DE1792819C2 - Tenebrimycin antibiotic VI and a process for its preparation - Google Patents

Description

The invention relates to the tenebrimycin antibiotic tenebrimycin VI with that specified in claim 1 Identification and a method for its production (claim 2).

Tenebrimycin VI can be made as a factor in a novel antibiotic complex that includes the seven Factors tenebrimycin I, Γ, II, III, IV, V and VI, each of which exhibits antibiotic activity. The complex is soluble as a free base in water and in methyl sulfoxide, slowly soluble in methanol and insoluble in most other organic solvents such as acetone, higher alcohols, dioxane, Ethyl acetate, diethyl ether, acetonitrile, methyl isobutyl ketone and hydrocarbon solvents. Of the free base complex is stable at freezing temperatures, at room temperature and at 37 ° C for the duration of at least 8 weeks within a pH range of up to 11, the distinguishable bands in the infrared absorption spectrum Over a range from 2 to μm are the following: 3.02 (board), 3.15, 5.82, 6.26, 7.43. 8.8 (wide) and 9.7 (very wide) μιη, positive reaction at the NInhydrin, Anthron and Elson-Morgan tests, the Lowry protein test, and a low positive reaction negative reaction to the Biuret and Sakaguchl tests. This antibiotic complex is in the German patent specification 16 17 451.

Tenebrimycin VI has titratable groups with pK'a values of about 5.6, 7.2, 8.2 and 9.3 as determined by electrometric titration in water.

The infrared absorption spectrum of tenebrimycin VI is shown in the figure. The distinguishable bands over a range from 2 to 15 μm are as follows 3.05, 3.19, 6.35, 7.47, 8.75, 9.8, 12.3 and 12.95 μm.

A 1% aqueous solution of the tetraacetyl derivative of tenebrimycin VI has a specific rotation of

ίο about + 95 ° when at 25 ° C in a 1% aqueous Solution is determined.

Acid addition salts of tenebrimycin VI can be prepared by conventional methods. It Either organic or inorganic acids can be used for salt formation. A convenient process making such salts consists in adding a solution of the salt-forming acid to one aqueous solution of the antibiotic. In the case of insoluble acid addition salts, the salt separates out the solution and can be easily separated by means of conventional working systems such as filtration or centrifugation.

If it goes wrong that the desired addition salt does not precipitate easily, you can promote development. By either reducing the solution to a smaller one Volume concentrated or a water-miscible solvent such as acetone or the like is added. The acid addition salt can easily be converted back to the free base form of the antibiotic, by passing an aqueous solution of the salt over an anionic exchange resin in the hydroxyl circuit. Tenebrimycin VI can depend on the various factors that form the aforementioned tenebrimycin complex, and distinguished from other antibiotics with similar properties by paper chromographic procedures become, such as B. by the following: The chromatography was in the conventional descending order Way carried out at 23 ° C ± 1 ° on Whatman No. 1 paper, size 190 χ 464 mm.

The results are shown in Table I. The constant of migration For each of the antibiotics listed, it is better to use a Λ, constant than to express it through the conventional Λ, value. The Λ ,, value expresses that Ratio of the distance traversed by the antibiotic with respect to the end of the sheet and not the solvent front.

This method of expressing the constant was chosen in view of the fact that the solvent front past the end of the leaf for the period that is used for most solvent systems was, had advanced.

Table I.

Paper chromatography of tenebrimycins and
known antibiotics with similar properties

Solvent system 1 and Rp values ABCDEFG

Tenebrimycin complex

Tenebrimycin I.
Tenebrimycin Γ
Tenebrimycin Il
Tenebrimycin 111

0.65 0.32 0 0

0.20 - - -

0.27 -

0.40 0.17 0.32 -

0.40 0.34 0.41 -

continuation

17 §2 819

continuation

Solvent system 1 and Rf values ABCDEFG

Tenebrimycin IV
Tenebrimycin V
Tenebrimycin VI
Kanamycin
Kanamycin B

Gentamycin

Catenulin
Neomycin

0.49 0.20 0.32 - -

0.55 0.22 0.35 - - - -

0.71 0.31 0.38 - - - -

0.35 0.35 0.41 0.65 0.42 0 0

0.57 0.26 0.32 0.70 0.29 0 0.05

0.51 0.39

0.83 0.75 0.62 - 0.51 0 0.1

0.95

0.44 0.25 0.34 0.65 0.35 0 0

0.60 0.12 0.19 - - 0 0.05

Test organism

minimal
Inhibitor concentration)

Tenebrimycin VI

The solvent systems used and the duration of the chromatography are given below.

A = n-butanol saturated with water, plus 2% p-toluene

sulfonic acid, for 40 hours B = S0% aqueous ethanol, plus 1.5% sodium chloride, for 40 hours, buffered on paper, with

0.95 m sulfate bisulfate
C = propanol. Pyridine, acetic acid and water

(15: 10: 3: 12) for 40 hours D = water saturated with methyl isobutyl ketone, plus

\% p-toluenesulfonic acid for 6 hours E = propanol and water (1: 1) for 24 hours

Paper buffered with 0.75 m phosphate, pH 4.0 F = n-butanol saturated with water, for 18 hours G = n-butanol saturated with water, plus 2% p-toluenesulfonic acid and 2% piperidine, for 18 hours.

These chromatographic data indicate the following: While for each individual solvent system some of the factors that make up the tenebrimycin complex as identical to or identical to each other Other known antibiotics appear, as evidenced by chromatography with a variety of solvent systems clearly their non-identity.

Tenebrimycin VI has an inhibitory effect on the growth of microorganisms, which are animal or plant life are pathogenic. The inhibitory concentrations of tenebrlmycin VI against a number of organisms are shown in the following table. The minimal inhibition concentrate ions were determined by the agar dilution method.

Table II

Test organism minimal Inhibitory concentration ") ^ g / ml) Tenebrimycin VI Staphylococcus aureus 3055 3.12 Bacillus subtilis <1.56 Mycobacterium avium <1.56 Streptococcus faecalis 3.12

Lactobacillus casei 12.5

Leuconostoc citrovorum 12.5

Escherichia coli No. 1 12.5

Escherichia coli No. 2 12.5

Proteus sp. No. 1 25

Proteus sp. No. 2 12.5

Pseudomonas sp. No. 2 6.25

Pseudomonas sp. No. 5 <1.56

Klebsiella-Aerobacter No. 14 6.25

Klebsiella-Aerobacter No. 15 6.25

Salmonella sp. No. 1 12.5

Vibrio metschnikovii 6.25

Agrobactcrium tumefaciens 50

Erwinia amylovora <1.56

Pseudomonas solanacearum 12.5

Xanthomonas phaseoli 3.12

The minimal inhibitory concentration was determined with the free base.

The actual toxicity of tenebrimycin VI was determined in mice.

The LD 50, expressed in mg / kg of free base, for tenebrimycin VI administered intravenously to mice is about 120.
The antibiotic complex containing tenebrimycin VI is produced by cultivating a certain actinomycete strain under aerobic conditions in a suitable cultivation medium until this medium contains substantial antibiotic activity. The tenebrimycin VI can be obtained by applying certain isolation and cleaning measures (see. Claim 2).

Because of the uncertainty of taxonomic studies regarding the genus Streptomyces, one always remains Element of doubt cling to the classification of a newly discovered organism. Nevertheless - as far as it is can be proven - is not the same as the organism used to manufacture the antibiotic a genus of Streptomyces described by Waksmann In »The Actinomycetes, "Vol II, The Williams and Wllkens Company (19oil), are described for culture comparisons to guarantee.

The organism is a spiral-shaped, heat-bearing, aerobic to microaerophller Streptomyces with elongated smooth-walled pores. It's unique in that he is

bo is inhibited by relatively low intensities of artificial light. Because of this property, the name Streptomyces tenebrarius sp. n. chosen for this organism.
The organism was isolated from a soil sample,

^b5 by suspending parts of the soil sample in sterile distilled water and spreading the suspensions on nutrient agar. The nutrient agar plates were incubated at 25 to 35 ° C until growth was secure.

was posed. Colonies became at the end of the incubation tent of the antibiotics-producing organisms are transferred onto agar slants using a sterile platinum inoculation loop. The agar slant was then incubated to provide the appropriate inoculation levels for the preparation of the antibiotic obtain. The strain of the organism used to make the antibiotic complex has been deposited with the American Type Culture Collection in Washlnton, D.C, under the culture number: ATCC 17 920.

The procedures used in the taxonomic experiments on S. tenebrarius ATCC 17 920 are the same, as is customary in the taxonomy of Actlnomycetes be handled. Cultivation characteristics were observed after incubation for one day. The Mor-! 5 Phology was determined on Czapek's peptone agar and Bennet's agar during a 2- to 7-day incubation. Effects on milk and the reduction of nitrates were observed after 7-14 days, hydrogen sulfide products after 24 to 48 hours and carbon recovery after 10 days. Unless otherwise stated, the cultures were incubated at 37 ° C. Carbon recovery studies were performed according to the procedure described by Prldham and Gottlieb, J. Bact., 56, 107 (1948).

The results of the taxonomic tests are summarized in the following sections. The payment in the brackets refer to the color blocks according to Maerz and Paul, "Dictionary of Color", McGraw Book Company (1950). The colors produced by the coloring blocks are symbolized, have been transferred to the ISCC-NBS color names as they are in the »Circular 553 ", U.S. Department of Commerce, National Bureau of Standards, 1955.

35

Microscopic morphology, breeding characteristics and physiology of S. tenebraris ATCC 17 920

Morphology:

Branched spore forms form on Czapek's peptone agar in irregular lumps on the aerial mycelium. Isolated spores are rare. The intact Aerial mycelium can easily be detached from the substrate. Mature spore chains usually form 5 to 6 open spirals. The spores are elongated to cylindrical. When viewed under the electron microscope, the spores appear smooth and measure 0.7 to 1.3 by 2.0 to 2.1 microns. ScIerotien were observed on Bennet's medium.

Colony characteristics on:

Growth rate low. Aerial mycelium poor; pale orange yellow (11-A2); Sporulation good: reverse pale orange-yellow Π1-Α2); soluble pigment light pink (1-sheet).

Czapek's peptone:

Growth abundant; Aerial mycelium abundant; light yellowish brown with white spots (11-A4); Profuse sporulation; Back light gray red (4-Hl); soluble pigment gray-pink (4 sheets).

Calcium malate agar

Moderate growth, moderate aerial mycelium, pale orange-yellow (11-A2); Moderate sporulation; Back gray-pink (4-Dl); soluble pigment gray-pink (4-sheets).

Tyrosin agar:

Growth sparse, aerial mycelium sparse; pale orange yellow (9-B2); Reverse pale red-yellow (10-B3); sparse Sporulation; no soluble pigment.

Inorganic Jialze Starch Agar:
Moderate growth, moderate aerial mycelium, brownish pink with white areas (11-A4); Profuse sporulation; Back pale yellow (HB 2), soluble pigment pale yellow (11-B2).

Glucose asparagine agar:

Growth moderate, aerial mycelium moderate, pale yellow (9-D2) with white areas (10-A1) moderate sporulation; back pale yellow (11-B2) no soluble pigment.

Tomato paste oatmeal:

Abundant growth, abundant aerial mycelium, light yellowish brown (12-B5); profuse sporulation; Back dark gray-reddish brown (40-J2); soluble pigment dark purple (47-hl).

Yeast extract:

Abundant growth, abundant aerial mycelium, pale orange-yellow (11-A2) with white spots; profuse sporulation; Back moderately yellow (11-J6); no soluble pigment.

Nutrient agar:

Poor growth, poor white (10-Al) aerial mycelium; poor sporulation; Reverse gray-greenish-yellow (12-12); no soluble pigment.

Bennet's agar:

Moderate growth; moderately white (10-Al to 10-Bl) Aerial mycelium; moderate sporulation; Reverse pale yellow (11-C2) D; no soluble pigment.

Effect on milk:

Dark yellow growth ring on the surface; Coagulation and peptonization were observed.

Nitrate reduction:

Positive.

HjS production:
Negative.
Nutrient gelatine - complete liquefaction after 14 days.

Temperature requirements for Czapek's peptone agar:
20 ° C no growth
26 ° C good growth, no aerial mycelium
30 ⁰ C growth and aerial mycelium moderate but none

Sporulation
37 ° C growth, aerial mycelium and sporulation very much

plentiful
43 ° C growth, aerial mycelium and sporulation very much

plentiful
50 ° C growth, aerial mycelium and sporulation very much

plentiful

55 ° C sparse growth
60 ° C no growth

Thermal dead center:

Spores from the spore suspension heated to 75 ° C remain viable for i5 rviiin.iang. When the spore suspension was heated to 100 ° C. for 15 minutes, the spores were no longer viable.
Oxygen tension:

Either aerobic or microaerophilic growth in stick cultures.
Iron ion effect:

A red soluble pigment is only produced in the presence of ferric ions and the intensity of the pigmentation is proportional to the concentration of ferric ions in a given area.
Effect of hydrogen ion concentration on Czapek's peptone agar observed:

No growth below pH 5.0; from pH 5.0 to 6.0 growth and aerial mycelium good; Growth and sporulation are plentiful at pH 7.0; from Ph 8.0 to 8.6, growth and aerial mycelia are good. The soluble pigment

is most intense at pH 5.0 to 6.0 and is low to absent from pH 6.5 to 8.6.
Light reaction observed on Czapek's agar:
Growth and sporulation are abundant in the dark. When plate cultures are incubated 38 cm from a 15 WaU standard fluorescent cold light source, growth will be sparse and no aerial mycelium present. Growth and aerial mycelium are moderate when the cultures are incubated 38 cm from a cooled, frosted 60 watt tungsten incandescent lamp.

In the table showing the results of the carbon recovery attempts summarizes that were carried out with S. tenebrarius ATCC 17920, the used characters have the following meaning:

+ = positive

(+) = likely

(-) = questionable

- = none (nothing)

Table III

Carbon recovery

from S. tenebrarius strain ATCC 17920

Carbon source on carbon source on

speak speak

15th

20th

25th

L (+) arabinose
L (+) rhamnose
D (-) ribose
D (+) xylose
D (-) fructose
D (-t-) mannose
D (+) glucose
Lactose
Maltose

Sanharose

D (+) trehalose

L (+) raffinose

Cellulose

Inulin

i-inositol

Mannitol

d-sorbitol

Salicin

Control <

(no carbon)

30th

35

45

S. tenebrarius ATCC 17920 also produces the well-known antifungal antibiotic caerulomycin, which also is produced by Streptomyces caeruleus (Cand.J.Microbiol. 5, 317 [1959]). Investigation of a breed of the The latter organism showed that it did not produce tenebrimycin under the conditions required for its Breeding have been described; even under changed conditions it cannot be induced to do so.

Cultivation media that are relatively simple are expediently used for the production of Tenebrimyclns VI Contain nutrient sources. For example, media useful for the production of tenebrimycin contain an assimilable source of carbon such as glucose, fructose, manose, maltose, starch, and the like. One a particularly preferred carbon source is glucose. In addition, the media that can be used include an assimilable one Nitrogen source such as peptones, hydrolyzed casein, yeast, amino acids and the like. In the present Traps are preferred as nitrogen sources: peptone, hydrolyzed casein, and glutamine.

Mineral salts, e.g. B. those which contain calcium, magnesium, sodium, potassium, chloride, sulfate, phosphate and carbonate ions can supply with good Success can be incorporated into the media, although an excess of phosphate should be avoided as it seems to reduce the yield of antibiotic. A source of growth factors, such as yeast or Yeast extract can also be added to the medium with good effect.

As is necessary for the growth and development of other microorganisms, important trace elements should also be included are added to the culture medium to promote the growth of the invention applied organisms. Such trace elements are mostly present as impurities, as random ones Accompanying substances when adding the other components of the medium.

Submerse aerobic culture conditions are especially preferred for the production of tenebrimycin. For the Shake flasks and surface cultures can be used to produce relatively small quantities. For the However, for large-scale production, submerged aerobic growth in sterile containers is preferable. That Medium in the sterile container can be inoculated with a sporulled suspension, but because of the growth retardation associated with the use of a sporulled suspension Suspension as inoculum, preference is given to the vegetative form of the culture. This is how you avoid them Growth retardation and achieves a more efficient utilization of the fermentation devices. Accordingly, it is advisable to first obtain a vegetative inoculum of the organism by inoculating a proportionately to produce a small amount of the culture medium with the spore shape of the organism, and when a young active vegetative inoculum is obtained became, this vegetative inoculum sterile In the great Transfer container. A fraction of the vegetative inoculum, around 4% of the mass, is useful of the medium into which it is introduced. The fermentation medium in which the vegetative inoculum is produced, can either be the same or different from the medium used for the large-scale Tenebrimycin production was used.

The organism grows within a relatively wide temperature range. Still, the organisms seem best to grow at temperatures between 30 and 50 ° C. An optimal production of tenebrimycin takes place at temperatures between 37 and 43 ° C. The organisms that produce tenebrimycin are sensitive to light and grow poorly if it is present.

Thus, fermentations in which the organisms are used are as far as possible without visible light to execute.

Consistent with common practice in submerged breeding operations, sterile air is passed through passed the growth medium. To an effective growth of the organisms and tenebrimycin production the air volume in the tank production of tenebrimycin is over 1.0 volume of air per Minute per volume of the growth medium, but is preferably much higher. Effective growth of the organisms and optimal yields of tenebrimycin are obtained if the amount of air used is at least one volume of air per minute per volume of culture medium.

The concentration of tenebrimycin activity in the fermentation medium can be conveniently monitored during the course of the fermentation by testing samples of the culture medium for their inhibitory activity

did check against the growth of organisms, which are known to be inhibited in the presence of tenebrimycin. Two of the organisms that do so have been applied in tracking the production of tenebrimycin are Klebsiella pneumonlae and Mycobacterium butyricum.

The former organism is chiefly involved in the well-known turbidity measurement, while the latter is used in the dish-plate method will.

In general, maximum production of the antibiotic occurs within 4 to 7 days of inoculation of the culture medium if submerse aerobic Cultivation or shake flask cultivation is applied, and after a somewhat longer period if surface kuiiuren were used.

The mycelium and undissolved residues are removed from the fermentation liquor by conventional means, removed such as filtration or centrifugation. The antlblotic Activity is contained in the filtered liquid and can be obtained from it through application of adsorption processes. The adsorbents are the cation exchange resins »IRC-50« (weak acidic polymethacrylate carboxylic acid resin).

In general, the procedure for obtaining the tenebrimycin from the fermentation liquor is as follows: The entire liquid is filtered with a filter aid, after the pH of the mixture has been lowered to about pH 2 by adding an acid such as Sulfuric acid, phosphoric acid, hydrogen chloride and the like. The pH of the filtrate is set to a value of about 5.5 adjusted by the addition of a concentrated base and then the mixture is filtered again. The filtrate is passed through an ion exchange column with IRC-50 in the ammonium circuit Is packed. The column is then filled with distilled water washed and the antibiotic activity eluted with dilute acid. The fractions which make up the anti-biotic Containing activity are pooled and concentrated to approximately 1/20 of the original volume. The pH the concentrate is increased to about 11 by adding concentrated base, and the basic concentrate is then poured into about 6 volumes of acetone, mixed well and refrigerated. The microbiologically inactive one Precipitate that separates is removed by filtration. The pH of the filtrate is brought to about 3.5 lowered by adding 20% sulfuric acid with thorough mixing. The tenebrlmycin complex falls from In the form of the sulfate salt, while the Caerulomycln as a by-product of fermentation in the supernatant Layer remains that is discarded.

Tenebrimycin Vi is obtained by further fractionation of the tenebrimycin complex. For the fractionation, the tenebrimycin is used as an acid addition salt, preferably in the form of the sulfate. Colored contaminants can be removed by stirring the solution with about 5% (weight / volume) activated carbon. Effective discoloration is usually obtained by stirring the mixture containing the charcoal for about an hour. The mixture is then filtered and the filtrate, which contains the tenebrimycin sulfate, is loaded into a column packed with IRC-50 in an ammonium cycle. The column is washed with water and the individual antibiotics that form the tenebrimycin complex are gradually eluted with 0.1 η ammonium hydroxide. In general, elution is extremely slow and a very large number of fractions are common.

20th

25 Hch needed to manage the fractionation of the complex. So requires z. B. the complete fractionation of about 250 g of tenebrimycin complex 700 fractions, each of which constitutes about 900 ml of the fluate. The first active fractions obtained contain tenebrimycin I, Γ and II. This is followed by a series of active fractions in which tenebrimycin 11 is the only antibiotic. A number of inactive fractions follow the elution of tenebrimycin II before the fractions containing tenebrimycin IV are eluted. The first fractions containing tenebrimycin IV also show traces of tenebrimycin III. Tenebrimycin V is recovered from the column in the active fractions that follow tenebrimycin IV. To obtain tenebrimycin VI, the column is further filled with a concentrated ammonium hydroxide solution after all of the tenebrimycin V has been recovered from the column. A suitable solvent for removing tenebrimycin VI is 0.3 η ammonium hydroxide.

example 1

Preparation of the tenebrimycin complex by
mentation from S. tenebrarius ATCC 17920

Dextrin 10 G Yeast extract 1 G hydrolyzed casein 2 G Meat extract 1 G CoCl ₂ • 6H ₂ O 0.01 G washed out agar 20th G delonlslertes water, on 1000 ml

A sporulated culture of S. tenebrarius ATCC 17920 is produced by growing the organism on modified Bennett's Schärg agar medium _J0 which has the following composition:

The inoculated vegetative medium is kept on a rotating shaker at 37 ° C for 16 hours incubated at 250 r.p.m. works and has a stroke of 63.5 mm.

A 50 ml portion of vegetative cultivation is used to inoculate a 44-1 seed box that

The pH of the medium is adjusted to pH 7 before autoclaving. The agar slant is inoculated with S. tenebrarius spores ATCC 17920 and incubated for five days at 37 ° C and without visible light. That Growth on the agar slant is covered with water and gently scraped to remove the spores and to obtain an aqueous spore suspension.

The spore suspension thus obtained is used to inoculate 800 ml of a medium, the following Composition has:

Dextrose 0.05 96 Nutrient meal (contains 35 to 45% 1.5 96 dispersible protein) Dextrin 700 1 96 (Potato dextrin with low chlorine content) Potassium chloride 0.1% hydrolyzed casein 0.3 96 KH ₂ PO, 0.05 96 MgSO ₄ · 7H ₂ O 0.5 96 CaCl ₂ · 2H ₂ O 0.025% deionized water

contains an aqueous medium of the following composition:

Dextrose

Soybean grains

KH ₂ PO ₄

MgSO ₄

CaCl ₂ • 2H: 0

Antifoam agents

1.5

0.05

0.5

0.1

0.025%

0.025%

The medium of the seed container is at about 120 ° C Sterilized for 30 minutes. Stirring at a rate of 370 r.p.m. one starts immediately after of inoculation, and aeration at a rate of 0.0226 m 'per minute. will be used throughout the incubation period maintain. At the end of the incubation period, the contents of the seed container are added used to inoculate a 1130 liter fermenter that contains a medium of the following composition:

Dextrose 4.0% refined soybean oil 3.0% Soy flour 3.0% NH ₄ CI 0.5% CaCl ₂ 0.3% MgSO ₄ 0.2% NH ₄ NOs 0.1% hydrolyzed casein 0.5% Antifoam agents 0.2% deionized water

Before inoculation, the fermentation medium is sterilized at 120 ° C. for 30 minutes. The fermentation will performed at 37 ° C with aeration at a rate of 0.48 m 'per minute throughout Time from inoculation to harvest. Stirring is stopped at 125 r.p.m. started and up to 180 r.p.m. increased after 12 hours. The fermentation continues for 5 days. The fermented cultivation liquid is filtered to remove the mycelium and other undissolved solids. The filtered liquid contains tenebrimycin at a concentration of 680 units per mol.

Example 2

Isolation of the tenebrimycin complex

The pH of 880 liters of antibiotic fermentation liquid, obtained as described in Example 1, is lowered to about pH 2 by adding approximately 10 to 20 liters of 20% aqueous. Sulfuric acid.

After 45 kg of a commercially available filter aid added to the acidified liquid is mixed thoroughly and filtered. The filter cake is washed with water and the washing solution is added to the filtrate.

The pH of the filtrate is adjusted to pH 5.5 by adding 50% strength aqueous sodium hydroxide. About 4 kg of a commercially available filter aid is added and the mixture is stirred thoroughly and filtered. The filtrate is passed through an IRC-50 column measuring 10.2 cm by 1.83 m. the The column is packed with the resin in the ammonium circuit up to a bed height of 112 to 114 mm. After this the entire filtrate has passed through the column, this is washed with about 200 liters of distilled water. The antibiotic activity is then about 150 to 15 hours over a period of 12 to 15 hours 175 liters of 0.1 η sulfuric acid elulert and the eluate In 10 liter fractons removed. Antibiotic activity Can be seen in the eluate when its pH value drops to around pH 4 or a little below.

As the elution proceeds, the pH of the eluate continues to drop, until it reaches a value of about pH 1.5 achieved.

The fractions which contain antibiotic activity are combined and placed under reduced pressure

ίο concentrated about '/ jo of the original volume.

The pH of the concentrated eluate is adjusted to pH 11 adjusted by adding 5096 ml of aqueous sodium hydroxide, and the basic concentrate then becomes 6 volumes of acetone added. The mixture is cooled and turned off to allow the precipitate to settle separates without antibiotic activity. The mixture is filtered and the filter cake, the inactive precipitate contains, is washed with water and discarded. The pH of the filtrate is reduced to pH 3.5 Thorough mixing of the addition of 20% aqueous sulfuric acid set. The tenebrimycin complex separates out as sulphate during the treatment and is practically completely separated out when the pH value of the solutions reached 4 or a value slightly below. The mixture is filtered and the filtrate, which contains cerulomycin as a by-product of fermentation, is discarded. The filter cake that the tenebrimycinsulfai; contains, is dissolved in the smallest possible amount of water and cooled. All solid components which do not dissolve easily are discarded, e.g. B. all solids during the Precipitate or crystallize during cooling. The clear one The solution of the tenebrimycin sulfate is basic over a Dowex 1 x 1 resin bed from 10.2 cm to 2.13 m Im Cycle guided. The antibiotic solution is added to the column and 40 to 50 liters are then distilled Water. The drain is taken in 10 liter fractions, the active fractions are pooled and under vacuum to a thick syrup

concentrated, which is dried and the dry one Tenebrimycln complex results.

Example 3 Separation of the tenebrimyln factors

Tenebrimycin sulfate is obtained by adjusting the pH of a 20% aqueous solution containing 258 g of des Tenebrimycin complex contains in free base form to 4.5 by adding concentrated sulfuric acid. The solution of the tenebrimycin sulfate thus prepared is stirred for about one hour with 5% (Weight / volume) activated carbon decolorized. The solution is filtered and the filtrate is passed through a column of 9 × 150 cm sent that with about 7 liters of IRC-50 resin in the ammonium circuit has been packed. After that, the filtrate containing the antibiotic salt pass through the column it is washed out with about 17 liters of deionized water. The elution of the retained on the column antibiotic η activity begins with 0.1 η ammonium hydroxide solution at a flow rate of about 20 ml per minute. The eluate is collected in fractions of about 900 ml, and each fraction is checked for their microbiological activity. Those containing the same antibiotic factor Fractions are collected and lyophilized, to obtain the individual dried tenebrimycins. The tenebrimvcin-VI appears as folet:

Factions present antibiotic I, Γ and II t manufacture. 1- 34 inactive II 35-53 Tenebrimycin 54-77 Tenebrimycin IV with a trace of 78-153 inactive III 154-172 Tenebrimycin IV Tenebrimycin 173-249 Tenebrimycin V 250-256 inactive 257-634 Tenebrimycin VI 635-668 * inactive At point the eluate changes to 0.3 η ammonium 669-698 Tenebrimycin hydroxide over. *) At this Of course you can also use tenebrimycin VI-Heliantha

Example 4
Final cleaning of the Tenebrimycln-Vl

4 g of tenebrimycin VI helanthate are mixed with about 200 ml with the addition of sulfuric acid adjusted to pH 2

slurried water. The mixture is stirred for approximately 4 hours and passed through a sintered glass funnel medium porosity filtered to remove the helianth acid. The filtrate is decolorized by it through a thin layer of activated carbon and then over a column 1 χ 32 cm, packed with Dowex 1x2 resin, leads in the Hyclroxylkreislauf. After the filtrate, distilled water is then added to the column to remove the Complete the elution of antibiotic activity. The effluent is collected in fractions, the active fractions are combined and lyophilized and result in a dry residue containing the antibiotic. The residue is about 15 ml Dissolved water and then passed through a column of

'S 1x15 cm, packed with resin that is made 1st by Polymerization of acrylic acid in a Dowex-1 resin. The antiblotisiche activity is distilled from the column Water is eluted, the active fractions are combined and lyophilized to give about 700 mg high purity tenebrimycin VI.

1 sheet of drawings

Claims (2)

Patent claims: 1. Tenebrimycin anti-blot VI, characterized by the following parameters titratable groups, determined by electrometric titration in water, with pK _a values of about 5.6; 7.2; 8.2 and 9.3, IR absorption spectrum as in the figure, which is part of this claim. 2. Process for the preparation of tenebrimycin VI according to claim 1, characterized in that one Streptomyces tenebrarlus ATCC 17 920 in a medium containing assimilable sources of carbon, Contains nitrogen and inorganic salts, submerged under aerobic conditions, then adjusted to pH 2 adjusted fermentation liquid filtered with a filter aid, the filtrate adjusted to pH 5.5 after filtration through an ion exchange column, which leads with IRC-50 in the ammonium circuit is packed, then eluted with dilute acid containing the Fractions containing antibiotic activity are combined and concentrated to adjust the pH of the concentrate sets to 11 and the basic concentrate in acetone pours and cools, the inactive precipitate formed is removed by filtration and the pH of the The filtrate is lowered to 3.5 by adding 20% sulfuric acid to precipitate the antibiotic complex as sulfate salt, with the sulfate salt or another acid addition salt of the antibiotic complex, optionally after removing colored impurities, a column packed with IRC-50 Im Ammonium cycle, loaded, washed with water, tenebrimycin 1, Γ, II with 0.1 η ammonium hydroxide, III, IV and V eluted, then the column with a concentrated Ammonium hydroxide solution and then the tenebrimycin VI with 0.3 η ammonium hydroxide eluted.