JPS6269992A - Antibiotic ss42227a and production thereof - Google Patents

Abstract

PURPOSE:To collect an antibiotic from a culture solution and to use it as a drug such as an antimicrobial agent, anti-cancer drug, etc., by cultivating a bacterium belonging to the genus Streptomyces, capable of producing the titled antibiotic. CONSTITUTION:Streptomyces sp. S42227(FERM P-8443) belonging to the genus Streptomyces is aerobically cultivated in a medium containing a carbon source, nitrogen source, inorganic substance, etc. After the cultivation is over, molds are removed and novel antibiotic SS42226A is separated from culture filtrate by solvent extraction method, ion exchange resin method, etc. The prepared antibiotic has properties of C30H33N3O10, 140 deg.C melting point, solubility in diethyl ether and chloroform, slight solubility in water, neutrality, colorless crystal, etc., antibacterial activity and antitumor action.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel antibiotic 5S42227A and a method for producing the same.

[Conventional technology]

A number of antibiotics produced by microorganisms have been known so far.

[Problem that the invention seeks to solve]

An object of the present invention is to provide a new antibiotic that is useful as a medicine.

[Failure to solve the problem]

The present inventors isolated numerous microorganisms from natural soil,
As a result of conducting various studies on the product, it was discovered that a strain isolated from the soil of Itakura-cho, Oura-gun, Gunma Prefecture produces a novel antibiotic 5S42227A having antibacterial and antitumor activities, and the present invention was completed.

That is, the present invention provides antibiotic 5S42227A and a method for producing the same.

842 producing the antibiotic 5S42227A' of the present invention
Strain 227 has the following mycological properties.

(1) Form Cultured at 28℃ on various agar medium for 10-14 days, 8422
The morphology of the 27 strains was observed using an optical microscope or an electron microscope, and the results showed the following characteristics.

The spore-forming hyphae are simply branched from the aerial hyphae, and their tips are spiral-shaped. This can be well observed on sucrose/nitrate agar, oatmeal agar, malic acid-lime agar, and the like.

No axle branching is observed. When matured conidia were observed on an oatmeal agar medium, 10 + [5 or more spore chains were observed, each solid was round to cylindrical in shape, and the size was 0.7 to 0.9 x 1. It is 0 to 1.3 μm. The planar structure of the spore is smooth. We observed 6 sporangia, flagellated spores, and flagellated spores on this strain grown on various agar media.
No special structures such as sclerotia were observed. Furthermore, no division of basal hyphae was observed.

(2) Growth status on various media The growth status of strain 342227 on various media is as shown in the following table. Observations were made after culturing at 28°C for 114 days. In addition, the description of colors is published by 1 Nippon Shikiken Business 4 (1981).
I used the systematic color names from the Color Name Dictionary. In addition, the number in parentheses in the table indicates the color standard number.

Blank space below (3) Physiological properties ■ Growth temperature range (yeast/malt agar medium, 14-day culture) Optimum growth temperature 24-29℃ Possible growth temperature 11-41℃ ■ Liquefaction of gelatin Positive ■ Hydrolysis of starch Positive ■ Coagulation of skim milk Negative Peptonization of skimmed milk Positive ■ Production of melanin-like pigments Negative ■ Reduction of nitrate Positive ■ Decomposition of cellulose Negative (4) Assimilation of carbon sources (Pridham-Godliep agar medium, cultured at 28°C for 14 days) L-arabinose, D-xylose, D-glucose,
D-fructose, D-mannitol, D-galactose, and salicin are used.

Inositol, L-rhamnose, cellulose, sucrose, and raffinose are not used.

(5) Diaminopimelic acid in all bacterial cells As a result of analyzing diaminopimelic acid in all bacterial cells, LL-
Diaminopimelic acid was detected.

The mycological properties of the 842227 strain described above can be summarized as follows.

The tip of the aerial hyphae is spiral-shaped, and its conidia consist of 10 or more spores linked together, and the surface of each spore is smooth. The color tone of the aerial mycelium is gray color series, the color of the underside is yellowish brown, and the soluble pigment is faint brown. No melanin-like pigment is produced.

Based on the above mycological properties and the presence of LL-diaminopimelic acid, strain 842227 was determined to be a strain belonging to the genus Streptomyces. In addition, since we did not find any known strains with the same properties, we used this strain as Streptomyces sp.
sp, 5422271, and was submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 8443 (FIRM p
-8443).

The antibiotic 5S42227A of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. As the antibiotic 5S42227A-producing strain, not only the above-mentioned 842227 strain, but also any artificial mutant strain or natural mutant strain thereof, as long as it has the ability to produce the antibiotic SS42227A, can be used in the present invention. can. The artificial mutant strain of the 842227 strain is similar to that of other actinomycetes, such as UV irradiation Kobal) 60
It can be easily obtained by irradiation, chemical mutagenic agents, etc.

Next, culturing of the bacterial strain in the production of antibiotic 5S42227A will be explained.

First, antibiotic 5S422 belonging to the genus Streptomyces
The 27A production strain is cultured using a conventional method for culturing actinomycetes. 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, fructose, mannitol, starch, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols,
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. and natural products such as soy flour, yeast extract, peptone, meat extract, dried yeast, cottonseed meal, etc. can be used. Groteose peptone, casamino acid, corn steep liquor, etc. are used alone or in combination. As the inorganic substance, for example, calcium carbonate, sodium chloride, copper sulfate, mango chloride, zinc chloride, etc. may be used alone or in combination. Furthermore, other substances that aid the growth of the 842227 strain and promote the production of 5S42227A, silicone oil, or a general foaming agent such as Adekanol (trade name) can also be appropriately added to the medium. As the culture method, methods commonly used for antibiotic production are employed, but liquid culture methods, particularly deep agitation culture methods, are most suitable.

Culture is carried out under aerobic conditions, and the appropriate temperature for culture is 2.
Although the temperature is 4 to 29°C, it is generally preferable to culture around 28°C. The production of antibiotic 5S42227A reaches its maximum after 2 to 4 days of culture in both shaking culture and deep agitation culture.

Next, when the amount of antibiotic 5S42227A accumulated in the culture solution reaches its maximum, the culture is stopped, and the target substance is separated and purified from the culture solution. Isolation and production of 5S42227A from the culture solution are carried out by using various methods alone or in appropriate combinations, taking into consideration the physicochemical properties of this antibiotic, as shown in Examples below. That is, since the antibiotic 5S42227A normally exists in the culture solution, the bacterial cells are separated from the culture by centrifugation or filtration, and the culture p solution is subjected to conventional separation methods such as solvent extraction, ion exchange resin method, gel Antibiotic 'J! Isolate and purify tSS42227A. Examples of preferable separation and purification include the following methods. After fermentation, the culture is centrifuged and separated into fermentation liquid and bacterial cells. The solution is extracted with a suitable solvent such as chloroform. The solvent of the extract was distilled off, and the residue was subjected to silica gel column chromatography. Next, after elution with an appropriate eluent such as chloroform, the active fraction is collected, concentrated under reduced pressure, and the residue is recrystallized with an appropriate solvent such as n-hexane-ethyl acetate mixture to obtain antibiotic 5S42227A.
colorless crystals are obtained.

The antibiotic 5S42227A obtained as described above has the following physicochemical and biological properties.

<Physical and chemical properties> ■Elemental analysis (C, H3, N, 0..・CH, C00C,
)I, as)HN Experimental value (%) 59.77 5.78 6.22
Theoretical value (chi) 59.73 6.04 6.15
■Molecular formula % Formula % ■Infrared absorption spectrum (KBr method) Figure 2 ■'H-NMR, x, vector (90MHz) 3rd
Measurement was carried out using TMS in chloroform solution as a standard substance.

■” C-NMRy, Hect # (215MHz 1
T M S f in deuterated chloroform solution was measured as a reference substance.

δ (ppm): 2016 (s), 17λ7 (s)
, 165.7 (sl.

163.8 (8), 163.2 (8), 156
．． 0 (s), 149.6 (s), 134.4
(s), 133.1 (s), 128.4 (!I
t.

127.7 (di, 126.9 (s), 125
．． 1 (d), 123.9 (d), 121.8 (
d), 120.1 (s), 108.6 (d).

84.0 (d), 76.9 (d), 76.7
(d), 56.0 (s l.

55.6 ((1), 53.7 (t l, 50.
6 (t l, 45.4 (d).

35.8 (t), 27.2 ((1), 20.
7 ((11,20,0(ql.

17.0 ((11) Solubility in solvents Soluble in diethyl ether, chloroform, ethyl acetate, acetone, and methanol.

n-hexane, poorly soluble in water.

OBasic/acidic/neutral distinction neutral Color and properties of 0 substances Colorless crystals (recrystallized from ethyl acetate-n-hexane) O color reaction Positive for Dragendorff reagent.

Biological Properties 5S42227A of the present invention has the following biological properties.

■Antibacterial activity The minimum inhibitory concentration (MIC) of 5S42227A against various microorganisms is shown in Table 1.

Table 1 - Antitumor Effect The therapeutic effect of 5S42227A on murine leukemia P-388 was tested by the following method. The results are shown in Table 2. In addition, the survival effect in the table is the number of survival days (C
The ratio of survival days (Tl) to the number of survival days (Tl) in the treatment group was expressed as a percentage.

Practical method: 1×10' P-388, cells as CDF,
After 24 hours, 5S42227A was implanted into the abdominal cavity of a mouse (Female 2 Nippon Charles River) once a day, for a total of 1 dose.
It was administered intraperitoneally 0 times.

Table 2 [Actions and effects of the invention] In view of these antibacterial and antitumor activities, the compound of the present invention 5
S42227A is useful as an antibacterial and anticancer agent.

〔Example〕

A liquid medium having a composition of 0.5% soluble starch and 0.5% cottonseed meal is adjusted to pH 7.0 and sterilized by dispensing 100 ml into a 500 ml Sakalo flask. This was combined with Streptomyces sp. 842227 strain (,
No. 443) and cultured with shaking at 28°C for 2 days to prepare a seed culture. Next, a liquid medium having a composition of 0.5% soluble starch and 0.5% cottonseed meal was adjusted to pH 7.0, and
Fill a 0.8-volume jar fermentor with 16 types, inoculate it with the above seed culture solution 160-, and culture for 70 hours at a culture temperature of 28°C, stirring rate of 450 rpm, and airflow rate of 16 p/min. After completion of the culture, the culture solution was centrifuged, and the resulting tear fluid was extracted with an equal volume of chloroform. The crude extract obtained by concentrating the solvent under reduced pressure was purified using silica gel (Merck [Ki
esel Gel 60.230-400 mesh) column chromatography (4,0 (φl x 40 c
ml, chloroform:methanol (100:2
) to obtain the active fraction.

After concentrating the target substance thus obtained to dryness, it was recrystallized from a mixture of n-hexane and ethyl acetate.
20 squares of colorless crystals of SS42227A were obtained.

This substance has the above-mentioned physicochemical properties.

[Brief explanation of drawings]

Fig. 1 shows the ultraviolet absorption spectrum of the antibiotic 5S42227A of the present invention, Fig. 2 shows its infrared absorption spectrum, and Fig. 3 shows its I-NMR spectrum. that's all

Claims (1)

[Claims] 1. Antibiotic SS4222 having the following physicochemical properties
7A. (1) Elemental analysis (C_3_0H_3_3N_3O_1_
0・CH_3COOC_2H_5) C H N Experimental value (%) 59.77 5.78 6.22 Theoretical value (%) 59.73 6.04 6.15 (2) Molecular formula C_3_0H_3_3N_3O_1_0 (3) FAB-mass spectrum (M+H )^+m/z596 (4) Melting point 140℃ (decomposition) (5) Ultraviolet absorption spectrum Figure 1 217 (
52,000) λ^M^e^O^H_m_a_xnm(ε) 248(
sh. ) 344(
5,000) (6) Infrared absorption spectrum (KBr method) Figure 2 (7)^1H-NMR spectrum (90 MHz) Figure 3 Measurement was carried out using TMS in deuterated chloroform solution as a reference substance. (8)^1^3C-NMR spectrum (22.5MHz
) Measurements were made using TMS in deuterated chloroform solution as a reference substance. δ (ppm): 202.6 (s), 172.7 (s),
165.7 (s), 163.8 (s), 163.2 (s
), 156.0 (s), 149.6 (s), 134.4
(s), 133.1(s), 128.4(s), 127
．． 7(d), 126.9(s), 125.1(d), 1
23.9(d), 121.8(d), 120.1(s)
, 108.6(d), 84.0(d), 76.9(d)
, 76.7(d), 56.0(s), 55.6(q),
53.7(t), 50.6(t), 45.4(d), 3
5.8(t), 27.2(q), 20.7(q), 20
．． 0(q), 17.0(q) (9) Solubility in solvents Soluble in diethyl ether, chloroform, ethyl acetate, acetone, and methanol. n-Hexane, poorly soluble in water. (10) Basic, acidic, neutral (11) Color and properties of the substance Colorless crystals (recrystallized from ethyl acetate-n-hexane) (12) Color reaction Positive for Dragendorff's reagent. (13) Thin layer chromatography carrier: Silica gel plate F_2_5_4 (manufactured by Merck & Co., Ltd.) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ 2. Cultivate SS42227A-producing bacteria belonging to the genus Streptomyces, and use the culture to produce antibiotic SS42227A
An antibiotic SS42227A characterized by collecting
manufacturing method. 3. The SS42227A substance-producing bacterium is Streptomyces
Streptomyces sp. S42
227 (KAIKOKEN BIYORI NO. 8443).