isolation agar medium, Actinomycete Isolation Agar (manufactured by D7CO) 15 m/ml, and this is added to the solidified agar surface. Furthermore, the above agar medium 4.5-, which had been dissolved by heating and cooled to about 50° C., was added on top of the agar medium, and the agar medium was mixed and allowed to stand. After drying the agar surface aseptically for 20 minutes in clean pliers, it was cultured at 28°C for one month.

Colonies generated by the above culture are transferred using a platinum loop to a slanted agar medium having the following composition, and cultured at 28°C for 14 days.

One platinum loop of the bacteria grown on the above culture medium was diluted 1,000 times with physiological saline, and 1 ml of it was added to the above actinomycete isolation agar medium;
It is mixed with 9vLl of agar (manufactured by D7CO) and cultured in a sterile petri dish at 28°C for 14 days, and it is confirmed visually and microscopically that the resulting colonies are not different from each other.

Of the above colonies, 101i1 colonies were each inoculated onto a slanted medium and cultured at 28°C for 14 days, and it was confirmed macroscopically and microscopically that the bacteria on the 10 slants were the same. It was confirmed that the properties and physiological properties on each medium of these 10 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 cultured bacteria in the 10 pots were monobacteria isolated from nature.

Next, a protective agent (10% skim milk and 1 g of sodium glutamate dissolved in water) was added to the pure cultured bacteria on the slant agar medium, and about 0.5 ml of the spore suspension on the slant was placed in an ampoule for freeze-drying. Dispense into portions and freeze-dry. The freeze-drying is carried out by quickly freezing the ampoule containing the spore suspension in dry ice-acetate, setting it in a freeze dryer, and setting the degree of vacuum to 0.03) or less. . Then, after vacuum sealing with a gas burner, 4°
Save with C. After storing the freeze-dried bacteria (standard sample) obtained in this way for 3 months, open the ampoule and transfer it to a sterilized test tube using a dental mini-spertill, and add condensate solution (i.e.
Niss Pea, Number One, Medium; ISP)/61
As a result of investigating 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 microorganism of the present invention can reliably reproduce the same results by subculture.

Reference example glucose 1%, soluble starch 2t, yeast extract 0.3%,
A liquid medium with a composition of 1% NZ amine, 1% corn-steep liquor, 0.31 calcium carbonate and pH 7.
, 0, and dispense 1 LOOml into a 500 ml volume slope flask to evaporate. This was inoculated with the Actinomadilla sp. 850732 strain (A Koken Bacteria No. 8613) obtained in Example 1, and cultured under shaking at 28° C. for 3 days to prepare a t4 culture solution. Next, a liquid medium having a composition of 1% glucose, 2 t of soluble starch, 2 t of cottonseed meal, 1% corn steep liquor, and 0.3% calcium carbonate was prepared as p)I6.8, and 304 volumes of jar 7 Pour 164 into an Amentor and sterilize.

This was inoculated with 160 rnl of the above-mentioned seed culture C, and cultured for 5 days under conditions of a culture temperature of 28° C., stirring at 40 rpm, and aeration of 1/16 [7 min]. In addition, to prevent the pH from increasing during culture, pi-17 was incubated with a mixture of acetic acid and citric acid.
, 8. The culture solution 166 thus obtained was adjusted to pH 2 with hydrochloric acid, stirred for a while, and then centrifuged to obtain a residue. To this residue, a chloroform/methanol (2:1) mixture 8e was added, stirred, and left to stir. Separate into layers and residual layers. Add 8 parts of a chloroform/methanol (2:1) mixture to the residue again, and after stirring, obtain a chloroform/methanol extract.
The solvent was distilled off under reduced pressure in combination with the previous extract to obtain a crude extract.

After washing this crude extract with n-hexane, silica gel (Merck iRKii = se1ge160.230-400) was used.
mesh) column chromatography (4,5 (φ)
33m). After first developing with 500 m7 of chloroform, 2 ml of chloroform/methanol (98:2) mixture
2, the SS50732C fraction is eluted first. Furthermore, if you develop it with the same (9:1) mixture 26, SS5073
The 2B fraction was eluted, and the same (4:1.) mixture 2, e
When developed, the SS50732A fraction is eluted.

(+) The purified SS50732A fraction of SS50732A was purified by Sephadex L) (20 (manufactured by Pharmacia) column chromatography (3,0φ
x 45 cm) and developed with a chloroform/methanol (2:L) mixture to obtain highly pure SS50732.
A fraction 750#? is eluted. Furthermore, this fraction was purified by silica gel (Merck Kieselge160, 230~
400 mesh) column chromatography (3,0φ
×30 cm) and chloroform/methanol/
High purity SS can be obtained by developing with acetic acid (80:20; 0.5)
The 50732A fraction was eluted. S
I got 350 copies of pure S50732A.

This substance was confirmed by thin layer chromatography and high performance liquid chromatography to be a single substance and to have the above-mentioned physical and chemical properties.

(II) Purification of SS 50732 B SS 507
The 32B fraction was subjected to Sephadex L) I20 (manufactured by Pharmacia) column chromatography (3, Oφx 45
cm) and chloroform/methanol (2:1
) Highly pure SS50732B fraction 8 when developed with a mixed solution
00 m9 is eluted. Furthermore, these two parts are silica gel (
Merck & Co., Ltd. Kiese1ge160.230-400 mesh) column chromatography (3,0φ x 30
cm ) and chloroform/methanol/acetic acid (9
0:10:0.5) to obtain high purity SS5073.
Fraction 2B was eluted. After distilling off both obtained solvents under reduced pressure, SS507 was washed with n-hexane.
I got a pure product of 32B, 3401 da.

It was confirmed by thin layer chromatography or high performance liquid chromatography that this substance is not a single substance and has the physical and chemical properties described above.

(III) Purification of SS50732C SS507
The 32C fraction was subjected to Sephadex L1 (20 (Pharmacia JR) column chromatography (3, Oφx 4.
5 cm) and chloroform/methanol (2:
1) If the mixture is developed, a highly pure 8S50732C fraction of 280% will be eluted. Furthermore, these two fractions were subjected to silica gel (Merck JRKieselge160.230-400 mesh) column chromatography (2,0φX3500).
Chloroform/methanol/vinegar! (9s:2:0
．． 5), a highly pure SS50732C fraction was eluted. After distilling off both the obtained solvents under reduced pressure, the pure product 1 of SS50732C was obtained by washing with n-hexane.
10η was obtained. This substance was confirmed by thin layer chromatography and high performance liquid chromatography to be a single substance and to have the above-mentioned physical and chemical properties.

[Brief explanation of drawings]

Figure 1 shows SS50732A, Figure 2 shows SS50732.
FIG. 3 of B is a drawing showing the ultraviolet absorption spectrum of SS50732C. Figure 4 shows SS50732A, Figure 5 shows SS50732.
FIG. 6 of B is a drawing showing the infrared absorption spectrum of SS50732C. that's all.

Claims (1)

[Claims] 1. SS50, a novel antibiotic belonging to the genus Actinomagilla
732A, B and C producing bacteria.