Pharmaceutically acceptable salts of the antibiotic C-19393 E.sub.5

Novel antibiotic C-19393 E.sub.5, which is produced by cultivating a microorganism belonging to the genus Streptomyces, has strong antimicrobial activity against gram-positive and gram-negative bacteria and, thus, are useful as a bactericide or disinfectant.

This invention relates to Antibiotic C-19393 E.sub.5, a new .beta.-lactam 
antibiotic compound, which has the formula (I): 
##STR1## 
and the salts thereof and to a method for producing said antibiotic and 
salts. The present inventors previously discovered and developed 
Antibiotic C-19393 S.sub.2 and Antibiotic C-19393 H.sub.2, both of which 
are novel .beta.-lactam antibiotics, and methods for producing them. These 
antibiotics and methods were described and claimed in Japanese Patent 
Laid-open Nos. 104296/1980 and 5496/1981. Thereafter, the present 
inventors discovered in the fermentation broths of C-19393 S.sub.2 - and 
C-19393 H.sub.2 - producing strains a new antibiotic compound which was 
quite distinct from said antibiotics, and designated it Antibiotic C-19393 
E.sub.5. Following that discovery, the inventors sought for a commercially 
useful method of producing Antibiotic C-19393 E.sub.5 and have ultimately 
developed the present invention. 
The present invention is directed to: 
(1) Antibiotic C-19393 E.sub.5, inclusive of its salts; 
(2) a method for producing Antibiotic C-19393 E.sub.5 characterized by 
cultivating a C-19393 E.sub.5 - producing strain of the genus Streptomyces 
in a culture medium to cause the strain to elaborate and accumulate 
C-19393 E.sub.5 in the resultant culture broth and harvesting the same 
antibiotic; and 
(3) a method according to (2) wherein said C-19393 E.sub.5 -producing 
strain is a strain which does not give normal growth on a medium 
containing inorganic sulfates as exclusive sources of sulfur but gives 
normal growth in the presence of at least one member selected from the 
group consisting of thiosulfates, cysteine and methionine. 
Hereinafter, Antibiotic C-19393 E.sub.5 will sometimes be referred to 
briefly as C-19393 E.sub.5.

Production of Antibiotic C-19393 E.sub.5 in accordance with this invention 
is carried out using a microorganism belonging to the genus Streptomyces 
which is capable of elaborating C-19393 E.sub.5. Such a microorganism will 
hereinafter be referred to briefly as a C-19393 E5-producing strain. 
As an example of such C-19393 E.sub.5 - producing strain, there may be 
mentioned Streptomyces sp. C-19393 [FERM-P No. 4774, IFO 13886, ATCC 31486 
NARL 15037. This microorganism was subsequently identified as Streptomyces 
griseus subsp. cryophilus C-19393]. Hereinafter, this strain will 
sometimes be referred to briefly as the C-19393 strain. The C-19393 strain 
elaborates Antibiotic C-19393 S.sub.2 and Antibiotic C-19393 H.sub.2 
[German Offenlegungsschrift No. 3003624] as well. 
In order that the present invention may be carried into practice more 
advantageously, it is desirable to use a strain belonging to the genus 
Streptomyces which is capable of elaborating C-19393 H.sub.2 and which 
would not give normal growth on a medium containing inorganic sulfates as 
exclusive sources of sulfur but gives normal growth in the presence of at 
least a member selected from the group consisting of thiosulfates, 
cysteine and methionine. The inorganic sulfates mentioned as sources of 
sulfur include alkali metal sulfate (e.g. Na.sub.2 SO.sub.4, K.sub.2 
SO.sub.4), alkaline earth metal sulfate (e.g. CaSO.sub.4, MgSO.sub.4), 
heavy metal sulfate (e.g. FeSO.sub.4, Fe.sub.2 (SO.sub.4).sub.3, 
CuSO.sub.4, ZnSO.sub.4, NiSO.sub.4), ammonium sulfate and other sulfates 
which would yield SO.sub.4.sup.-- ions and which would not inhibit the 
growth of microorganism. On the other hand, the thiosulfates may for 
example be sodium thiosulfate, potassium thiosulfate, ammonium 
thiosulfate, etc. 
The test of growth of the C-19393 E.sub.5 -producing strain is performed on 
synthetic agar medium. The screening procedure may for example be as 
follows. First, two kinds of media are prepared. Medium (1): 10 g of 
glycerol, 1 g of L-asparagine, 1 g of dipotassium phosphate, 0.2 g of 
magnesium sulfate, 20 g of agar (Difco Lab., U.S.A.) and 1000 ml of 
distilled water (pH 7.1). Medium (2): the same medium as (1) plus 50 mg of 
sodium thiosulfate, L-cysteine or L-methionine. Each medium is 
steam-sterilized and put into a Petri dish. After the agar is set, the 
dish is inoculated with a suspension of the spores or mycelia of the 
C-19393 H.sub.2 -producing strain (When any carry-over of growth factors 
from the preculture is suspected, the spore suspension is washed with 
distilled water once) and the inoculated dish is incubated at 28.degree. 
C. for 2 to 7 days. There will be an abundant growth on plate (1), whereas 
either no growth or only a sparse growth will be found on plate (2). 
As examples of useful C-19393 E.sub.5 -producing strains, there may be 
mentioned Streptomyces griseus subsp. cryophilus K-4, K-101 and SR-135 
(hereinafter sometimes referred to briefly as K-4 strain, K-101 strain and 
SR-135 strain, respectively) which have been obtained by subjecting the 
parent C-19393 strain to mutagenic treatment. These and other mutants can 
be derived, for example by the following methods. 
(A) Selection from among the mutants requiring for growth at least a member 
selected from the group consisting of thiosulfates, cysteine and 
methionine. 
The suspension of spores prepared by mutagenic treatment of the C-19393 
strain is spread on Bennet's agar medium, for instance, to cultivate 
colonies (about 10 to 100 per plate). These colonies are replicated onto 
(i) a minimal medium [e.g. 10 g of glycerol, 1 g of L-asparagine, 1 g of 
dipotassium phosphate, 0.2 g of magnesium sulfate, 20 g of agar and 1000 
ml of distilled water; pH 7.1] and (ii) a screening medium [the minimal 
medium supplemented with 50 to 500 mg of thiosulfate, L-cysteine or 
L-methionine] and each of the media is incubated at 24.degree.-30.degree. 
C. for 2-5 days. The mutant which gives as good growth as the parent 
strain on the screening medium but gives either no growth or only sparse 
growth on the minimal medium is selected. The mutant is cultured on a 
liquid medium and its elaboration product is examined. The desired mutant 
strain is accordingly selected. 
(B) Selection of a selenate-resistant strain 
The suspension of spores of the C-19393 strain subjected to mutagenic 
treatment is spread on a minimal agar medium containing 0.025 to 0.2% 
(preferably 0.05-0.2%) of a selenate (e.g. sodium selenate, ammonium 
selenate) at a high concentration and incubated at 24.degree.-30.degree. 
C. for 2-14 days. If the concentration of the selenate is low, a thin 
growth will be formed all over the agar surface. The strain giving a large 
colony on the plate is picked and reinoculated on an agar medium 
containing the selenate in the same concentration to purify the colony. 
The resultant grown colony is picked and incubated on a slant medium. The 
selenate-resistant strain thus obtained is subjected to liquid culture and 
its elaboration product is examined. In this manner, the contemplated 
mutant strain can be selected. 
The mechanism by which the desired mutant strain can be obtained with 
efficiency by method (A) or (B) remains yet to be fully elucidated but it 
is likely that a defect was produced in the organism in connection with 
the incorporation or metabolism of inorganic sulfate ions. 
The mutagenic treatment mentioned hereinbefore may be any suitable chemical 
or physical treatment that is known, such as UV light irradiation and a 
treatment with NTG(N-methyl-N'-nitro-N-nitrosoguanidine), ethyl 
methanesulfonate, nitrous acid or the like. 
The three strains of Streptomyces griseus subsp. cryophilus K-4, K-101 and 
SR-135 have been deposited with Institute for Fermentation, Osaka, Japan, 
Fermentation Research Institute, Agency of Industrial Science and 
Technology, Tsukuba, Japan, The American Type Culture Collection, U.S.A. 
and the U.S. Department of Agriculture, Northern Regional Research 
Laboratory, U.S.A. under the following IFO numbers, FERM-P numbers and 
ATCC number since Oct. 16, 1980, Oct. 17, 1980, Oct. 28, 1980, and Aug. 2, 
1982 respectively. 
______________________________________ 
IFO FERM-P No. ATCC NRRL 
______________________________________ 
K-4 14089 5750 31740 15115 
K-101 14090 5751 31741 15116 
SR-135 14091 5752 31742 15117 
______________________________________ 
The bacteriological characteristics of these strains, except for 
biochemical properties and antibiotic producing ability, are substantially 
similar to those of the parent C-19393 strain. However, compared with the 
parent strain, the K-4 strain features a considerably sparse production of 
aerial mycelium. 
(a) Morphological characteristics 
The aerial mycelium, about 1.mu. in length, extends from a well-branched 
substrate mycelium and branches out monopodially to present side branches 
each carrying straight or slightly curved chains of spores in the manner 
of rectus flexibilis. The spores are cylindrical 
(0.35-0.55.mu..times.0.7-1,4.mu.), each having a smooth surface. There are 
no other special organs such as spherical sporangium, flagellum, 
sclerotium, etc. 
(b) Cultural characteristics 
The cultural characteristics of the present strain on various media are 
shown in Table 1. Unless otherwise indicated, these are observations after 
incubation at 28.degree. C. for 2 weeks. 
TABLE 1 
______________________________________ 
Aerial Soluble 
Medium Growth Mycelium Reverse 
Pigment 
______________________________________ 
(1) Sucrose nitrate 
Moderate White Colorless 
None 
agar 
(2) Glucose aspara- 
Slight White Colorless 
None 
gine agar 
(3) Glycerin Moderate White Colorless 
None 
asparagine agar 
(4) Starch agar Moderate None Ocher None 
(5) Nutrient agar 
Moderate White Ivory None 
(6) Tyrosine agar 
Moderate None Colorless 
None 
(7) Yeast malt agar 
Moderate None Grayish 
None 
yellow 
(8) Oatmeal agar 
Moderate White Colorless 
None 
______________________________________ 
Note 
Media (1), (2), (3), (4) and (6) were each supplemented with 50 mg/l of 
sodium thiosulfate. 
(c) Physiological characteristics 
(1) Temperature range for growth: 
lower limit: lower than 15.degree. C. 
upper limit: 32.degree. to 35.degree. C. 
optimal temperature: 26.5.degree. to 30.degree. C. 
(2) Gelatin: liquefied 
(3) Starch: hydrolyzed 
(4) Skim milk: peptonized but not coagulated 
(5) Melanoid pigment: 
Tyrosine agar: not produced 
Peptone yeast extract iron agar: nor produced 
(6) The carbohydrate assimilation spectrum of the strain (Pridham-Gottlieb 
agar with 50 mg/l of sodium thiosultate) is shown in Table 2. 
TABLE 2 
______________________________________ 
Carbon Sources Assimilation 
______________________________________ 
Glycerin + 
i-Inositol .+-. 
D-Mannitol - 
D-Xylose .+-..about.+ 
L-Arabinose .+-..about.+ 
D-Glucose + 
D-Galactose + 
D-Fructose + 
Maltose + 
Sucrose - 
Rhamnose + 
Raffinose - 
Starch + 
Cellulose .+-. 
Control (No addition) 
- 
______________________________________ 
-: no growth; 
+: growth 
.+-.: doubtful growth; 
(d) Biochemical characteristics 
All of K-4, K-101 and SR-135 strains require for growth at least one of 
sodium thiosulfate, cysteine and methionine. 
The cultivation of such strain for the production of C-19393 E.sub.5 in 
accordance with this invention is conducted using a culture medium 
containing nutrients which the strains may utilize for growth. Among 
useful medium components are such sources of carbon as glucose, starch, 
glycerin, dextrin, sucrose, millet jelly, molasses, etc. and such sources 
of nitrogen as meat extract, dried yeast, yeast extract, soybean flour, 
corn steep liquor, wheat germs, cottonseed flour, ammonium sulfate, 
ammonium nitrate, etc. As additional components, such inorganic salts as 
calcium carbonate, sodium chloride, potassium chloride, phosphates, etc. 
as well as organic and inorganic components which will either assist in 
the growth of the strains or contribute to an increased output of C-19393 
E.sub.5 may be incorporated into the culture medium. 
If necessary, heavy metals such as ferrous sulfate, copper sulfate, etc. 
and vitamins such as vitamin B.sub.l, biotin, etc. are also added to the 
medium. It will also be beneficial to incorporate in the medium an 
antifoaming agent or surfactant such as silicone oil or a polyalkylene 
glycol ether. Other organic and inorganic substances which aid the growth 
of microorganism and promote the production of C-19393 E.sub.5 can be 
added to the medium in suitable amounts. 
Cultivation of the strain can be conducted by the conventional procedure 
for the microbial production of antibiotics using solid or liquid media. 
When, for example, a liquid medium is employed, cultivation may be carried 
out by any of such methods as stationary culture, stir culture, shake 
culture, aerobic culture and so on, although submerged aerobic culture is 
preferable. The preferred range of cultivation temperature is about 
15.degree. C. to about 32.degree. C., the preferred range of medium pH is 
about 4 to about 8, and the cultivation time is about 8 to about 168 
hours, preferably about 24 to about 144 hours. 
Since C-19393 E.sub.5 is mostly produced extracellularly, it is a preferred 
procedure to subject the culture broth to centrifugation or filtration to 
obtain a supernatant and isolate the desired antibiotic from the 
supernatant. It is, however, also possible to isolate and purify the 
antibiotic directly from the culture broth. 
The potency assay of the product thus obtained may be performed against 
Comamonas terrigena IFO 13299 as the test organism and using C-19393 
E.sub.5 -producing strain as the standard by the cylinder-plate method or 
by the paper employing a bouillon agar and TSA (trypticase soy agar; 
Baltimore Biologicals, Co., Ltd., U.S.A.) as the assay medium. 
For the isolation of C-19393 E.sub.5, the procedures conventionally 
employed for the harvest and isolation of microbial metabolites can be 
utilized to advantage. For example, since C-19393 E.sub.5 is a 
water-soluble acid substance and is mostly produced extracellularly, it is 
generally isolated by subjecting the culture broth to centrifugation or 
filtration to separate the microbial cells and isolating and purifying the 
activity from the filtrate. Thus, for example, various separation methods 
such as those utilizing differential solubilities or distribution 
coefficients in solvents, differential rates of precipitation or 
differential adsorption affinities, ion-exchange chromatography, molecular 
sieve chromatography, concentration in vacuo, lyophilization, etc. can be 
utilized in a suitable combination and in any order or in repetition. By 
way of illustration, activated carbon, adsorbent resins, anion-exchange 
resins (anionic type), cellulose powder, silica gel, etc., as well as 
those supports functioning as molecular sieves can be employed. Elution 
from such adsorbents or supports may be effected with an eluent 
appropriate to the particular adsorbent or carrier used, such as 
water-soluble organic solvents such as acetone, methanol, ethanol, 
propanol, butanol, isopropyl alcohol, isobutanol, etc., aqueous solutions 
of such water-soluble solvents, acid, alkali or buffer solutions, or 
aqueous solutions of inorganic or organic salts. 
More specifically, the first step in the separation and purification 
procedure comprises filtering the culture broth in the presence of a 
filter aid to remove the microbial cells. Since Antibiotic C-19393 E.sub.5 
is an acidic compound, the Cl.sup.- or ACO.sup.- forms of anion-exchange 
resins such as Amberlite IRA-400, 402, 410 (Rohm and Haas Co., U.S.A.), 
Dowex-1 (Dow Chemical Co., U.S.A.) and Diaion SA-21 A and C (Mitsubishi 
Kasei K.K., Japan) can be utilized to advantage in further purification. 
In this case, the adsorbed antibiotic is eluted with an aqueous solution 
of sodium chloride or a buffer solution. The eluate is then passed through 
a high-porous adsorbent resin, e.g. Diaion HP-20 (Mitsubishi Kasei K.K., 
Japan), and the adsorbed activity is eluted with water. Desalination of 
this eluate is carried out by activated carbon chromatography using an 
aqueous alcohol or the like as an eluent. The eluate is concentrated at 
reduced pressure and low temperature to remove the alcohol, followed by 
addition of sodium chloride to a concentration of 5%. The solution is 
subjected again to HP-20 chromatography and elution is carried out with a 
5% aqueous solution of sodium chloride, further followed by desalting with 
activated carbon. The eluate obtained using an aqueous alcohol or the like 
is concentrated at low temperature to remove the alcohol or the like. The 
residue is passed through a resin such as Diaion WA-30 (acetate-form, 
Mitsubishi Kasei K.K., Japan) and elution is carried out with a 1M aqueous 
solution of sodium chloride. The eluate is desalted with activated carbon 
and, as the eluent, aqueous isobutanol. Satisfactory results can be 
obtained if dilute aqueous ammonia is added to the eluent so as to carry 
out the elution under neutral or slightly basic conditions. The eluate 
containing the antibiotic activity is concentrated under reduced pressure, 
acetone is then added to the residue, and the resulting precipitate is 
recovered by filtration. This powdery precipitate can be further purified 
by column chromatography, for example by using an adsorbent resin HP-20 or 
XAD (Rohm and Haas Co., U.S.A.) and DEAE or QAE-Sephadex (Cl-form, 
Pharmacia, Sweden) in combination. Thus, the above powder is dissolved in 
a small amount of water and chromatographed on a column of XAD-II, elution 
being carried out with water. The active fractions are pooled and 
concentrated, and the concentrate is further passed through a 
QAE-Sephadex-25 (Cl.sup.31 -form). Elution is carried out with 0.04M 
phosphate buffer and the eluate is desalted by activated carbon 
chromatography in the same manner as described above. The eluate is 
concentrated and the concentrate is chromatographed on a column of HP-20, 
elution being carried out with water. The fractions giving a single peak 
on liquid chromatography described hereinafter are pooled and concentrated 
to dryness at reduced pressure and low temperature. To the concentrate is 
added acetone, for instance, whereby C-19393 E.sub.5 is obtained. This 
compound is capable of forming metal or ammonium salts. The metal salts 
include sodium, potassium, lithium and other salts. 
The physical and chemical properties of the sodium salt of C-19393 E.sub.5 
prepared in accordance with Example 1, which appears hereinafter, are as 
follows. 
(1) Appearance: White powder 
(2) Thin-layer chromatography: 
Cellulose f (Tokyo Kasei Co., Ltd., Japan) and a developer solvent system 
of n-propanol/water (4:1): Rf=0.57.+-.0.1. 
DEAE-cellulose (Tokyo Kasei Co., Ltd., Japan) and a developer solvent 
system of 0.02M aqueous NaCl/n-propanol (1:1): Rf=0.44.+-.0.1. 
(3) High-voltage paper electrophoresis: Electrophoretic mobility on Whatman 
No. 1 paper [W and R Balston, Ltd., England] in 0.02M phosphate buffer (pH 
6.9) at a potential gradient of 45 V/cm for 50 minutes: 11 cm.+-.2 cm 
toward the anode. 
(4) Molecular formula: C.sub.13 H.sub.15 N.sub.2 O.sub.6 SNa 
(5) High-performance liquid chromatography (Waters Associates Inc., 
U.S.A.): HPLC on Microbondapak C.sub.18 using 0.02M phosphate buffer with 
4% methanol (pH 6.3) and 0.02M phosphate buffer with 6% methanol (pH 6.3) 
at the flow rate of 2 ml/min. showed the retention times of 4.2 min. and 
2.8 min., respectively. 
(6) Ultraviolet absorption spectrum: The UV spectrum of C-19393 E.sub.5 in 
aqueous solution is depicted in FIG. 1 and the maximum values are as 
follows: .lambda..sub.max.sup.H.spsb.2.sup.O(E.sub.1 cm.sup.1%)=244.+-.2 
nm (374.+-.20) and 286.+-.2nm (313.+-.20). 
(7) Infrared absorption spectrum: The IR spectrum (KBr disk) of C-19393 
E.sub.5 is depicted in FIG. 2. Main absorptions (wave-numbers) are as 
follows: 3400, 2980, 1765, 1700, 1620, 1505, 1380, 1260, 1160, 1100, 1055, 
1000, 970, 940, 870, 810, 790, 690, 640, 600, 520, 450 (cm.sup.-1) 
(8) Circular dichroism spectrum (in H.sub.2 O): The spectrum shows a 
positive Cotton effect at 234 nm and negative Cotton effects at 210, 260 
and 290 nm. 
(9) Color reactions: 
Ehrlich: positive 
Potassium permanganate: positive 
Ninhydrin: negative 
(10) Solubility: Readily soluble in water; insoluble in benzene, ethyl 
acetate and acetone. 
(11) Proton magnetic resonance spectrum: The characteristic chemical shifts 
of C-19393 E.sub.5 (sodium salt) in D.sub.2 O at 100 MHz are as follows: 
1 ca. 1.38 ppm, doublet (J=ca. 6 Hz) 
##STR2## 
2 ca. 2.16 ppm, sharp singlet (CH.sub.3 --CO--NH--) 3 ca. 2.8-4.0 ppm, 2H 
and 1H, multiplet 
##STR3## 
4 ca. 4.0-4.6 ppm, 2H, multiplet 
##STR4## 
5 ca. 6.44 ppm, doublet (J=14 Hz) (--N--CH=) 6 ca. 7.60 ppm, doublet (J=14 
Hz) (--S--CH=) 
Based on the above physical and chemical properties, C-19393 E.sub.5 is 
assumed to be a compound having the structural formula (I). 
The properties of C-19393 E.sub.5 described above are in good accord with 
those of the compound obtained by oxidation of Epithienamycin B. 
The biological properties of C-19393 E.sub.5 are as follows. The 
antibacterial spectrum of the sodium salt of C-19393 E.sub.5 is shown in 
Table 3. It is apparent from the data that C-19393 E.sub.5 is a broad 
spectrum antibiotic which is highly active against both gram-positive and 
gram-negative bacteria. 
TABLE 3 
______________________________________ 
Antimicrobial Spectrum of Antibiotic C-19393 E.sub.5 
Sodium Salt 
Minimum Inhibitory 
Test Organism Concentration (.mu.g/ml) 
______________________________________ 
Escherichia coli NIHJ 
0.78 
Salmonella typhimurium IFO 12529 
0.78 
Klebsiella pneumoniae IFO 3317 
6.25 
Proteus vulgaris IFO 3988 
1.56 
Proteus mirabilis ATCC 21100 
3.13 
Serratia marcescens IFO 12648 
6.25 
Alcaligenes faecalis IFO 13111 
1.56 
Pseudomonas aeruginosa IFO 3080 
6.25 
Comamonas terrigena IFO 13299 
0.78 
Staphylococcus aureus 209P 
0.78 
Sarcina lutea IFO 3232 
0.2 
Bacillus subtilis PCI 219 
0.78 
______________________________________ 
Note 
Medium: Bouillon agar 
As shown in Table 3 above, C-19393 E.sub.5 obtained in accordance with the 
present invention has strong antimicrobial activity against gram-positive 
and gram-negative bacteria and, thus, can be used in the treatment of 
bacterial infections in mammalian animals (e.g. mouse, rat, dog, human 
being) and domestic fowls (e.g. poultry, duck). 
To use C-19393 E.sub.5 as a drug for the treatment of Escherichia coli 
infections, for instance, it can be formulated into an injectable solution 
in physiological saline and administered parenterally, e.g., 
subcutaneously or intramuscularly at the daily dose level of 0.1 to 50 
mg/kg or preferably 0.5 to 20 mg/kg. It may also be formulated into an 
encapsulated preparation together with lactose, for instance, and 
administered in the daily dose of 1 to 100 mg/kg or preferably 5 to 50 
mg/kg (as C-19393 E.sub.5). 
C-19393 E.sub.5 can be used as a disinfectant. For example, it can be 
dissolved in distilled water to a concentration of 0.01 to 0.1 w/v % so as 
to provide a liquid disinfectant or formulated with a base such as 
vaseline or lanolin to provide an ointment containing 0.2 to 20 mg or 
preferably 1 to 10 mg of C-19393 E.sub.5 per gram. The solution and 
ointment can each be externally applied as a bactericide or disinfectant 
for the extremities, eyes, ears, etc. of said animals. 
Antibiotic C-19393 E.sub.5 is also a very promising compound as an 
intermediate for the synthesis of new pharmaceutical agents. The aqueous 
solution of C-19393 E.sub.5 is stable in the neutral pH region. 
EXPERIMENT 1 
The spores (10.sup.7 /ml) of Streptomyces griseus subsp. cryophilus C-19393 
(IFO 13886, ATCC 31486) were suspended in 10 ml of 0.05 M tris-maleic acid 
buffer (pH 8.9). To the suspension was added 
N-methyl-N'-nitro-N-nitrosoguanidine at the rate of 1 mg/ml and, then, the 
mixture was shaken at 30.degree. C. for 60 minutes. The reaction mixture 
was subjected to centrifugation (10,000.times.g, 15 min.), to remove the 
supernatant, followed by addition of 10 ml of sterile distilled water to 
the residue to wash the spores well. The mixture was again centrifuged to 
collect the spores. The spores thus obtained were suspended in 10 ml of 
sterile distilled water. The suspension was diluted with sterile distilled 
water to 10.sup.2 -10.sup.4 of spores. A 0.1 ml each portion of the 
suspension was spread on Bennet's agar medium (composition: 1 g of yeast 
extract, 1 g of meat extract, 2 g of NZ amine A, 10 g of glucose, 1000 ml 
of distilled water and 20 g of agar; pH 7.1) and incubated at 28.degree. 
C. for 4 days. 
The 20 plates were selected from among those cultivated with about 20-100 
colonies per plate. These colonies were inoculated by the replicate method 
onto a minimal medium (composition: 10 g of glycerol, 1 g of L-asparagine, 
1 g of dipotassium phosphate, 0.2 g of magnesium sulfate, 20 g of agar and 
distilled water; pH 7.1) and a screening medium A (the minimal medium 
supplemented with 50 mg/l of L-cysteine) and each of the media was 
incubated at 28.degree. C. for 3 days. Another 20 plates were selected 
from among those cultivated with about 20-100 colonies per plate. These 
colonies were replicated on a minimal medium (the same as said 
composition) and a screening medium B (the minimal medium supplemented 
with 50 mg/l of sodium thiosulfate) and each of the media was incubated at 
28.degree. C. for 3 days. 
In comparison of the growth of colonies on the minimal medium with that on 
the screening medium, K-4 strain was selected from colonies which gave 
only sparse growth on the minimal medium but gave good growth on the 
screening medium A, and K-101 strain was selected from colonies which gave 
abundant growth on the screening medium B. 
These strains were purified on the respective screening media. 
The strains thus obtained were incubated on minimal media containing sodium 
thiosulfate, L-cysteine or L-methionine in various concentrations at 
28.degree. C. for 4 days. The results of growth are shown in Table 4. 
TABLE 4 
__________________________________________________________________________ 
Concentration of Compound (mg/ml) 
Compound 
Strain 
0 3.125 
6.25 
12.5 
25 50 100 
__________________________________________________________________________ 
Sodium K-4 .-+. 
+ + ++ +++ +++ +++ 
thiosulfate 
K-101 
.-+. 
.+-. 
.+-. 
.+-. 
+ ++ +++ 
C-19393 
+++ +++ +++ +++ +++ +++ +++ 
L-Cysteine 
K-4 .-+. 
+ + ++ +++ +++ +++ 
K-101 
.-+. 
+ ++ +++ +++ +++ +++ 
C-19393 
+++ +++ +++ +++ +++ +++ +++ 
L-Methionine 
K-4 .-+. 
+ ++ ++ +++ +++ +++ 
K-101 
.-+. 
+ ++ +++ +++ +++ +++ 
C-19393 
+++ +++ +++ +++ +++ +++ +++ 
__________________________________________________________________________ 
- No growth, 
+ Growth, 
.-+. Sparse growth, 
++ Good growth, 
.+-. Moderate growth 
+++Abundant growth 
EXPERIMENT 2 
The suspension of spores prepared by mutagenic treatment similar to 
Experiment 1 was diluted to 10.sup.0 -10.sup.2 of spores. A 0.1 ml portion 
of the suspension was spread on a plate of agar medium which was added to 
a minimal medium (composition: same as Experiment 1) with 500 mg/1 of 
sodium selenate and incubated at 28.degree. C. for 7 days. A scant growth 
was observed all over the agar surface. The strain giving a large colony 
on the plate was selected and reinoculated on an agar medium containing 
sodium selenate to purify the colony. The resultant grown colony was 
picked out and incubated on a slant medium. In this manner, SR-135 strain 
was selected. 
SR-135 and C-19393 strains were incubated on a minimal medium containing 
sodium thiosulfate, L-cysteine or L-methionine at 28.degree. C. for 4 
days. 
The results of growth are shown in Table 5. 
TABLE 5 
__________________________________________________________________________ 
Concentration of Compound (mg/l) 
Compound 
Strain 
0 3.125 
6.25 
12.5 
25 50 100 
__________________________________________________________________________ 
Sodium SR-135 
.+-. 
+ ++ ++ +++ +++ +++ 
thiosulfate 
C-19393 
+++ +++ +++ +++ +++ +++ +++ 
L-Cysteine 
SR-135 
.+-. 
++ +++ +++ +++ +++ +++ 
C-19393 
+++ +++ +++ +++ +++ +++ +++ 
L-Methionine 
SR-135 
.+-. 
++ +++ +++ +++ +++ +++ 
C-19393 
+++ +++ +++ +++ +++ ++ + 
+++ 
__________________________________________________________________________ 
(.+-..about.+ ++ have the same meaning as defined in Table 4) 
The growths (28.degree. C., 7 days incubation) of strains on the minimal 
medium containing sodium selenate in various concentrations are shown in 
Table 6. 
TABLE 6 
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Compound 
Sodium selenate (mg/l) 
Strain 0 62.5 125 250 500 1000 2000 
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C-19393 
+++ +++ ++ .+-. .-+. - - 
SR-135 + ++ ++ ++ ++ ++ + 
______________________________________ 
(-.about.+++ have the same meaning as defined in Table 4) 
As shown in Table 6, the growth of C-19393 strain was inhibited by addition 
of sodium selenate not less than 250 mg/l. 
The following examples are intended to illustrate the invention in further 
detail and should by no means be construed as limiting the scope of the 
invention. In these examples, all percents are by weight/volume unless 
otherwise indicated. 
EXAMPLE 1 
Streptomyces sp. C-19393 (IFO 13886, ATCC 31486) was grown on 200 ml of a 
medium comprising 2% oatmeal, 2% tomato paste, 0.2% bovril (Bovril, 
England) and 2% agar (pH 7.0) in a one-liter conical flask to cause 
sporulation. The spores were suspended in sterile water to a viable count 
of 1.2.times.10.sup.8 /ml. The spore suspension was diluted 10-fold with 
sterile water and 1 ml of the dilution was used to inoculate 40 ml of a 
seed medium in a 200-ml conical flask, which was inocubated at 28.degree. 
C. on a rotary shaker for 2 days. The resulting culture fluid was 
transferred to a 2-liter Sakaguchi shake flask containing 500 ml of the 
same seed medium as above and cultivated at 28.degree. C. on a 
reciprocating shaker for 2 days. The culture was further transferred to a 
200-liter stainless steel fermenter containing 100 liters of said seed 
medium supplemented with 50 ml of Actocol (Takeda Chemical Industries, 
Ltd., Japan) and cultivated at 28.degree. C., 70 liters/min aeration and 
150 r.p.m. for 2 days. Then, the culture was transferred to a 6-m.sup.3 
fermenter containing 4 m.sup.3 of a main culture medium and grown at 
30.degree. C., 2800 liters/min. aeration and 150 r.p.m. for 3 days. The 
seed medium mentioned above contains per liter 20 g of glucose, 30 g of 
soluble starch, 10 g of raw soybean flour, 10 g of corn steep liquor, 5 g 
of Polypepton (Daigo Nutritive Chemicals, Ltd., Japan), 3 g of sodium 
chloride and 5 g of precipitated calcium carbonate (adjusted to pH 7.0 
before sterilization). The main culture medium contained per liter 30 g of 
glucose, 30 g of soluble starch, 15 g of raw soybean flour, 15 g of 
cottonseed flour, 0.25 g of potassium dihydrogen phosphate, 0.6 g of 
potassium monohydrogen phosphate, 0.002 g of cobalt chloride and 0.5 g of 
Actocol (adjusted to pH 7.0 before sterilization). These media were all 
steam-sterilized at 120.degree. C. for 20 minutes. 
The fermentation broth thus obtained was filtered with Hyflo-Supercel 
(Johns Manville Co., U.S.A.) and the filtrate (4000 l) was adjusted to pH 
6.3 and passed through a column of Amberlite IRA-402 (Cl.sup.- -form). 
After washing the column with 200 l of 0.02 M aqueous NaCl, elution was 
carried out with 1000 l of 1.5 M NaCl. The eluate was passed through a 
column of HP-20 (70 l) and the antibiotic activity was eluted with 280 l 
of water. The eluate was passed through a column of activated carbon (15 
l) and after washing the column with 45 l of water, the antibiotic 
activity was eluted with 60 l of 7% isobutanol. The eluate was 
concentrated to 10 l and 500 g of NaCl was added to the residue. The 
mixture was passed through a column of HP-20 (6 l) and elution was carried 
out with 36 l of 5% NaCl. The eluate was passed through a column of 
activated carbon (3 l). The column was washed with 7.5 l of water and, 
then, elution was carried out with 8% isobutanol. The eluate was 
concentrated to 8 l under reduced pressure and, then, passed through a 
column of WA-30 (acetate-form) (500 ml). The column was washed with 2.5 l 
of 0.2 M acetic acid-sodium acetate buffer and the activity was eluted 
with 5 l of 1 M NaCl in the same buffer. 
The eluate was passed through a carbon column (500 ml). After the column 
was washed with 5% NaCl, elution was carried out with 2.5 l of 5% aqueous 
NaCl-methanol (4:1). The methanol was distilled off under reduced pressure 
and the residue was passed again through a carbon column (200 ml). The 
column was washed with 600 ml of H.sub.2 O and 600 ml of 20% aqueous 
methanol, followed by elution with 600 ml of 8% isobutanol. The eluate was 
concentrated under reduced pressure, the residue was treated with acetone 
and the resultant powder was collected (580 mg). The powder was dissolved 
in a small amount of water and passed through a column of Amberlite XAD-II 
(100-200 mesh) (360 ml). Fractional elution was carried out with water and 
the active fractions were pooled, concentrated to dryness, and treated 
with acetone to give 100 mg of powder. The powder was dissolved in a small 
amount of water and passed through a column of QAE-Sephadex A-25 (Cl.sup.- 
-form) (40 ml). Elution was carried out with 0.04 M phosphate buffer and 
the active fractions were pooled and subjected to liquid chromatography as 
described hereinbefore. The fractions giving a single peak were pooled and 
passed through a column of 10 ml activated carbon. The carbon column was 
washed with 30 ml of water and the activity was eluted with 50 ml of 8% 
isobutanol. The eluate was concentrated to dryness and acetone was added 
to the concentrate. The above procedure gave 20 mg of C-19393 E.sub.5 
sodium salt. 
Example 2 
From slant cultures of Streptomyces griseus subsp. Cryophilus K-4, (IFO 
14089, ATCC 31740), K-101 (IFO 14090, ATCC 31741) and SR-135 (IFO 14091, 
ATCC 31742) and Streptomyces sp. C-19393 (IFO 13886, ATCC 31486), a 
loopful each was taken and inoculated into 40 ml of a seed culture medium 
in a 200 ml conical flask. The flask was incubated on a rotary shaker at 
28.degree. C. for 2 days. A 1 ml portion of the culture was transferred to 
a 200 ml conical flask containing 40 ml of a main culture medium and 
cultivated at 28.degree. C. for 4 days. The seed culture medium contained 
per liter 20 g of glucose, 30 g of soluble starch, 10 g of raw soybean 
flour, 10 g of corn steep liquor, 5 g of Polypepton, 3 g of NaCl and 5 g 
of precipitated calcium carbonate (adjusted to pH 7.0 before 
sterilization). The main culture medium contained per liter 15 g of 
glucose, 45 g of soluble starch, 15 g of cottonseed flour, 15 g of corn 
steep liquor, 0.25 g of potassium dihydrogen phosphate, 0.6 g of potassium 
monohydrogen phosphate and 0.002 g of cobalt chloride (adjusted to pH 7.0 
before sterilization). All media were steam-sterilized at 120.degree. C. 
for 20 minutes. 
After the above cultivation, the cells are removed and the supernatant is 
assayed for C-19393 E.sub.5. The results obtained in accordance with the 
above procedures are shown below. 
______________________________________ 
C-19393 E.sub.5 in supernatant 
Strain (.mu.g/ml) 
______________________________________ 
K-4 3 
K-101 2 
SR-135 1 
C-19393 (parent) 
0.2 
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