NL194396C - Antibiotics producing microorganisms and method for preparing antibiotics. - Google Patents

Description

1 194396

Antibiotics producing microorganisms and method for preparing antibiotics

The invention relates to a pure culture of an Actinomadura sooft capable of producing antibiotic-active acidic polycyclic ethers.

A culture of Actinomadura verrucosospora is known from U.S. Pat. No. 4,195,079. This Actinomadura species produces an antibiotic referred to in the patent as compound 51,532 belonging to the acidic polycyclic ethers; further structure data of compound 51,532 are missing in the patent. The free acid of compound 51,532 has an optical rotation [a] 0 ^ of -11.0 ° in chloroform, while the sodium salt has a [a] D25 of -29.9 ° in chloroform.

European patent application 0 035 119 discloses antibiotic-active acidic polycyclic ethers, designated herein as X-14868A, X-14868B and X-14868C, which can be obtained by fermentation of the strain Nocardia X-14868 ATCC 31585.

A new Actinomadura species has now been found, capable of producing other polycyclic ether antibiotics. The pure culture according to the invention is characterized in that the Actinomadura species the Actinomadura yumaense sp. Nov. which produces the antibiotics such as X-14868A, X-14868B and X-14868C

The invention also relates to a method for preparing the antibiotics such as X-14868A, X-14868B and / or X-14868C by aerobic fermentation and recovery of the antibiotics formed in the fermentation medium, which method is characterized in that the microorganism is Actinomadura yumaense 20 ECTS Nov. breeds

The antibiotic X-14868A has the formula formula formula, wherein R1 and R2 each represent a methyl group. The symbol R in this compound stands for a hydrogen atom. The ammonium salt of this compound is now under the name maduramicin. The gross formula is the antibiotic X-14868C has the structure of the formula sheet wherein R and R 1 each represent a hydrogen atom and R 2 represents a methyl group. The gross formula of X-14868C is ^ 4β ^ 7βΟΐ7 ·

The antibiotic X-14868B is also a polycyclic ether that satisfies the formula sheet formula, wherein R, R1 and R2 each represent a methyl group. The gross formula is Ο ^ Η ^, Ο ^.

The antibiotics X-14868A, X-14868B and X-14868C mentioned in European patent application 35 119 have an optical rotation [a] D in chloroform of resp. + 40.6 °, -1-46.1 ° and + 49.6 °. According to that publication, they can be obtained by fermentation of the strain Nocardia X-14868 ATCC 31585. This strain produces an aerial mycelium of rope-like tufts, where no traces were found; the cell wall also contains arabinose. The new Actinomadurasoori differs from these 35 Nocardias in both respects. The consumption of carbon sources is also different.

The antibiotics X-14868A, X-14868B and X-14868C have been found to be active against a number of gram-positive bacteria, coccidia and nemathodes. They are also formed during the cultivation under controlled conditions of Actinomadura yamaense sp. Nov., which is the subject of the present invention.

A representative strain of this microorganism was isolated from a soil sample collected in Yuma County, Arizona 40 and is kept in the culture collection of the Lerie Laboratories Division, American Cyanamid Company, Peart River, New York, under cult number X-14868. A vigorous culture of this representative strain has been deposited at the Culture Collection Laboratory, Northern Regional Center, US Department of Agriculture, Peoria, Illinois 61604, and has been added to the permanent collection under accession number NRRL12515.

45

Taxonomic characterization of NRRL 12515

The strain NRRL 12515 is taxonomically characterized and identified as the strain type of a new species of the genus Actinomadura referred to as Actinomadura yumaense sp. Nov.

Observations were made on the cultural characteristics, physiological characteristics and morphological characteristics of the representative strain NRRL 12515 using methods of E.B. Shirting and D. Gottlieb, "Methods for characterization of Streptomyces species," Internat J. Syst. Bacteriol. 16: 313-340 (1966), and R.e. Gordon et al, "Nocardia coeliaca, Nocardia autotrophica, and the nocardial strain," Internat J. Syst. Bacteriol. 24: 54-63 (1974). Media used in the study were selected from the media recommended by TG Pridham et al., "A selection of media for maintenance and taxonomy study of 55 Streptomycetes", Antibiotics Ann., biz. 947-953 (1956/1957); GF Gauze et at, "Problems in the classification of antagonistic actinomycetes", State Publishing House for Medical Literature, Medgiz, Moscow (1957), and RE Gordon et al. (See above), for the taxonomic investigation of actinomycetes and soil bacteria.

194396 2

The chemical composition of the cell walls of the microorganism was determined using the method of H.A. Lechevalier et al., "Chemical composition as a criterion in the classification of actinomycetes," Adv. Appl. Microbiol. 14: 47-72 (1971). Phospholipid patterns were determined using the method according to M.P. Lechevalier et al., "Chemotaxonomy or aerobic actinomycetes: 5 phopholipid composition," Biochem. Syst. Ecol. 5: 249-260 (1977). Details are given in Tables 1-6, and a general description of the culture is given below. The underlined descriptive colors are taken from K.L. Kelly and D.B. Judd, "Color, Universal Language and Dictionar of Names," US Nat. Bur. Stand., Spec. Publ. 440, Washington, DC (1976) and the accompanying Inter-Society Color Council, Natl. Bur. Stand. Centroid Color Charts.

The data observed for this new species, as represented by the strain NRRL 12515, can be compared with the data published for the known species of the genus Actinomadura (M. Goodfellow et al., Numerical Taxonomy of Actinomadura and related actinomycetes, "J. Gen. Microbiol. 122: 95-111 (1979); LH Huang," Actinomadura macra sp. Nov., the producer of antibiotic CP-47.433 and CP-4734 ", Internat. J. Syst. Bacteriol 30: 565-568 (1980), J. Meyer, "New species of the genus Actinomadura", Z. Allgem Mikrobiol. 19: 37-44 (1979), H. Nomura and Y. Ohara, Distribution. or actinomycetes in soil XI. Some new species of the genus Actinomadura, Lechevalier, et al. ", J. Ferment. Technol. 49: 904-912 (1971); and TP Preobrazhenskaya et al." "Key for identification of the species of the genus Actinomadura ”, The Biology of Actinomycetes and Related Organisms 12: 30-38 (1977). The culture NRRL 12515 has a slight resemblance to Actinomadura pelletieri, but does not resemble any other species described and differs in a number of properties from A. pelletieri. Therefore, strain NRRL 12515 is designated as the type strain of a new species, called Actinomadura yumaense, sp. Nov., named after the collection site of the soil sample from which the strain type was isolated.

Micro-morphology Tracks are formed in short spiral chains (maximum length about 20 tracks per chain) on lacquered, almost wreath-like aerial tracks. The spores are egg-shaped, 0.6-0.8 µm at 1.0 to 1.4 µm, with a smooth surface.

Cell wall composition Whole cell hydrolysates of this culture contain madurose (3-O-methyl-D-galactose) and the meso isomer of diaminopimelic acid (DAP). The culture also has a type P-1 phospholpid pattern and no diagnostic phospholipid other than any phosphatidyl glycerol. These traits are all very typical of members of the genus Actinomadura.

35 Amount of growth

Good growth is observed on Bennett’s agar, Gauze No. 2 agar, NZ amine starch glucose agar (ATCC medium 172), tomato puree oatmeal agar, and yeast extract malt extract agar; moderate growth was observed on Benedict's agar, Czapek's sucrose agar, glycerol asparagine agar, Hickey-Tresner agar, and oatmeal agar; poor growth is observed on calcium malate agar, Gauze No. 1 agar, and 40 inorganic salt-starch agar.

Vegetative mycelium

On media on which good growth took place, it was observed that the vegetative mycelium was rising and rolling up, and that it generally had yellowish-gray hues.

45

Aerial mycelium and spore color

The aerial mycelia and / or the trace masses had a white to 264 light gray color. The aerial mycelia production is light on most media.

50 Soluble pigments

Absent on many media; yellow pigment on Benedicts and glycerol asparagine agars; yellow-green pigment on calcium malate agar, greenish-brown pigment on NZ amine starch glucose agar; orange pigment on Bennett’s and yeast extract-malt extract-agars. 1 i

Physiological reactions

No melanin pigments on peptone-iron agar and tyrosine agar (ISO-7); strong peptonization of litmus milk; strong proteolysis of diet gelatin; moderate reduction of nitrate; no hydrolysis of adenine or 3 194396 guanine; strong hydrolysis of hypoxanthine, tyrosine and xanthine; weak hydrolysis of starch; hydrolysis of esculin; variable hydrolysis of urea. No growth at 4 ° C, 10 ° C or 55 ° C; moderate growth at 25 ° C and 45 ° C; good growth at 32 ° C and 37 ° C. Carbohydrate use, as determined by the method according to T.G. Pridham and D. Gottlieb, "The utilization of carbon compounds by some actinomycetals as an aid for 5 species determination", J. Bacteriol. 56: 107-114 (1948): good use of glucose, glycerol and trehalose; moderate use of maltose and sucrose; poor use of fructose, galactose, inositol, mannose, and melezitose; no use of adonitol, arabinose, dulcitol, lactose, mannitol, melibiosis, raffinose, rhamnose, salicin, sorbitol and xylose. Acid production from carbohydrates according to the Gordon method , et al. (see above): good acid production from glucose, glycerol, maltose, sucrose and trehalose; weak acid production from galactose, inositol and mannose. Use organic acids according to the method of Gordon et al., (see above) : use of acetate, malate, propionate, pyruvate, succinate and tartrate, no use of benzoate, citrate, lactate, mucate and oxalate TABLE 1 15 ---

Cultural characteristics of Actinomadura yumaense NRRL12515

Incubation: 14 days Temperature 28 ° C

Medium Amount Air Myce - Soluble Reverse color 20 growth lium and / or pigment spores

Benedict's agar moderate to flat, yellowish 89. pale yellow poorly powdery colonies; aerial mycelia white to 264. light gray 30 Bennett's agar good no orange 81. dark gray aerial mycelia; yellowish brown rolled up vegetative growth 93.

35 yellowish gray to 80. grayish yellowish brown calcium malate agar poor flat growth; yellow green 90. greenish scarce yellow 40 white mycelia

Czapek's sucrose agar poor to flat growth; none 89. pale yellow moderate moderate aerial mycelia 45 vegetative growth 90. grayish yellow

Gauze No. 1 agar poor colorless no colorless flat growth; 50 scarce white aerial mycelia 194396 4 TABLE 1 (continued)

Cultural characteristics of Actinomadura yumaense NRRL 12515 5 Gauze no. 2 agar well raised 72. colonies dark orange-yellow rolled up; vegetative mycelia 93.

yellowish gray; moderate aerial mycelia, 92. yellowish white glycerol paragin agar poor to flat, yellow 89. pale yellow moderate powdery colonies; white aerial mycelia 20 Hickey-Tresner agar moderately flat none 90 grayish yellow waxy colonies; 90. grayish yellow; scarce air mycelia, white to 264. light gray inorganic salts-starch-poorly flat, no colorless agar colorless, powdery colonies; white aerial mycelia NZ amine starch glucose agar good strong greenish brown 78. dark 35 rolled-up yellowish brown growth, 61. grayish yellowish brown to 65. brownish black 40 moderate aerial mycelia, white to 264. light gray oatmeal agar moderately flat none 90. grayish yellow 45 waxy growth, 90. grayish yellow; moderate aerial mycelia, 50 white 5 194396 TABLE 1 (transport)

Cultural characteristics of Actinomadura yumaense NRRL12515 5 tomato paste oatmeal agar good flat none 90. grayish yellow waxy growth; 91. dark grayish yellow; trace 10 of white air myceia yeast extract-malt extract agar well-up 78. dark, yellowish-brown waxy, rolled-up colonies, 93. yellowish gray to 80. grayish yellowish brown; 20 none

Air myceia TABLE 2 25 Micromorphology of Actinomadura yumaense NRRL 12515

Medium Air myce - Trace shape Traces · Traceium and / or surface area bearing structures

Czapek's sucrose-agar air-sporoidal 0.6-0.8 µm smooth; branched, x nearly 1.0-1.4 µm 33 crowned; carry relatively short spiral chains 40 of mature spores 1 TABLE 3

Physiological reactions of Actinomadura yumaense NRRL 12515 gQ Medium Incubation period Quantity Physiological growth reaction pepton-iron-agar 7 days good light brown coloring 14 days good light brown coloring tyrosine agar 7 days moderate no pigment gg 14 days good yellowish pigment litmus milk 14 days good good peptonization

, "I

194396 6 TABLE 3 (continued)

Physiological reactions of Actinomadura yumaense NRRL 12515 5 28 days good strong peptonization food gelatin 14 days good light proteolysis 28 days good total proteolysis nitrate medium 14 days good very weak reduction 10 28 days good moderate reduction adenine agar 14 days good no hydrolysis 21 days good no hydrolysis guanine agar 14 days good no hydrolysis 21 days good no hydrolysis 15 hypoxanthine agar 14 days good total hydrolysis 21 days good total hydrolysis tyrosine agar 14 days good strong hydrolysis 21 days good strong hydrolysis xanthine agar 14 days good moderate hydrolysis 20 21 good hydrolysis days of NZ amine with soluble starch and 5 days of poor or no growth at 4 ° C, 10 ° C and glucose agar (ATCC Med. No. 172) 55 ° C; moderate growth at 25 ° C; 28 ° C and

45 ° C; good growth at 32 ° C and 37 ° C

urea medium 28 days good decomposition variable 25 esculin medium 14 days good hydrolysis 28 days good hydrolysis starch-agar 5 days good no hydrolysis 10 days good no hydrolysis 30 TABLE 4

Consumption of carbon sources by Actinomadura yumaense NRRL 12515 on ISP-9 carbohydrate medium

Incubation: 28 days Temperature. 28 ° C

35 Carbon source Use adonitol l-arabinose dulcitol - fructose bad * 0 d-galactose bad d-glucose good glycerol good

Hnositol poor lactose 45 matose reasonable d-mannitol d-mannose poor d-melezitose poor d-melibiosis 50 d-raffinose l-rhamnose salidne sorbitol sucrose reasonable 33 d-trehalose good 7 194396 TABLE 4 (continued)

Consumption of carbon sources by Actinomadura yumaense NRRL 12515 on ISP-9 carbohydrate medium 5 d-xylose - negative control ~ TABLE 5 10 Acid production from various carbohydrates by Actinomadura yumaense NRRL 12515 on Gordon's basic inorganic nitrogen medium

Incubation: 28 days Temperature 28 ° C

Carbon source Acid production 15 7 days 28 days adonitol - “l-arabinose - - duldtol - - fructose - - 20 d-galactose - + d-glucose +++ +++ glycerol ++ +++ i-inositol - + lactose 25 maltose - +++ d-mannitol - - d-mannose - + d-melezltose - - d-melibiosis 30 d-raffinose - - l-rhamnose - - salidne - - sorbitol - - sucrose - +++ 35 d-trehalose - + ++ d-xylose negative control 40 +++ = strong positive response ++ = moderate positive response + = low positive response - - negative response 1 2 3 4 5 6 7 8 9 10 11 TABLE 6 2 y. _ 3

Use of organic acids by Actinomadura yumaense NRRL 12515 on Gordon's modification of 4

Koser's basic agar (Koser's clatata tagar) 5 _____ - 6

Incubation: 28 days Temperature 28 ° C

7

Carbon source Use 8 acetate + 9 benzoate 10 citrate 11 lactate malate + 194396 8 TABLE 6 (continued)

Use of organic acids by Actinomadura yumaense NRRL 12515 on Gordon's modification of Koser * s basic agar (Koser's citrate agar) 5 --- slime acid oxalate propionate + pyruvate + 10 sucdnate + tartrate + + = positive response - = negative response 15 It is noted that the term Actinomadura yumaense is not limited to the strain Actinomadura yumaense NRRL 12515 or to strains that fully meet the above growth characteristics and microscopic characteristics, which are given for illustrative purposes only. The Actinomadura yumaense described here includes all strains of Actinomadura yumaense that produce the antibiotics X-14868A, B and C and which cannot be distinguished sharply from Actinomadura yumaense NRRL 12515 and its subcultures, including mutants and variants thereof. The term "mutants" includes the natural (spontaneous) mutants of this organism as well as induced mutants derived from this organism by mutagenic agents known to those skilled in the art, including exposure to X-rays, ultraviolet radiation, nitrogen mustard, actinophages and nitrosamines. also desirable and contemplated to include inter- and intra-specific genetic recombinants obtained by genetic techniques known to those skilled in the art, for example, by conjugation, transduction, and genetic engineering techniques.

Culturing Actinomadura yumaense can be performed with a variety of solid or liquid culture media. Media useful for the production of antibiotics X-14868A, B and C included an assimilable source of nitrogen such as protein, protein hydrolyzate, polypeptides, amino acids, corn soaking fluid, etc., and inorganic anions and cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc. are supplied as contaminants to other components of the media. Aeration in tanks and bottles is achieved by passing sterile air through or on the surface of the fermentation medium. A mechanical stirrer ensures further agitation in tanks. An anti-foaming agent such as pork lard oil or a silicone defoamer can be added as required.

Actinomadura yumaense is grown and stored on oblique agar, for example Bennett's agar, yeast malt agar, or ATCC medium 172. ATCC medium 172 is preferred. The agar is inoculated with a culture of Actinomadura yumaense and incubated at 28-37 ° C, preferably at about 32 ° C, for about 7 days. These starting cultures can be stored on fresh agar by serial transfers, or an amount of mycelia agar from the well-grown agar can be used to inoculate liquid media.

Grafting material for Actinomadura yumaense shake is prepared by inoculating 100 cm 3 of sterile liquid medium into 500 cm 3 bottles with spores obtained by scraping or washing an agar culture. Examples of suitable graft medla are: 45

Medium A

beef extract 0.3% 50 Bacto® trypton1 0.5% glucose 1.0% yeast extract 0.5% water q.s. 100% 55 (1 a peptone, branded product from Difco Laboratories, Detroit, Michigan.)

The pH is adjusted with a diluted base, e.g. sodium hydroxide set to 6.8-7.2.

194396 g

Medium B

glucose 1% starch 2% 5 yeast extract 0.5% NZ amine A®2 0.5% calcium carbonate 0.1% water 100% 10 (2 Casein digest, branded product of Sheffield Chemical Co., Div. Naf1 Dairy Products Corp., Norwich, NY)

Medium B is preferred.

The bottles are incubated at a temperature of 25-35 ° C, preferably at 32 ° C and shaken vigorously on a rotary shaker for 1-4 days. This seeding material is then used to inoculate a fermentation culture; this culture can also be frozen and stored to provide seed material for later seed cultures.

The following media are examples of media suitable for the fermentation of Actinomadura yumaense for the production of the antibiotics X-14868A, B and C.

Medium C

glucose 1.5% glycerol 1.5% soybean meal3 1.5% calcium carbonate 0.1% sodium chloride 0.3% water q.s. 100% (3 Can be replaced with cottonseed flour or soluble meat components with equal effect.) 30

Medium D

glucose 3% 35 soybean meal 1.5% calcium carbonate 0.1% sodium chloride 0.3% water q.s. 100% 40

Medium E

starch 1% molasses 2% 45 soybean meal 1.5% calcium carbonate 0.1% water q.s. 100% 1

Medium F

glucose 3% soluble meat components 2.5% sodium chloride 0.2% 55 calcium carbonate 0.1% water q.s. 100% 194396 10

Medium G

glucose 3% 5 soybean meal 0.5% ammonium sulphate 0.3% sodium chloride 0.2% calcium carbonate 0.1% 10 MediumH, 00% glucose 3% ammonium sulphate 0.3% dibasic potassium phosphate 0.1% sodium chloride 0.2% calcium carbonate 0.1% water qs 100%

Medium D is preferred.

The fermentation can be carried out in 100 cm 3 of media in a 500 cm 3 bottle inoculated with 3-10% (v / v) of a food culture prepared as described above and incubated for 24-72 hours under aeration at 25- 35 ° C, preferably at about 32 ° C. Samples from the fermentation culture can be frozen and stored for later use as seeding material for seed cultures.

On the other hand, the fermentation can be carried out in larger fermentation tanks, which are provided with aeration and agitation agents.

Each tank is inoculated with 3-10% (v / v) grafting material prepared in the manner described above. Aeration is carried out at a rate of 0.5-2.0 dm3 of sterile air per dm3 of fermentation medium per minute and the fermentation medium is passed through a stirrer at a speed of 200-400 rpm. stirred. The temperature is kept at 25-35 ° C, preferably at 32 ° C. The fermentation is continued until antibiotic accumulates in the fermentation medium, usually after 100-150 hours, after which the antibiotic is harvested.

The antibiotic can be harvested and purified according to the methods described in U.S. Patent No. 4,278,663, or according to the following procedure:

The crude fermentation mixture, which contains whole cells prepared in the manner described above, is mixed with an equal volume amount of an organic solvent immiscible with water. Preferred are methylene chloride and ethyl acetate. The organic phase is separated and concentrated under reduced pressure to an oily syrup.

The oily syrup is dissolved in methylene chloride and added to a column of silica gel, alumina, Sephadex LH-20® (Pharmacia Fine Chemicals Div. Of Pharmacia, Inc., Piscataway, N.J.), or 40 magnesium aluminum silicate. Examples of suitable solvents for developing the column are diethyl ether, ethyl acetate and mixtures of methylene chloride with diethyl ether, acetone, lower alcohol (preferred is methanol), acetonitrile or dioxane. Methylene chloride: ethyl acetate 1: 1 (v / v) is preferred. Fractions are collected and checked for the presence of antibacterial activity by bioanalysis against a sensitive organism, e.g. Bacillus subtills. Active fractions are combined and concentrated under reduced pressure to a residue. This residue is dissolved in an organic solvent, e.g. tert-butanol (preferred), kenzene, or p-dioxane, and freeze-dried.

The freeze-dried solid is dissolved in a suitable organic solvent, e.g., methylene chloride, hexane, methylene chloride: ethyl acetate, diethyl ether, hexane: ethyl acetate, hexane: chloroform, or hexane: ether. Diethyl ether is preferred. This solution is shaken with water and the pH is adjusted to 1.5-4.0, preferably about 2.5, with any dilute inorganic acid. The organic phase is separated, washed with water to remove any excess acid, dried over a suitable drying agent, filtered, and concentrated under reduced pressure.

The residue is dissolved in a suitable solvent and the solution is allowed to evaporate slowly, preferably at about 4 ° C. Examples of suitable solvents are methylene chloride, hexane, ethyl acetate, diethyl ether, chloroform and mixtures thereof. Hexane: ether 5: 2 (v / v) is preferred. The obtained crystals are collected and washed, preferably at about 40 ° C, with any hydrocarbon boiling at a moderate temperature, for example hexane or heptane, and air-dried 11 194396 tsneiPidc a! SindprCuliCi the antibiotic VOi 111 V £ u1 höt vrijö £ UUf ιθ VörKrijQöl ·

If the product is desired in the form of a salt, the free acid can be converted by treatment with the appropriate cation, preferably in the form of a diluted inorganic base, in accordance with the procedures known to those skilled in the art.

The following examples illustrate the invention. The media A, B, C etc. are as defined above. Unless otherwise indicated, all procedures were performed at room temperature (about 22 ° C) and at a pressure of 1 atm.

Example 1 Washed traces of an oblique agar from Actinomadura yumaense NRRL 12515 were used to inoculate a 500 cm 3 bottle containing 100 cm 3 of sterile medium B. The bottle was incubated at 28 ° C for 2 days on a rotary shaker.

A 5% seed material from this culture was then transferred to 100 cm 3 of sterile medium C in a 500 cm 3 bottle and incubated for 5 days at 28 ° C on a rotary shaker.

The presence of antibiotic activity was monitored daily by bioanalysis against Staphylococcus aureus ATCC 6538 P, Bacillus subtilis, and Streptococcus faecalis, and anthelmintic activity was monitored by bioanalysis against the free-living nematode Caenorhabdltis elegans.

EXAMPLE II

Seven 500 cm3 bottles were prepared, each containing 100 cm3 from one of the following sterile media: Bottle 1: 100 cm3 medium C

bottle 2: 100 cm3 medium C with 1.5% cottonseed flour instead of soybean meal bottle 3: 100 cm3 medium C with 1.5% soluble meat constituents instead of soybean meal bottle 4: 100 cm3 medium D 25 bottle 5: 100 cm3 medium E bottle 6: 100 cm3 medium F bottle 7: 100 cm3 medium G.

These bottles were each inoculated with a 5% inoculum of Actinomadura yumaense NRRL 12515, which had grown in medium B. The bottles were then incubated for 4-6 days at 28 ° C on a rotary shaker.

Each of the above cultures was found to be active in analysis for antibiotic activity as in Example 1 and in analysis for anticoccidial activity against Eimeria tenella in tissue cultures of chickens. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

EXAMPLE III

2

Frozen fermentation culture cells from Actinomadura yumaense NRRL 12515 were used to make a 500 3

cm 3 of a bottle containing 100 cm 3 of sterile medium B. The bottle was then left at 32 ° C for 4 days

4 incubated on a rotary shaker.

5

A 5% seed material from this culture was then transferred to 100 cm 3 of sterile medium H in a 6 500 cm 3 bottle and incubated for 5 days at 28 ° C on a rotary shaker.

7

Antibacterial activity was confirmed in the manner described in Example I and anticoccidial activity was confirmed in the manner described in Example II.

9

The presence of antibiotic activity was also demonstrated by thin layer chromatography on silica gel plates developed in ethyl acetate chloroform 70:30 (v / v).

11 12

EXAMPLE IV

13

100 cm 3 of sterile medium A was inoculated into a 500 cm 3 bottle with washed traces of an agar culture of 14

Actinomadura yumaense NRRL 12515. The bottle was incubated for 2 days at 32 ° C on a rotary shaker.

16

A 5% seed material from this culture was then transferred to 100 cm 3 of sterile medium H in a 500 cm 3 bottle and incubated for 2 days at 32 ° C on a rotary shaker.

A 5% inoculum of this culture was then transferred to a 500 cm3 bottle containing 100 cm3 of fresh sterile medium H and incubated for 6 days at 28 ° C on a rotary shaker.

The antibacterial activity was confirmed in the manner described in Example I.

194396 12

Example V

Two 500 cm3 bottles, each containing 100 cm3 of sterile medium A, were inoculated with a frozen seed culture of Actinomadura yumaense NRRL 12515 and incubated for 2 days at 32 ° C on a rotary shaker.

The contents of the two bottles were then combined and added to 12 dm 3 of fresh sterile medium A in a 20 dm 3 bottle. This culture was then incubated for 2 days at 28 ° C with aeration.

The contents of this bottle were then transferred to a 300 dm3 seed tank containing 288 dm3 sterile medium A and this culture was aerated and agitated for 25 hours at 32 ° C.

At the end of the 25-hour incubation, the 300 dm 3 seed culture was transferred to a 1500 dm 3 fermentor containing 1200 dm 3 of sterile medium C. This culture was incubated under aeration and agitation for 115 hours at 28 ° C.

The fermentation mixture was analyzed for antibiotic activity by thin layer chromatography on silica gel plates developed in ethyl acetate: chloroform 70:30 (v / v). Alternatively, several of the developed plates were subjected to bioanalysis against Bacillus subtills, demonstrating the presence of antibiotic activity.

Example VI

A medium for growing the primary seeding material was formulated in accordance with the following formulation: beef extract 0.3%

Bacto®-trypton 0.5% glucose 1.0% yeast extract 0.5% Bacto®-agar 0.15% water q.s. 100%

Spores and mycelia obtained by washing or scraping an agar culture of Actinomadura yumaense NRRL 12515 were used to inoculate a 500 cm 3 bottle containing 100 cm 3 of the above sterilized medium. The bottle was placed on a rotary shaker and shaken vigorously for 48 hours at 32 ° C. The resulting inoculum (100 cm 3) in the bottle was used to inoculate 1 dm 3 of the same sterile medium into a 2 dm 3 bottle. This seeding material was aerated with sterile air while the culture was continued for 48 hours at 28 ° C. This 1 dm 3 culture was then used to inoculate a 30 cm fermenter tank containing the same sterile medium, and this tank was aerated with sterile air and incubated at 28 ° C for 42 hours.

EXAMPLE VII

A fermentation medium was prepared in accordance with the following formulation: dextrose 1.5% glycerol 1.5% soybean meal 1.5% calcium carbonate 0.1% sodium chloride 0.3% water q.s. 100%

The pH was adjusted to 7.0 with 6 N sodium hydroxide and the medium was sterilized. A 30 dm3 portion of the seeding material prepared according to Example I was used to inoculate 250 dm3 of the medium described above in a 300 dm3 fermentor. The fermentation mixture was aerated sterile and 50 at 220 rpm. stirred. The fermentation was carried out at 32 ° C for 97 hours, after which the solid material was harvested

EXAMPLE VIII

A fermentation medium was prepared in accordance with the following formulation: dextrose 3.0% soybean meal 1.5% 13 194396 dextrose 3.0% calcium carton> 0.1% sodium chloride 0.3% water q.s. 100%

The pM was adjusted to 7.0 with 6 N sodium hydroxide and the medium was sterilized. A 300 dm1 portion of the seeding material prepared according to Example I was used to inoculate 3000 dm1 of the above medium into a fermenter. The fermentation mixture was aerated sterile and at 100 rpm. stirred. The fermentation was carried out at 32 ° C for 115 hours after which the solid material was harvested.

EXAMPLE IX

1. The solid fermentation material (100 dm1) was adjusted to pH 4.0 with 6 N HCl. The acid mixture was then stirred for 1-2 hours with an equal volume amount of methylene chloride. The mixture was then filtered using diatomaceous earth as a filter aid. The methylene chloride extract was filtered off with suction and concentrated under reduced pressure to about 2 ml of partially purified antibiotics.

2. Chromatography of the partially purified antibiotics on silica gel. A glass column with a diameter of 7 cm was filled to a height of 91 cm with Woelm silica gel TSC. The crude concentrate (see above under 1) with a volume of about 2 dm 1 methylene chloride, followed by methylene chloride: ethyl acetate (1: 1 volume ratio), whereby a total of 126 fractions was obtained, each with a volume of 80 cm 1. The column was then further developed using ethyl acetate-ethanol (7: 3) to give an additional 87 fractions.

Fractions 58-87, which were rich in X-14868A, were combined and concentrated under reduced pressure to a residue weighing 90.4 g. This residue was taken up in a mixture of 600 ml of diethyl ether and 600 ml of hexane. The suspension was filtered to give a clear filtrate. The clear filtrate was concentrated under reduced pressure until crystals started to form. After standing overnight, the crystalline X-14868A was collected on a filter funnel, washed with cold ether and air-dried to give 33.1 g of crystalline X-14868A. An additional amount of X-14868A of 12.4 g was obtained by further volume reduction of the combined wash and filtrate.

The fractions 177-203 of the elution with ethyl acetate-ethanol, which were rich in X-14868C, were combined and concentrated under reduced pressure, whereby 6.7 g of partially purified X-14868B were obtained, as described above. was treated.

Fractions 204-213 of the elution with ethyl acetate-ethanol, enriched in X-14868B, were combined and concentrated under reduced pressure to a paste and extracted several times with methanol. The methanol extract was extracted with methylene chloride, which was placed on a column containing Woelm silica gel. The column was washed with methylene chloride and then eluted with 40 methylene chloride: ethyl acetate (2: 3). The fractions were followed by thin layer chromatography and the active fractions were combined, treated with charcoal, concentrated and extracted several times with heptane. The final heptane extract was cooled at 0 ° C for 48 hours and the crystals were separated from the mother liquor by filtration.

This mother liquor was dissolved in methylene chloride and placed on a glass column packed with Woelm silica gel. The column was then eluted successively with 2 ml of methylene chloride, 8 ml of methylene chloride: ethyl acetate (1: 1) and 4 ml of ethyl acetate: methanol (9: 1). The eluate was collected in fractions and the fractions were tested for their antibiotic composition. The active fractions were combined and concentrated under reduced pressure to give a residue containing X-14868B.

50

Example X

Further purification of X-14868C from silica gel chromatography.

Sephadex® LH 2 O was allowed to swell in a mixture of hexane-methylene chloride-methanol (10: 5: 1).

A glass column with a diameter of 5 cm was filled with the gel to a height of 86 cm. The batch, 3 g of purified X-14868C, was dissolved in 10 cm 1 of methylene chloride, 1 cm 1 of hexane and was introduced into the gel column. The column was then developed with the same solvent mixture. Using

Claims (4)

194396 14 a collector, fractions were collected. The first 23 fractions each had a volume of 17 cm 3. Fractions 17-26 contain X-14868C according to thin layer chromatography. These fractions were combined and concentrated under reduced pressure to give pure amorphous X-14868C in an amount of 1269 mg. Example XI Crystallization of X-14868C as sodium salt Purified X-14868C (2.4 g) prepared as described in Example IX was dissolved in a mixture of 500 cm 3 of diethyl ether, 160 cm 3 of hexane and 25 cm 3 of methylene chloride. The resulting solution was transferred to a separatory funnel containing 300 cm 3 of distilled water. Diluted HCl (1 N) was added dropwise until the pH reached 2.0-2.5 after shaking and settling. The acidic aqueous phase was discarded and the acid-treated organic layer was washed three times with 400 cm 3 of water each time. The washed organic layer was stirred with a teaspoon for 15 minutes with Darco 15 G 60 powder and filtered through Ceite. The decoloured organic layer was placed in 500 cm 3 of distilled water and 5 N NaOH was added dropwise until the pH reached 11.0-11.5 after shaking and settling. The basic aqueous phase was discarded and the remaining organic layer was washed twice with 400 ml portions of water. The washed organic layer was dried over sodium sulfate and then concentrated under reduced pressure to a volume of 150 cm 3. This concentrate was left in a fume cupboard for several hours. The crystalline X-14868C was collected on a funnel, washed with cold hexane and air-dried to give 402 mg of the crystalline salt of X-14868C. Analysis: calculated for C ^ H ^ O ^ Na: C 59.70; H, 8.33; O 29.46; ash 2.49%; Found: C, 61.55; H, 8.79; ash 3.31%; N, O. MP. (Fisher-John's apparatus = 174 [FDMass Spec) = 924 [α] 2 ^ = + 3.2 in methanol. Example XII Purification of X-14868B. A Waters Prep. 500A HPLC (high performance liquid chromatography) instrument was provided with a silica gel column under a pressure of 38 bar. The batch, which was 5.0 g of the crude material from Example 11a, was dissolved in 50 cm 3 of heptane: ethyl acetate (45:55) and injected into the column. The column was then developed with the same solvent mixture at a flow rate of 200 cm 3 / min where 40 fractions of 400 cm 3 were collected. Fractions 21-32 were combined, concentrated to a residue, treated with hexane and evaporated, yielding pure X-14868B. The above procedure was repeated four times to give a total amount of 280 mg of amorphous X-14868B. A 90 mg portion of amorphous X-14868B was dissolved in 10 cm 3 of diethyl ether, mixed with 10 cm 3 of water, and adjusted to pH 2.5 with 0.1 N hydrochloric acid. The ether layer was washed with water, with 0.11 N sodium hydroxide adjusted to a pH of about 11, separated and washed again with water. The ether phase was dried over sodium sulfate, filtered, and concentrated to a residue. A solution of this residue in ether-hexane was allowed to evaporate slowly, yielding an amorphous white solid. This amorphous white solid had the following properties: elemental analysis: C 61.79; H, 8.84; axis 0.32; [α] 6 D = +27 (C 0.724, methanol); field desorption mass spectrometry: (M + Na) + = mIe 953, the calculated molecular weight is therefore 930. 50 A pure culture of an Actinomadura species capable of producing an antibiotic-active polycyclic ether, characterized in that the Actinomadura-soori Actinomadura yumaense sp. Nov. 55 which produces the antibiotics X-14868A, X-14868B and X-14868C. Pure culture according to claim 1, characterized in that the Actinomadura is yumaense NRRL 12515. 3. Process for the preparation of the antibiotics X-14868A, X-14868B and / or X-14868C by aerobic fermentation 194396 and recovery of the antibiotics formed in the fermentation medium, characterized in that in the aerobic fermentation, Actinomadura yumaense sp. . Nov. used Method according to claim 3, characterized in that the Actinomadura yumaense is the strain Actinomadura yumaense NRRL 12515. Hereby 1 sheet drawing