DE1227194B - Process for the manufacture of new antibiotics Sp-30 - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. α .:

C12d

German class: 30h-6

Number: 1227194

File number: Sch 35872IV a / 30 h

Filing date: November 13, 1962

Opened on: October 20, 1966

The invention relates to the use of microorganisms of the genus Mjcromonospora for production new antibiotics Sp-30 by microbiological means as well as their enrichment, isolation, Purification and reconditioning.

The new antibiotics are made by getting a certain species of the Micromonospora genus (Order Actinomycetales), in particular the kind Micromonospora halophytica, breeds until antibiotic have formed active substances and isolated the antibiotic from the culture, if necessary broken down into its components and processed.

The new antibiotics obtained according to the invention by incubating suitable Micromonospora species are referred to as "antibiotics of the Sp-30 group".

Cultures of the preferably used according to the invention Microorganisms were found in the culture collection of the Department of Agriculture (Department of Agriculture) of the United States of America, Northern Utilization Research and Development Division, Peoria, Illinois. ■ A strain of the species Micromonospora halophytica n ·. sp. has NRRL number 2998 and was originally made from the mud at the bottom of one Salt pond in Syracuse, N.Y., USA, isolated. - M. halophytica is characterized by a long, ramified one Mycelium with an average diameter of 0.5 μ and abundant spore formation. The spurs are ellipsoidal to spherical and have a longitudinal axis or a diameter of 1.2 μ. In older cultures appear smooth-walled and dark in color. The spores appear to be statistically uniform To be distributed all over the mycelium and carried by short or long sporophores; occasionally they are also sessile. The vegetative mycelium does not normally divide into polymorphic elements, but seems to remain a whole. The mycelium is not acid-fast and gram-positive.

To isolate the mentioned microorganisms, some of the dried sludge sample is used in sterile distilled water, and after making appropriate dilutions, the suspension is spread on an agar medium. The medium expediently contains 0.5% tryptose, 2.0% soluble Starch, 0.3% calcium propionate and 2.0% agar, all in distilled water.

The cultures are tested for antibiotic activity by first growing them for up to 60 days at 26 ° C. in the following medium: 0.3% beef extract, 0.5% tryptose, 0.1% dextrose, 2.4% soluble Starch, 0.5% yeast extract, 1.5% agar, all in tap water. Then one extracts the process of making new ones
Antibiotics Sp-30

Applicant:

Scherico Limited, Lucerne (Switzerland)

Representative:

Dr.-Ing. A. ν. Kreisler, Dr.-Ing. K. Schönwald,

Dr.-Ing. Th. Meyer and Dr. J. F. Fues,

Patent attorneys, Cologne 1, Deichmannhaus

Named as inventor:

George M. Luedemann, Bloomfield, N. J .;

Marvon J. Weinstein,

East Brunswick. N. J. (V. St. A.)

Claimed priority:

V. St. v. America July 23, 1962 (211 806) -

whole aqueous agar with butanol and concentrate the butanol-water extract. Through the butanol-water mixture enough antibiotic is extracted to deliver a concentrate that works in one Disc test prevents the growth of Staphylococcus aureus and Bacillus subtilis. An antibiotic Activity against the same organisms can be observed after 96 hours in an aqueous Medium incubated submerged containing appropriate nutrients; e.g. a nutrient medium containing 1% NZ amine Contains type A (a pancreatic casein hydrolyzate) and 5% soluble starch.

M. halophytica is characterized by good growth in the pH range 6.8 to 7.8. Your colonies on many natural agar media are initially orange, but take on some as they age (20 days) Shades of brown; Light brown ("tan"), orange brown and brown are common color variants. The colony rarely turns dark brown, never black. The mentioned microorganism forms abundant spores, especially in older, brown-colored colonies. When using a 0.5% yeast extract and 1% one the following sugars: galactode, lactose, laevulose, mannose, raffinose and trehalose Agars usually form a reddish brown diffusible pigment.

Table I compares the new described above with certain known Micromonospora species. If there are dashes in individual table fields

609 707/380

3 4

find, it means that. appropriate data incubated. From the position of the zones of inhibition

either missing or not listed in the tables - on the R _F values of the antibiotic active components

were taken. ten close.

Table I compares the characteristic properties. Table II shows the solvent groups used by colonies of the preferred systems according to the invention and the R _F values determined therein for the antibiotic components and some known species of the genus; it is indicated in each case whether Micromonospora is a descending or ascending solution agent after 14 days of incubation at 26 ° C. when different substances are used. The data are medium. The isolated antibiotics can be processed in the usual manner for the known types, eg. B. are compared in their derivatives according to used, are out of io to be overfeed.

Bergey's "Manual of Determinative Bacteriology", It is already known to be an antibiotic under the

7th edition, 1957, edited by Williams & name Micromonosporin by cultivating one

Taken from WilMns Company, Baltimore. Microorganism of the genus Micromonospora

The microorganisms which can be used according to the invention. W a k s m a η et al. have the properties

Men supply this antibiotic in Soil Science, 54, pp. 281 to 296 with the aid of the 15 described below

Fermentation Process Antibiotic Active Substan- (1942), and Journal of Bacteriology, 53, pp. 355-357

Zen. After breeding, these can be found both in (1947), described. This description shows that the

Mycelium as well as in the broth; after separation of the micromonosporin, all properties are

Mycelium from the broth is identified as a source to its area by the antibiotics of the SP-30 group.

The broth is preferably used for extraction. You divorce. From the first work, table VI shows

initially holds mixtures of antibiotics. on page 291 and from the text in the last paragraph

From paper chromatic investigations it has been found on page 290 that Micromonosporin is opposite

show that that caused by M. halophytica and their B. Mycoides and B. Cereus is inactive. In contrast

Equivalents of antibiotic substances formed from Table IV shows that the antibiotics SP-30

seem to consist of at least four components. 25 are effective against these bacteria. On page 291

The antibiotic mixture formed by M. halophytica of the first-mentioned reference is stated that

is referred to below as SP-30 and the micromonosporin with ether at pH values

named four components with SP-30A, SP-30B, between 3 and 8.5 cannot be extracted.

SP-30C or SP-30D. In contrast, the SP-30 antibiotics with ether are good

For the formation of the antibiotic substances, 30 can be extracted at pH values between 2 and 9.5,

the microorganisms in the usual way at a Finally, in the second reference on

Temperature of about 25 to 40 ⁰ C under submerged page 357 indicated that it is Micromonosporin

aerobic conditions in an aqueous nutrient medium is a highly pigmented, stable protein,

cultivated, which is an assimilable carbon and The antibiotics of the SP-30 group are neither pigmen-

Contains nitrogen source. Suitable carbon sources are still of protein character,

are carbohydrates such as starch, dextrin, certain methods. The following examples are suitable methods

Sugar and the like. Suitable nitrogen sources can be used for the fermentation of M. halophytica, for extra-

Both organic and inorganic in nature here are the antibiotic substances from the broth

but they are preferably organic products, and to separate these substances into theirs

such as soybean meal, peptones and the like explains 40 main components. In some of these

The fermentation takes place at a pH in the range games are test values of the antibiotic in question

from about 6 to 8 over a period of about 96 to substances, expressed in units per milligram,

120 hours; towards the end of this period is given as a measure of their activity. The exam

maximum antibiotic formation achieved. Separation takes place microbiologically according to a standardized one

now mycelium and broth from each other and isolate the 45 agar diffusion test method ("disc type"; cf. Donald

Antibiotics as described below: C. Grove and William A. Randell, "Assay

Since the vast majority of them are antibiotic Methods of Antibiotics - A Laboryator Manual «,

active substances is in the broth, the New York [1955]) using staphylococ-

Mycelium filtered off and discarded. The antibiotic cus aureus (ATCC 6538P) as a test organism. It

Substances are extracted from the broth by means of a solution.

agent extraction isolated. You can counteract the dosage of the concerned by distribution-targeted effect

chromatographic methods in their components antibiotic substance (diluted in phosphate buffer

be separated. at pH - 8) in the following medium:

The antibiotic substances are initially Perrton '' 0 6 ° /

from the extract obtained from the broth through 55 pancreatic cesein hydrolyzate '.'. '.'. ' 0.4%.

Evaporation of the solvent in the form of a jerk yeast extract 0 3 ° /

Stand separated. The residue becomes paper beef extract ΎΥΥΥΥΥΥΥΥ. o | l5%

chromatographically investigated what could be a clue dextrose 015 ° /

is used for partition chromatography. The paper aear 15 ⁰ i

Chromatograms are in different solution 60 jj ^ g ⁰

by means of systems and the R _F values for '

the individual components according to bioautographic The suspension of the test organism used

Standard methods determined, which consist in that (Staphylococcus aureus) is standardized in such a way that

develop a paper chromatogram and dry it in a colorimeter light with one wavelength

net and then through a Staphylococcus aureus 65 of 660 πιμ, to 20%. The quantitative

inoculated agar plate. After a period of contact, the respective antibiotic sample becomes effective

of 15 minutes, the paper is determined from the plate from the reference curve and in units per

separately, which one then expressed 16 to 20 hours at 37 ° C milligrams; such a unit counts as a unit

Amount of test substance required to form an 18 mm zone of inhibition on a 13.5 mm diameter disc.

For example

Obtaining crude SP-30 by incubating M. halophytica on a laboratory scale

A. Germination

A lyophilized culture of M. halo- ίο is brought phytica in a 300 ml shake flask containing 100 ml of the following sterile culture medium:

Bacto beef extract 3 g

Tryptose 5 g

Dextrose Ig ^1S

Soluble starch 24 g

Yeast extract 5 g

Tap water 1000 ml *)

* In this and similar recipes throughout the description The solvent (mostly water) is given through it once as an absolute amount, another time with the prefix »ad«. Although these are, strictly speaking, different indications, they can be within the scope of these recipes required accuracy, these two expressions can be exchanged for each other as required.

25th

The flask and its contents are incubated for 5 days at 37 ° C. on a rotating shaking machine (280 revolutions per minute, 5 cm shaking movement).

B. Fermentation (incubation)

Transfer 25 ml of the inoculum obtained by the above germination to four 2-1 flasks, each of which contains 500 ml of the following medium:

Soluble starch 50 g

NZ-amine type A 10 g

Soft water ad 11

pH 7.45 after sterilization

The flasks and their contents are kept for 96 to 120 hours at 28 ° C. on a rotating shaker incubated.

C. Isolation of SP-30 from the
Fermentation broth

35

45

Diatomaceous earth is added to 3.51 of the combined substrates from Part B of this example as a filter aid, filtered, washed the filter cake with several 100 ml of water and combined the filtrate and washing liquid (Total volume about 3600 ml). An equal volume of ethyl acetate is then added and the mixture is stirred For 30 minutes, separate the ethyl acetate layer and extract the aqueous layer again with a equal volume of ethyl acetate. The ethyl acetate extracts are combined and placed on a thin film evaporator evaporated in vacuo to a residue weighing about 4 g and on agar plates a antibiotic activity against Staphylococcus aureus ATCC 209P down to a dilution of 1 ^ g / ml shows.

The lyophilized culture of M. halophytica according to Part A of this example can be obtained by a corresponding Culture to be replaced by M. halophytica var. Nigra.

Example 2

Obtaining crude SP-30 by fermentation of M. halophytica on an industrial scale
A. Identifier

As in example 1, A.

B. Preparation of an inoculum

Each 25 ml of the first inoculum obtained by the above germination is transferred to four 2-1 flasks, each containing 500 ml of the sterile medium used for germination, incubate the Flask and its contents for 5 days at 28 ° C on a rotating shaker (280 revolutions per minute, 5 cm Shaking movement) and unites the contents of the flask. 25 ml each of the second inoculum thus obtained transfers the flasks are incubated on ten 2-1 flasks, each containing 500 ml of the above sterile medium including contents 3 to 5 days at 28 ° C on a rotating shaker (280 tours per minute, 5 cm Shaking movement), combines the contents of the flask and aseptically transfers them to a sterile inoculum bottle with side extension tube (total volume: approx. 51).

C. Tank fermentation

Transfer the 51 of the part B of this example obtained inoculums aseptically into an approximately 1301 capacity fermenter, which is approximately 901 of the following sterile medium contains:

Soluble starch, 4500 g

NZ-Amin type A 900 g

Defoaming agent 100 ml

Soft water ad 901

Incubate for 55 to 65 hours (until the compact cell volume is about 4.5 to 5.0%, as can be seen through Centrifugation at 2800 rpm for 5 minutes determined) aerobic under the following conditions:

Temperature 26 ° C

Supply of sterile air ... about 76 to 80 l / min

Pressure about 0.5 to 0.6 atmospheres

Stirring motion 160 to 165 rpm

The following data are typical of the fermentation process:

To
hours temperature Air supply pressure Around
rotations PH Compact
Cell volume Disc inspection
against Incubation ° C l / min atü per minute % Staph. aureus 0 26th 76 0.49 160 6.80 8th 26th 76 0.55 160 6.95 1.0 - 16 26th 76 0.53 160 6.85 1.0 - 23 26th 76 0.52 160 7.10 1.5 14 mm zone 31 26th 76 0.53 160 7.35 1.5 38 26th 79 0.49 165 7.60 2.5 - 46 26th 79 0.54 165 7.75 2.5 21 mm zone 54 26th 79 0.52 165 7.90 4.5 - 65 26th 79 0.51 165 7.80 5.0 25mm zone

D. Isolation of SP-30 from the
Tank fermentation

Filter the mycelium from the broth using a filter aid, adjusts the pH of the filtrate to 7.0 and extracts the present 901 with 2 volumes of ethyl acetate. The extract is concentrated in a vacuum to 31, which is removed when standing separating aqueous layer and then concentrated further to about 200 ml. Then left over ι ο Standing in the cold at night, filtered from the precipitate from, the filtrate pours into ten times the volume of a mixture of petroleum ether and ethyl ether (1: 1) and nitrates the precipitated Precipitation also from. Wash it with several portions of the solvent mixture and combines the washing liquid with the filtrate. The combined fluids that make the antibiotic Containing substances are evaporated in vacuo to a residue of 18 g of a dark brown oil. This, the raw SP-30, prevents the growth of Staphylococcus aureus 209 P on agar in Dilutions down to 1 μg / ml.

..- ■ · -

. - ■ -. '' Example 3

Separation of SP-30 into its components

^: The separation of the crude SP-30, as it is obtained according to Examples 1 and 2, into its components. “Is carried out by column chromatography using purified diatomaceous earth as the inert carrier.

A glass column with a diameter of about 12.7 mm and a height of about 305 mm is packed with a suspension of Chromosorb W in formamide. Then you mix a small amount of Chromosorb W with 1 ml of a methanolic solution of 408 mg SP-30 (obtained according to Example 2, D), dries vacuum the mixture to remove the methanol and carefully place it on top of the column on.

First elute in sequence with 100 ml of each of the following solvent mixtures:

•

a) Benzene-chloroform (95: 5), ■

.saturated with formamide,

b) benzene-chloroform (90:10),
saturated with formamide,

c) benzene-chloroform (75:25),
saturated with formamide,

d) benzene-chloroform (50:50),.

saturated with formamide,

e) benzene-chloroform (25:75), '
saturated with formamide,

then with 600 mg of chloroform and finally with 1000 ml of methanol. A total of 84 fractions of 25 ml each are obtained in this way. Each of these fractions is bioautographically tested against Staphylococcus aureus ATCC 6538 P, and based on the test results the fractions with similar antibiotic activity and composition increase Groups united. Each of the groups thus formed is evaporated in vacuo, mixed with water and extracted with chloroform. The chloroform extracts are separately evaporated to dryness, the obtained Residues, as described above, bioautographically tested against Staphylococcus aureus and their components identified by the location of the spots in the paper chromatogram. The activity is made according to the standardized disc method using a disc with a diameter of 6.3 mm, which was impregnated with Staphylococcus aureus, was determined. The results of this Tests are shown in Table III.

Physical and chemical properties of the antibiotics obtained according to the above examples

The antibiotics of the SP-30 group are in most of the common organic solvents, such as Ethyl acetate, chloroform, methylene chloride (as well as other halogenated hydrocarbons !!) and low Alkanols (e.g. methanol and ethanol), soluble, very poorly soluble in water. At a pH im Range from 2 to 9.5 they are obtained from the fermentation broth by butanol, ethyl acetate, methylene chloride, Chloroform, carbon tetrachloride, benzene or ether easily extracted.

In qualitative tests on specific groups, the SP-30 behaves as follows:

Ninhydrin and Sakaguchi test

negative (both the crude SP-30 and all of the individual 'fractions);

On reducing sugar

(ammoniacal silver nitrate)
positive (of the individual components, component A reacts strongly positive, components B, C and D react negatively;

Reduction of ferricyanide

positive (of the individual components, component A reacts negatively, components B, C and D positive).

SP-30 and its individual components, when dissolved in methanol, show the following absorption behavior against ultraviolet light (in the range from 220 to 400 ηιμ):

The raw SP-30 has a rather difficult to describe absorption curve with inflection points at 260 and 290 ΐημ, the inflection point at 260 ηαμ having an E ¹⁰ / »of about 28; the component SP-30 A has a similar curve with inflection points at 258 πιμ (E ¹⁰ I " about 70) and 295 ΐημ; SP-30 B shows a maximum at 268 ηιμ (E ¹⁰ / o about 111); SP-30C shows no maximum, but turning points at 265 ηιμ (E ¹⁰ / o about 181) and at about 290_πιμ; SP-30D shows a maximum at 263 ΐημ (E ¹⁰ O about 115).

The infrared spectrum of SP-30 and its components SP-30 A, SP-30 B, SP-30 C and SP-30 D, suspended in Nujol *) are shown in FIGS. 1 through 5 of the drawings. The absorption maxima (schw = weak, m = medium, m-st = medium to strong, st = strong, sst = very strong, Sch = shoulder) and Other characteristics of the spectra are at the following wavelengths:

*) ■ Nujol is a trademark for a mineral oil based on hydrocarbons.

Raw SP-30 (see Fig. 1)

λ (μ) intensity

2.98 st

5.78 st

6.00 m-st (wide)

6.58 black

6.81 m-st

7.02 black (Sch)

7.22 m

7.71 m-st (Sch)

7.83 st

8.32 black (Sch)

8.86 m-st

9.28 m-st

9.54 wide

9.75 wide

13.42 m

14.20 black (wide)

SP-30A (see Fig. 2) λ (μ) intensity

2.95 st (wide)

3.42 m

5.82 st (Sch)

5.89 st

6.15 m (Sch)

6.60 black

6.80 black

7.16 m-st

7.56 m- (wide)

8.50 black

9.22 black

9.46 black

SP-30B (see Fig. 3) λ (µ) intensity

2.98 black (wide)

5.72 m-st

5.80 m-st

6.79 m-st

7.15 m-st

7.21 m-st

7.88 st

8.70 st (wide)

10.00 st (wide)

11.51 m-st

12.00 st (wide)

13.00 st (wide)

SP-30G (see Fig. 4) λ (µ) intensity

2.95 black (wide)

5.74 st

6.20 black (Sch)

6.79 m-st

7.20 m

7.70 st (Sch)

7.82 st

8.85 st

9.28: st

9.56 m

9.60 m (wide)

9.90 m (wide)

11.50 black (wide)

12.00 m-st (wide)

12.80 m-st (wide)

13.35 black (wide)

14.15 _w black (wide)

SP-30D (see Fig. 5)

λ (μ) intensity

2.95 m

5.75 st

6.00 m (Sch)

6.16 black (Sch)

6.30 black (Sch)

6.56 black (Sch)

ίο 6.78 m-st

7.20 m

7.28: ... black (Sch)

7.70 st (Sch)

7.80 st (Sch)

7.88 st

8.32 black

8.52 black (Sch)

8.88 ... st

9.28 st

ao 9.42 m-st (wide)

9.56 m-st (wide)

9.72 m-st (wide)

10.44 black

11.30 black (wide)

11.60 black (wide)

11.82 black (Sch)

12.42 st

13.32, .. m (wide)

13.48 m (wide)

14.20 m (wide)

The stability of the antibiotics of the SP-30 group is different for the different components. Fraction A is stable at room temperature when mixed with an aqueous buffer at a pH range of 2 to 10. When heated to 100 ^° C., its antibiotic activity drops when the pH is less than 6. The activity is lost on heating at 100 ° C. for 30 minutes at pH = 2, whereas it is not reduced under similar conditions at a pH of 7 and more. - Fraction B is stable within a pH range of 2 to 10 at room temperature. At an elevated temperature (100 ^° C.) it is stable down to a pH of 2 for 30 minutes; then the activity decreases. Fraction C is still ^{stable for 30 minutes even at 100 °} C. within a pH range of 2 to 10. Fraction D is stable for 30 minutes at room temperature within a pH range from 2 to 10, at 100 ^° C. within a pH range from 4 to 10.

Biological properties of the antibiotics obtained according to the above examples

The antibiotics of the SP-30 group have a wide range of effects against gram-positive organisms. They are of particular value for treating diseases caused by penicillin resistant Microorganisms were caused

(see Table IV). They are also used to treat caused by Staphylococcus aureus, Diplococcus pneumoniae and diseases caused by pathogenic microorganisms are useful.

Table IV gives the in vitro activity of SP-30

⁶ S against a number of gram-positive organisms. The susceptibility of the microorganisms to the antibiotics was determined using standardized tube dilution methods: The inoculum was used throughout

609 707/380

dilution at 10 ~ ⁵ a 24 hour old broth culture used were the 24-hour incubation at 37 Endpunkte.nach ⁰ C was added; the nutrient medium consists of tryptose phosphate broth. (The antibiotic material used for this determination is that obtained from Example 1, G, which has a potency of 4000 dilution units per mg; that is, if 1 mg of the product is dissolved in 1 ml of solvent and diluted to 4000 times, then under the applied test conditions still prevent the growth of Staphylococcus aureus JATCC 6538P] zone was measured after a disc impregnated with the antibiotic had been placed on an agar plate inoculated with the test organism and ^{incubated for 24 hours at 37 0} G, an in vitro activity against certain microorganisms, as set out in Table V. The in vivo activity against certain bacterial infections in mice was also tested. The mice were infected with an inoculum of the bacteria in question by intraperitoneal injection twice daily and were then ^he aqueous carboxymethyl cellulose, was used with subcutaneous injections of SP-30, wherein the above material from Example 1, C, suspended in 0.25% strength, treated. A 100% ^e r protection of Staphylococcus aureus infected animals was thereby achieved using 10 mg (= 40000 dilution units) per mouse per day, a 100 ° / oig ^e r protect with Diplococcus pneumoniae infected animals using 15 mg (= 60000 dilution units) per mouse and day, administered over 2 days. - The acute toxicity LD ₈₀ of the same SP-30-preparation, in a conventional manner determines (dosage form: suspension in 0.25% strength aqueous ^he carboxymethylcellulose), was found to be> 7500 mg per kg mouse by subcutaneous,> 7500 mg per kg Mouse when administered intraperitoneally and 800 mg per kg mouse when administered intravenously.

Table I.

Used
medium M. halophytica
NRRL 2998 M. chalcea M. fusca aerobic M. parva aerobic M. globosa aerobic M. coerulea Glucose--
Asparagine
Agar Growth: medium
moderate to poor;
Colony flat. Colour:
Orange-g 4 LA,
Intense orange-50 no aerial mycelium;
Growth: extremely good;
does not form soluble pig
ment. Color: pale pink
to deep orange with
brownish to greenish
black spore layer Growth: extremely good.
forms soluble brown
Pigment. Color: changes
from orange to deep
brown with gray-black
to brown-black spo
reschicht Growth:
bad Growth:
bad Growth:
bad gelatin liquefaction liquefaction Weak liquefaction liquefaction liquefaction rapid
liquefaction milk is yerdaut Occasional peptone formation
lich coagulation. is digested slowly,
not coagulated remain
unchanged Coagulation
and peptone
education can koagu
be lured
digestion
very weak Sucrose is consumed is inverted is inverted won't
inverted will
inverted will
inverted strength is hydrolyzed is hydrolyzed is hydrolyzed will
hydrolyzed will
hydrolyzed will
hydrolyzed Cellulose is decomposed Is rapidly decomposed is weakly attacked will
not decomposed- will
not decomposed will
not decomposed Njtrat-
reduction Formed from nitrates
hen nitrites arise from nitrates
Nitrites changeable no
reduction from nitrates
develop
Nitrites no
reduction temperature Good growth at
18 to 40 ⁰ C, no
Growth at 50 ° C optimal growth between
between 30 and 35 ⁰ C --- - - · - growth
(aerobic or
anaerobic) aerobic aerobic aerobic
£

Table II.

Chromatographic Studies.

on those formed by incubation of M. halophytica antibiotic substances

60

Solvent used Rf values of the main _: system stains A. descending 0.0, 0.35, 0.60, 0.72 B. ascending 0.02, 0.11, 0.95 C. ascending 0.95 D. ascending - 0.95 E. ascending 1.0 F. ascending. 1.0 G. ascending 1.0 H. descending., - 0.01, 0.44, 0.65, 0.83

65 Systems used

A. Benzene-chloroform (93: 7 parts by volume), saturated with formamide. Chromatographic paper in 25% methano-fruited formamide before use immersed, extinguished and for 5 minutes to remove the methanol. air dried.

B. Benzene-Methanol (9: 1).

C. methanol-water (80:20) at 3 ° / _o sodium chloride content. Before developing, the paper was buffered with a 0.95 molar sodium sulfate and 0.5 molar sodium sulfate solution and dried.

D. Propanol-acetic acid-water (50: 5:40).

E. butanol-acetic acid-water (4: 1: 5).

F. Propanol-pyridine-acetic acid-water
(15: 10: 3: 12).

G. Phenol-Water (80g: 20ml).

H. methylene chloride-carbon tetrachloride (80:20), saturated with formamide. The chromatographic paper was in 50% strength before use Dipped methanolic formamide, extinguished and to remove the methanol on for 5 minutes air dried.

The observed R _F values differentiate the antibiotics of the SP-30 group from all known antibiotics including oleandomycin, novobiocin, penicillin G and erythromycin.

Table III

Chromatography of SP-30 group antibiotics on diatomaceous earth

Eluate fraction
No. group
No. Contains components 1 A. no 2 to 5 B. D (track) 6 to 16 C. D, C (track) 17 to 23 D. C, D (track) 24 to 43 E. C. 44 to 46 F. C ₅ B 47 to 57 G B, C (track) 58 to 80 H no 81 to 84 I. A, B (track) C (track)

Table IV
In vitro activity of SP-30

tribe
description ATCC 7064 Minimal Systematic name DA 39 Inhibi-
functional
focus the microorganisms used DA 30 tration Bacillus cereus var. Mycoides DA 31 µg / ml Bacillus cereus var. Mycoides DA 36 6.0 Bacillus cereus var. Mycoides DA 35 0.75 Bacillus megatherium DA 32 12.0 Bacillus megatherium DA 33 0.25 Bacillus megatherium DA 999 3.0 Bacillus sphaericus DA 40 6.0 Bacillus sphaericus ATCC 6538 2.0 Diplococcus pneumoniae ... ATCC 6538P 1.0 Staphylococcus albus ATCC 12715 0.075 Staphylococcus aureus ATCC 1163 0.75 Staphylococcus aureus ATCC 9996 0.25 Staphylococcus aureus 0.25 Staphylococcus aureus 2.0 Staphylococcus aureus 1.0 Staphylococcus aureus var. DA 141 0.75 Gray Staphylococcus aureus vai. DA 2026 0.25 Smith DA 2027 Staphylococcus auieus 0.25 (penicillin-resistant clinical isolates) 0.25 0.75

Table IV (continued)

tribe
description DA 2028 Minimal Systematic name DA 2029 Inhibi-
functional
focus the microorganisms used DA 2030 tration DA 2031 µg / ml DA 2032 1.5 DA 2033 0.75 DA 2034 2.0 DA 2035 1.0 DA 41 0.75 ATCC 9341 0.75 DA 60 0.75 Staphylococcus epidermidis DA 21 1.0 Sarcina lutea ATCC 10541 0.25 Micrococcus flavus DAIlO 0.75 Streptococcus pyogenes C. DAlIl 0.05 Streptococcus fecalis DA 150 0.025 Escherichia coli 16.0 Escherichia intermedia 16.0 Mycobacterium smegmatis ^AV J ^U
2.0 4.0

Table V
In vitro activity of the SP-30 group antibiotics

30 Used 35 SP-30A 40 SP-30 B 45 SP-30 C " Applied Inhibition zone (mm) against Strepto
coccus Bacillus Anti
bioticum Conc
tration Staph
coccus pyogenes subtilis V-S aureus 29 12th SP-30 1000 28 19th + 50 SP-30 D 100 19th 9 0 10 13th 0 0 1 9 31 9 1000 28 12th 0 100 17th 0 0 10 + 0 0 1 0 35 11 1000 28 20th 8th 100 20th + 0 10 11 0 0 1 0 33 14th 1000 28 24 8th 100 22nd 14th 0 10 17th + 0 1 9 19th 0 1000 19th 10 0 100 13th 0 0 10 + 0 0 1 0

"+" Means the presence of a small one, numerically 55 poorly definable inhibition zone.

Claims (1)

Claim: The use of microorganisms of the 60 genus Micromonospora, especially the species M. halophytica (NRRL 2998) for the production of new antibiotics SP-30 by cultivation, isolation from the culture, optionally decomposition into its components SP-30A, SP-30B, SP-30C 65 and SP-30 D and work-up. 1 sheet of drawings 609 707/380 10.66 © Bundesdruckerei Berlin