CH626628A5 - Process for the preparation of an antibiotic - Google Patents

Description

The present invention further shows a process for the preparation of MM 13902 and its salts, which consists in the cultivation of strains of Streptomyces producing MM 13902, with subsequent separation of MM 13902 or its salts from the culture medium.

It has been found that strains producing MM 13902 for this process are strains of Streptomyces olivaceus and related organisms of the type described below.

The most suitable organisms are strains of Streptomyces olivaceus such as ATCC 21379, 21380, 21381, 21382 and 31126 or mutants thereof. A particularly suitable organism for the production according to the invention is Streptomyces olivaceus ATCC 31126.

The term “cultivation” used here means the intended aerobic cultivation of organisms in the presence of assymilizable sources of carbon, nitrogen, sulfur and mineral salts. Such aerobic rearing can take place in a solid or semi-solid nutrient medium or in a liquid medium in which the nutrients are dissolved or suspended. The cultivation can take place on a ventilated surface or in submerged culture. The nutrient medium can be composed of complex nutrient substances or of chemically defined compounds. It has been found that media containing complex nutrients, such as yeast extract, soybean meal and the like, are particularly suitable. It has also been shown that the addition of cobalt ions, sulfate ions and calcium carbonate has a beneficial effect.

A temperature suitable for cultivation is 28 ± 2 ° C, with acceptable antibiotic yields being obtained. A suitable time for introducing the cultivated product is two to three days after the start of the fermentation.

The term “mutant” used here encompasses any mutated strain which arises spontaneously or is formed by the action of an external agent, which agent can be used intentionally or in another way. Suitable methods for producing mutant strains are those as described by H.I. Adler in Techniques for the Development of Microorganisms in "Radiation and Radioisotopes for Industriai Microorganisms", Prodeed-ings of an Symposium, Vienna, 1973, page 241, International Atomic Energy Authority and these methods include:

1. Ionizing radiation (such as X-rays and gamma rays), UV light plus a light-sensitive agent (such as 8-methoxypsoralen), nitrous acid, hydroxylamine, pyrimidine-base analogues (such as 5-bromouracil), acridines, alkylating agents (such as Mustard gas, ethyl methanesulfate), hydrogen peroxide, phenols, formaldehyde, heat, and

2. Genetic techniques such as recombination, transformation, transduction, lysogenization, lysogenic conversion and selective techniques for spontaneous mutation.

It has been found that the use of a mutation-promoting agent can lead to the production of organisms that have the ability to obtain increased levels of the desired antibiotics. For example, irradiation of cultures of Streptomyces olivaceus ATCC 21379, followed by separation of the strain obtained, which appears to produce the greatest zone of activity in the KAG test, leads to the isolation of Streptomyces olivaceus ATCC 31126, which is a preferred strain is for the purposes of this invention. ATCC 31126 was deposited in the Netherlands under the designation CBS 155.75 '. The aforementioned KAG test will be described below.

As a rule, all insulation and cleaning processes for the desired products mentioned should take place at not elevated temperatures, e.g. below 20 ° C and even better not above 12 ° C.

The desired product is usually obtained predominantly from the culture filtrate, so that the first isolation step to be used preferably is the separation of the solid material from the fermentation product, e.g. by filtration.

An impure preparation of MM 13902 or a salt thereof can be obtained from the clarified culture filtrate by absorbing MM 13902 or its salt on an active material such as activated carbon or the like, and then eluting the desired substance from the active material using a solvent. like aqueous acetone. Usually this procedure is used with the dibasic salt of MM 13902.

Alternatively, an impure preparation of MM 13902 or its salt can be obtained from the culture filtrate by s

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Extraction using a lipophilic ammonium salt and a water-immiscible solvent. This procedure is often more effective than the process described above (the MM 13902 can be obtained as the substituted ammonium salt by evaporating the organic solvent under reduced pressure). Preferably, the solution of the substituted ammonium salt of MM 13902 initially obtained in this way is subsequently extracted back into an aqueous phase using a solution of an alkali metal iodide, such as sodium iodide. This latter process variant generally leads to the production of an aqueous solution of an impure dibasic salt of MM 13902.

In the following, the impure forms of MM 13902 or their salts are expediently and generally subjected to the prescribed chromatographic processes in order to convert the material into a suitable purity.

Streptomyces olivaceus and related organisms have the following characteristics:

a) they have trace mycelia in air, either in the yellow or gray series;

b) they have a spore carrier shape, either rectiflexible or spiral;

c) they have spores with a smooth surface;

d) they do not produce melanin.

Species that have the above characteristics are those listed in Tables 5 to 9.

Description 1

Gust determination

The iso value is the proportion of material which is required to produce a 50% inhibition of the hydrolysis of ampicillin by the β-lactamase from Escherichia coli B1, an organism which contains an R-factor-controlled β-lactamase. This β-lactamase is classified as a type IIIa enzyme according to the classification by Richmond and Sykes (Adv. In MicrobiologicalPhysiology, 9,31 [1973]).

The extent of ß-lactamase hydrolysis of ampicillin into its penicillic acid can be followed by an iodine starch test, in which the amount of penicillic acid is measured by monitoring the decolorization of an iodine-starch complex. This method and the preparation of the β-lactamase are described in a publication by Cole M., ElsonS., And Fullbrook P.D. (Biochemical Journal 1972, 127, 259-308). A slight modification of the above method was used in that the sample of the inhibitor was pre-incubated with the enzyme for 15 minutes at 37 ° C before the addition of the substrate ampicillin. The procedure was as follows:

Reagents

Buffer: 0.05m potassium phosphate buffer, pH 7

Starch / iodine solution: prepared according to Novick, Biochemical

Journal (1962) 83,236 Substrate: Ampicillin 40 (xg / ml in buffer

β-lactamase enzyme: prepared from E. coli B1, as described by Cole and co-workers, Biochemical Journal (1972) 127,295. The dilution of the enzyme preparation in the buffer was such that it dropped approximately 0.3 units of optical density in 100 seconds in the uninhibited reaction. Other strains of ß-lactamase from E. coli can be used, particularly those which have an R-factor of e.g. B. have RTEM.

conditions

The reaction was carried out in a 1 cm cuvette at 37 ° C. in a Pye Unicam spectrophotometer SP 800. This instrument can carry four cuvettes and the corresponding blank samples at the same time. The first cuvette was used to control the reaction and contained no inhibitor. The second, third and fourth cuvettes were used for the various dilutions of the inhibitor.

Reagents

sample

Blank test

Cuvette

Cuvette

Starch / iodine reagent

1.0 ml

1.0 ml

E. coli β-lactamase

0.1 ml

-

Buffer inhibitor

0.1 ml

0.1 ml

buffer

0.3 ml

0.4 ml

Substrate (supplied after incubation

the above mixture during

15 minutes at 370 C)

1.0 ml

1.0 ml

The progress of the reaction was monitored by registering the optical density at 590 nanometers and measuring the speed of the reaction by changing the optical density for three to six minutes at 100 second time intervals. The inhibitor sample was diluted until a dilution was reached which gave 50% of the reaction rate of the comparison test without inhibitor.

Description 2

The KAG test

The KAG test is a method for determining the presence of β-lactamase inhibitor in fermentation solutions or during the stages of isolation. Melted nutrient agar is germinated at 45 ° C. with a suitable β-lactamase-producing strain from Klebsiella aerogenes and subsequently mixed with a sufficient proportion of a sterile solution of penicillin G, so that a concentration of 6 μg / ml penicillin G is obtained in the agar The agar is then poured into a petri dish and, after solidifying at uniform distances, provided with cylindrical holes using a sterile metallic cutting tool. The solutions to be tested are filled into these holes. The dish is then incubated at a constant temperature between 27 and 37 ° C. During the incubation period, an inhibitor, which may be present in the test solutions, diffuses from the hole into the agar and inhibits the action of β-lactamase, which is produced by the Klebsiella cells, thus protecting the penicillin G from being destroyed by β-lactamase and is present in sufficient concentration to do that To prevent growth of Klebsiella. In those parts of the agar into which the inhibitor was unable to diffuse in sufficient concentration, the penicillin G is destroyed by the β-lactamase, and consequently a dense growth of the Klebsiella develops. Clear circular inhibition zones of the adhesive growth around the holes containing inhibitor are thus formed, the size of the zones being dependent on the concentration of the inhibitor in the solution tested. The potency (arbitrary units per ml) of the test solutions is obtained by plotting the logarithm of the inhibitor concentration against the zone diameter. Such a test system can also be used for bioautography.

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Description 3 Preparation of MM 4550 (complex), comparable to that according to British Patent No. 1,363,075 Streptomyces olivaceus ATCC 21379, was grown for seven days at 28 ° C. on a solid agar layer in a Roux vessel. The agar medium had the following composition:

component

Proportion (g / 1)

Yeast extract

10.0

Glucose monohydrate

10.0

Agar

15.0

Tap water

1 liter

(The yeast extract used was "Yeatex" supplied by Bovril Food Ingrédients, PO Box 18, Wellington Road, Bur-ton-on-Trent, UK, and the agar was supplied by Oxoid Limited, Southwark Bridge Road, London, SEI, UK ).

The pH of the medium was adjusted to 6.8 before sterilization. 50 ml of sterile deionized water containing 0.02% "Tween 80" (registered trademark, Tween 80 is a polyoxyethylene sorbitol monooleate) was added to the Roux vessel culture and the spores suspended in it by shaking. The spore suspension was added as an inoculum to 75 liters of sterilized seed medium in a 100 liter stainless steel fermenter. The composition of the seed medium was as follows:

component

Proportion (g / 1)

Soy flour

10.0

Glucose monohydrate

20.0

Tap water

1 liter

(The soybean meal was "Arkasoy 50" supplied by British Arkady Co., Ltd., Old Trafford, Manchester). To curb foam formation, 50 ml of a 10% by volume solution of "Pluronic L81" (registered trademark) in soybean oil was added to the fermentation medium before sterilization. (Pluronic L81 »was supplied by Jacobsen van den Berg U.K. Ltd., 231 The Vale, London, W.3, U.K., and is a block polymer of ethylene oxide and propylene oxide).

The medium was sterilized in the fermenter for 20 minutes at 120 ° C. The seed culture was stirred at 340 revolutions per minute with a 21 cm paddle stirrer, whereby 1501 / min, sterile air was blown through the open end of an inlet tube. The culture vessel was provided with baffles. The temperature was regulated at 28 ° C. and after incubation under the conditions mentioned for 45 hours, 7.5 liters of this germ culture were added as inoculum to 150 liters of sterile fermentation medium in a fermenter with a capacity of 300 liters made of stainless steel. The fermentation medium had the following composition:

Part component (g / 1)

Soy flour («Arkasoy 50») 10.0

Glucose monohydrate 20.0 lime (precipitated calcium carbonate) 0.2 cobalt chloride (C0CI2 • 6H2O) 0.001 tap water per 1 liter

300 ml of a 10% solution of "Pluronic L81" in soybean oil was added to prevent foaming. The

Fermentation product was harvested after 48 hours and clarified by centrifugation. The clarified solution gave 50% inhibition in the enzyme test in a dilution of 1: 100,000. 100 liters of the clarified solution were stirred with 12 kg wet weight “Whatman DE32” (registered trademark), an ion exchange cellulose in the acetate form. (Supplied by H. Reeve Angel & Co., 14 New Bridge Street, London E.C. 4, U.K .; the product is a microcrystalline cellulose with diethylaminoethyl groups as a substituent).

The slurry was filtered and the MM 4550 (complex) eluted from the cellulose with 12 liters of 0.5 M potassium sulfate. The extract was concentrated to 6 liters in a thin film evaporator under vacuum and below 30 ° C. The majority of the potassium sulfate was precipitated by adding 12 liters of acetone. The solution was filtered and concentrated to 200 ml by evaporation under vacuum below 30 ° C. The concentrate was applied to a column of 76 mmX2 mouse "AmberliteXAD-4" resin (registered trademark) (manufactured by Rhöm & Haas Co., Philadelphia, USA; the material is a nonionic polystyrene resin). The column was eluted with deionized water and the eluate collected in fractions of 140 ml each. The active fractions, discovered with the KAG test, were pooled (2.2 liters) and concentrated to 275 ml by ultrafiltration using «Amicon- UM-05 membrane »(150 mm in diameter) (registered trademark) (supplied by Amicon Ltd., 57 Queen's Road, High Wycombe) under a nitrogen pressure of 3.6 kp / cm2. The concentrate was freeze-dried to give 2.2 g of a brown powder (I50 = 0.004 ng / ml). 1 g of the powder was dissolved in one liter of 0.2 M sodium sulfate and mixed with one liter of 2% tetra-n-butylammonium hydrogen sulfate (supplied by AB Astra, Sodertalje, Sweden) in dichloromethane. The dichloromethane phase was separated off, cooled to -70 ° C., filtered to separate off ice and concentrated by evaporation to 20 ml. 400 ml of petroleum ether with a boiling range of 40 to 60 ° C. were added to the concentrate and the precipitate was centrifuged off. The precipitate was dissolved in 10 ml dichloromethane and extracted with 10 ml water containing 80 mg barium iodide and 70 mg barium carbonate. The phases were separated, the aqueous phase was filtered and adjusted to pH 6.5. The solution was freeze-dried to give a yellow powder. The solid substance was washed with acetone to extract excess barium iodide and the light yellow solid substance was centrifuged and dried in vacuo. The yield was 13 mg (I50 = 0.0004 (xg / ml).

Description 4 Demonstration of the Benefit of Activated Carbon Treatment of the Culture Filtrate for the Production of a Material According to British Patent No. 1 361 075 The culture filtrate obtained according to Description 3 gave a zone diameter of 17 ml in the hole of an agar plate diffusion in the test against Klebsiella aerogenes, a zone diameter of 38 mm in the KAG test and an iso value at a dilution of 1: 100,000. The clarified culture filtrate (170 liters) was perculated at 5 ° C in the upward flow through a column of 23 cm in diameter and 40 cm in height Activated carbon ("Farnell BO", supplied by Dearborn Chemicals Ltd., Dilton, Widnes, Lancs., The grains 0.25 to 0.18 mm, prewashed with HCl and buffered to pH 6 with phosphate) at a flow rate of 800 to 1000 ml / minute. The broth was washed down from the coal with 10 liters of deionized water and the column afterwards eluted with 20% acetone at 20 ° C. The active fractions in the amount of 10 liters were concentrated by evaporation to 6 liters and freeze-dried under 30 ° C.,

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whereby 282 g of crude MM 4550 complex was obtained. The complex showed an iso value of 0.05 (ig / ml. The yield of enzyme-inhibitory activity was 22%.

Alternatively, with similar results, an activated carbon called "Darco granular carbon" can be used (supplied by Honeywill-Atlas Ltd., Mill Lane, Carshalton, Surrey).

Description 5 Usefulness of ion exchange resin chromatography of a culture filtrate for the production of material according to British Patent No. 1 363 075 A column of 1 cm inner diameter was packed to a height of 6 cm (bed volume = 4.7 ml) with “Dowex 21K »Ion exchange resin (0.84 to 0.30 mm grit in chloride form) (« Dowex 21K »was supplied by BDH Chemicals Ltd., Poole, Dorset, UK and is a polystyrene-divinylbenzene resin with basic groups). The resin bed was then washed with approximately 4 X 4.7 ml of 5% aqueous methanol, followed by 1X 4.7 ml of distilled water. Two liters of culture filtrate were obtained essentially according to description 3 and placed on the column. The resin was then washed with 5% aqueous methanol to remove contaminants.

The MM 4550 (complex) was eluted from the resin with 5% NaCl in 50% aqueous methanol. Table 1 below shows the results in unit values of activity. The MM 4550 (complex) content of the culture filtrate was arbitrarily set at eight units per ml. The eluted fractions from the column were tested using an antibacterial diffusion method against Klebsiella aerogenes, the units of action being calculated using a standard curve prepared by plotting the zone diameter against units per ml for different concentrations of the culture filtrate (ie the culture filtrate was used as standard ).

It can be seen that the column is an effective means of concentrating the MM 4550 (complex). Fractions 2-5 contained 60% of the activity in a total volume of only 37 ml. The total dry weight of these fractions was approximately 210 mg, whereas 35 g of solid substances were applied to the column.

Table 1 (continued)

Table 1

Results of the chromatography on «Dowex 21K»

Sample pH

volume

Units / ml

Total units

(ml)

Eluate 7

7.6

14.2

66

937

Eluate 8

7.6

20.0

33

660

Total

13141

Sample pH

Volume (ml)

Units / ml

Total units

Culture filtrate

6.5

1970

8th

15760

Percolat 1

6.9

550

<1

<100

Percolat 2

7.0

525

<1

<100

Percolat 3

7.0

595

<1

<100

Percolat 4

7.0

300

<1

<100

Eluate 1

7.8

7.0

84

588

Eluate 2

7.8

7.0

328

2296

Eluate 3

7.7

8.5

440

3740

Eluate 4

7.7

10.0

320

2200

Eluate 5

7.6

11.5

160

1840

Eluate 6

7.5

11.0

80

880

10th

Description 6 Use of the ion pair extraction of the culture filtrate to produce a material according to British Patent No. 1363 075 15 248 g of sodium sulfate was added to 10 liters of a clarified culture filtrate which had been prepared as described in description 3 and the solution was extracted with 2% tetrahydrofuran. n-butyl-ammonium hydrogen sulfate in 10 liters of dichloromethane by stirring for 30 minutes. The phases were separated and the dichloromethane phase was cooled to 2 ° C. A small proportion of suspended water was filtered off through a “Whatman No. 1 PS filter”. The dichloromethane solution was concentrated to 20 ml by evaporation in vacuo below 30 ° C. Adding 400 ml of petroleum ether 25 (40 to 60 ° C) precipitated a resin containing the MM 4550 (complex).

The resin was centrifuged off, dissolved in 50 ml of dichloromethane and extracted with 50 ml of water containing 1 g of barium iodide and 1 g of barium carbonate by shaking for 30 minutes. The existing solid substances were filtered off and the two phases were separated. The aqueous phase, which contained the MM 4550 (complex), in the form of the barium salt, was brought to pH 6.5 and freeze-dried. 317 mg of a crude preparation of MM 4550 35 (complex) with an iso value of 0.001 [ig / ml) were obtained.

Description 7 Preparation of partially purified antibiotic complex containing M 4550 and MM 13902 using tetra-n-butylammonium hydrogen sulfate 12.5 g of a crude preparation MM 4550 (complex), prepared according to description 7, was dissolved in 125 ml Water and extracted with 125 ml of 10% tetra-n-butyl-ammo-45 nium hydrogen sulfate solution in dichloromethane. The two phases were separated and the dichloromethane was extracted back with 100 ml of water at 2 ° C., this water containing 190 mg of sodium iodide, by slowly hydrogenating and adjusting the pH of the aqueous phase to 6.4 with 2% sodium bicarbonate. The solution was freeze-dried and the dry solid substance washed with acetone. 88 mg of mixed sodium salts of MM 4550 and MM 13902 with an iso value of 0.0002 (ig / ml against Escherichia coli-β-lactamase were obtained. The recovery 55 of antibiotics was 70%.

The antibacterial activity of the mixtures of ampicillin and a material prepared according to description 7 was determined using the dilution series method in nutrient. The minimal inhibitory concentrations obtained are listed in Table 2.

Table 2

Synergistic effect of ampicillin and a material as described 7

organism

Ampicillin alone

Ampicillin + MM 4550 (complex) at

0.1 ng / ml

1 [ig / ml

10 [ig / ml

E. coli B1 E. coli JT417

> 500 250

> 500 250

500 250

50 50

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Table 2 (continued)

organism

Ampicillin

Ampicillin + MM 4550 (complex) at

alone

0.1 (ig / ml

1 ng / ml

10ng / ml

E.coliJT39

> 500

> 500

500

25 *

Shigella sonrei S239

125

125

125

25 *

Klebsiella aerogenes A

125

25th

5 *

> 0.1 *

Klebsiella aerogenic IP 282

50

50

12.5

0.5 *

Proteus mirabilis 889

> 500

> 500

50

0.5

Proteus mirabilis 247

> 500

125

5.0

0.5

Proteus morganü F

50

50

25th

0.5

Proteus rettgeri R110

250

125

25 *

0.25 *

Staph. aureus (Russell)

250

125

> 0.1

0.25 *

Staph. aureus (Russell H)

> 500

> 0.1

> 0.1

> 0.1

* partial inhibition by MM 4550 (complex) alone at these concentrations; Similar synergistic effects have been observed for mixtures of amoxycillin and MM 4550 (complex).

Description 8 Preparation of a partially purified antibiotic complex containing MM 4550 and MM 13902 using cetyldimethylbenzylammonium chloride. A freeze-dried product with an iso value of 0.02 [ig / ml from a "Farnell" coal column, eluted with Ace-ton / Water according to description 4, was dissolved in distilled water to a concentration of 13 mg / ml. The pH of the solution was adjusted to 6.5.

100 ml of the solution was extracted with an equal volume of 0.1% cetyldimethylbenzylammonium chloride in dichloromethane. The phases were separated by centrifugation and the organic phase back extracted with 100 ml of 0.05% sodium iodide solution. The phases were separated and dichloromethane remaining in the aqueous phase was removed by applying reduced pressure for 10 minutes. The aqueous solution was freeze-dried and the freeze-dried product was washed three times with 50 ml of acetone. The product washed in this way was dried in a vacuum desiccator and gave a light brown powder weighing 4.3 mg with an iso value of 0.002 [ig / ml.

Description 9 Production of a partially cleaned antibiotic complex containing MM 4550 and MM 13902 under

Use of gel filtration 1 g of crude MM 4550 (complex) with an iso value of 0.2 (ig / ml, prepared according to the method according to description 4, was chromatographed on "Sephadex G25" (fine) using acetone / water 4: 6 as eluent The column dimensions were 2.5 cm X 32 cm and elution was carried out at a throughput rate of 1.5 ml per minute The active fractions were combined, concentrated in vacuo below 30 ° C. and freeze-dried to give 22 mg amorphous leather-colored solid substance with an I50 value of 0.005 ng / ml. The yield was 88% and the cleaning was carried out 40 times.

Description 10 Preparation of a purified antibiotic complex containing MM 4550 and MM 13902 using cellulose chromatography 610 mg MM 4550 (complex), prepared according to method 7, were chromatographed on a column of 2.5 cm × 32 cm on cellulose (" Whatman CC 31 ») and eluted with a mixture of isopropanol / water 7: 7 V / V at a flow rate of 1.5 ml per minute. The antibacterial active fractions were pooled, concentrated in the

Vacuum below 30 ° C and freeze-dried. This gave 40 mg of a brown amorphous solid material, Antibiotic complex with an iso value of 0.00007 [xg / ml. The very low iso value indicates that it is a 25 active material of improved purity.

On another occasion, the aforesaid method yielded a material with an iso value of 0.001 (xg / ml). This material showed an antibacterial activity according to Table 3, determined using a standard microtitration 30 method in "Oxoid" sensitivity nutrient solution using a light inoculum (1% rearing overnight).

Table 3

Antibacterial activity of a mixture of MM 4550 35 and MM 13 902 with an iso value of 0.001 (xg / ml

organism

MIC in (ig / ml

Staphylococcus aureus (Oxford)

7.5

Staphylococcus aureus (Russell)

7.5

Streptococcus faecalis

125

Bacillus subtilis

0.9

Escherichia coli (10418)

3.7

Escherichia coli (Bll)

15

Klebsiella aerogenes

1.8

Enterobacter cloacae

62

Proteus mirabilis

7.5

Providentia stuartii

7.5

Acinetobacter anitratus

0.9

Pseudomonas aeruginosa

250

Serratia marcescens

7.5

Salmonella typhimurium

15

Shigella sonnei

7.5

ss description 11

MM 4550

MM 4550 can be recognized by its properties of the following type: MM 4550 is an acidic, solid substance which has the following characteristics 60 in the form of a sodium salt:

1. It is highly soluble in water, soluble in methanol and essentially insoluble in chloroform, diethyl ether and hydrocarbons.

65 2. In aqueous solution it shows a characteristic ultraviolet spectrum with absorption maxima at around 238 nanometers and around 287 nanometers.

3. In a concentration of 0.4 wt .-% and in one

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freshly made KBr disc it shows a characteristic infrared spectrum with absorption maxima and others at about 3450, 2950, 1765, 1695, 1510, 1390 and 1260 cm-1. If the same KBr disk is taken up again after a week, the spectrum shows considerable changes, e.g. such that the previously noted strong peak at 1765 cm-1 is significantly reduced or completely absent.

4. The substance shows a characteristic NMR spectrum, recorded in D2O, which i.a. has a pair of low field doublets centered at approximately 2.45 T and 3.65 T with coupling constants of approximately 15 Hz, a doublet centered at approximately 8.55 T and a sharp singlet at approximately 7.95 T. .

5. The following approximate Rf values result from thin-layer chromatography on cellulose with the eluents specified below:

a) butanol / isopropanol / water 7: 7: 6 V / V; Rf = 0.6

b) isopropanol / water - 7: 3 V / V; Rf = 0.7

c) n-butanol / ethanol / water - 7: 7: 6 V / V; Rf = 0.7

d) n-propanol / water-4: 1 v / v; Rf = 0.6.

6. The substance has antibacterial activity against various species, including against strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Acinetobacter anitratus, Serratia marcescens and Shigella sonnei.

7. The substance has enzyme-inhibitory activity against the ß-lactamases, which are produced by different strains, including Escherichia coli, Klebsiella aerogenes and Staphylococcus aureus. It has an iso value (as defined above) of less than 0.0001 (xg / ml against the β-lactamase from Escherichia coli BH.

8. In a mixture with ampicillin, the substance exerts a synergistic effect against organisms including Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Proteus morganii and Staphylococcus aureus Russell.

9. Amino acid analysis indicates that the material is not a polypeptide or protein. No a-aminoadipic acid is found after acidic hydrolysis.

10. The substance reacts with Ehrlich's reagent (300 mg of 4-dimethylaminobenzaldehyde, dissolved in a mixture of 54 ml of n-butanol, 9 ml of ethanol and 9 ml of concentrated hydrochloric acid) to form blue color on paper chromatograms and DCS plates.

11. The substance is not a general enzyme poison and does not inhibit the following enzymes at concentrations in excess of those necessary to inhibit Escherichia coli β-lactamase, monoamine oxidase, carboxylic anhydrase, dopadecarboxylase, trypsin, chymotypsin or urease.

example 1

Production of MM 4550 (complex) and separation into MM 4550 and MM 13902

The insulation procedures in the previous sections can be used in a number of ways. A particularly suitable consequence is adsorption on coal, ion pair extraction and cellulose chromatography in the manner described below:

340 liters of culture filtrate, prepared according to description 3, was percolated in an upward flow at 800 to 1200 ml / minute through a column of 22.5 cm in diameter and 53 cm in height, packed with “Farneil BO” coal. The coal was previously used to adsorb MM 4550 (complex) and was regenerated by washing with the following reagents: 0.5-n NaOH, 0.2-n NaOH / acetone (3: 2), water; n HCl, water, phosphate buffer pH 6, water. The column was washed with 20 liters of water to displace the culture filtrate and eluted downwards with acetone / water 2: 8 V / V at 200 to 250 ml throughput per minute. It was separated into one liter fractions. The fractions containing the MM 4550 (complex), determined by means of the antibacterial diffusion test using Klebsiella aerogenes, were combined (11 liters) and concentrated to 5.5 liters by evaporation in vacuo at 30.degree. The yield of MM 4550 (complex) at this stage was 20%. The concentrate was cooled to 2 ° C., 5.5 liters of a 2 vol.% By weight tetra-n-ammonium hydrogen sulfate solution in dichloromethane were added at -5 ° C., and the two phases were mixed together for 2 minutes . The dichloromethane phase was separated and added to one liter of sodium iodide solution (0.6%) at 0 ° C. The two phases were stirred and the aqueous phase was gradually adjusted to pH 6.5 to 7.0 with 2% aqueous solution of NaHCO 3. The aqueous phase was separated and freeze-dried. The dried solid was extracted with dry acetone to dissolve excess sodium iodide and the acetone was removed from the insoluble residue in vacuo. The yield of a leather-colored powdery substance was 1.1 g. The overall yield of MM 4550 (complex) was 10% at this stage. The calculated accumulation of total dissolved solid substances in the culture filtrate was 125 times.

A column with a diameter of 2.5 cm was packed with cellulose powder ("Whatman CC 31") in isopropanol / water 7: 3 V / V at a height of 32 cm. 500 mg of the leather-colored powder was dissolved in 2 ml of isopropanol / water 7: 3 V / V and chromatographed using the same solvent at a flow rate of 1.5 ml per minute. 3 ml fractions were collected from the column and those which showed ultraviolet absorption maxima at approximately 285 nanometers were pooled. They were concentrated and freeze-dried to give MM 4550 (complex). Preliminary experiments had shown that the fractions at about 285 nanometers absorbent contained the enzyme-inhibiting antibacterially active material.

The yield of freeze-dried MM 4550 (complex) was 35 mg. It was a brown amorphous substance with an iso value of 0.0001 (ig / ml. The yield of active material at this stage was 3% overall and the cleaning effect was 600 times.

The antibacterial activity of this preparation was determined by the standard microtiter method in “Oxoid” sensitivity test solution using a light inoculum. The minimum inhibitory concentration obtained was in similar ranges as listed in Table 3 above.

Analysis of the substance by means of thin layer chromatography on cellulose (Eastman Kodak "Chromagram" plates) developed with n-butanol: isopropanol: water 7: 7: 6 revealed two active substances, one with an Rf value of approximately 0.58, which corresponds to that MM 4550 and one with an Rf value of 0.72, attributed to MM 13902. Both of these materials were determined by bioautography on a variety of organisms including B. subtilis, Staphylococcus aureus (Oxford), Staphylococcus aureus (penicillin resistant ), Escherichia coli (sensitive and resistant to penicillin), Salmonella typhimurium, Proteus mirabilis, Proteus morganii and Klebsiella aerogenes. Some of the results obtained are shown in Table 4.

In a further experiment, the two substances were separated on a thin layer plate of cellulose, developed with isopropanol: water 8: 2 and extracted from the cellulose with phosphate buffer. Each extract was tested for ß-Lacta-

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inhibition of mas and both substances proved to be inhibitors of Escherichia coli-β-lactamase. Both substances also showed synergistic effects with benzylpenicillin against Klebsiella aerogenes.

Table 4

Antibacterial activity of MM 4550 and MM 13 902, determined by means of bioautography (thin layer chromatography on cellulose with butanol / isopropanol / water 7: 7: 6 V / V)

Organism zone diameter in bioautography

MM 4550 zone in

MM 13 902 zone in

mm at Rf 0.58

mm at Rf 0.70

Klebsiella aerogenes

20.0 mm

20.00 mm

Proteus mirabilis

17.5 mm

13.5 mm

Proteus morganü

16.00 mm

9.5 mm

Salmonella typhi

19.5 mm

21.00 mm

Escherichia coli 10418

20.0 mm

13.5 mm

Escherichia coli Bll

17.5 mm

10.5 mm

Enterobacter aerogenes

6.5 mm no zone

Styphylococcus aureus

(Oxford)

13.0 mm

4.0 mm

Staphylococcus aureus

(Russell)

12.0 mm

4.0 mm

Bacillus subtilis

24.0 mm

15.0 mm

Example 2

Production of MM 4550 (complex) and separation into MM 4550 and MM 13902

1100 liters of a culture filtrate from the fermentation of Streptomyces olivaceus ATCC 31126 at pH 6.5 and 5 ° C were percolated at 3.2 liters / minute through a column (0.3 m X1 m), packed with «Darco» coal ( the coal was already used to adsorb MM 4550 [complex] and was regenerated before use by washing with the following reagents: 0.5-n NaOH; 0.2-n NaOH / acetone 3: 2; water; n HCl; Water; phosphate buffer, pH 6; water). The column was washed with 60 liters of water to displace the culture filtrate and then eluted with water / acetone 2: 8 V / V at 30 ° C at a flow rate of 1.1 liters per minute. First, 60 liters were collected, followed by fractions of 5 X10 liters. These fractions, which contained the MM 4550 (complex), determined by means of the antibacterial diffusion test using Klebsiella aerogenes, were combined (40 liters) and concentrated in vacuo below 30 ° C. to 32 liters. The yield of MM 4550 (complex) was 15% at this stage.

The concentrate was cooled to 5 ° C, 16 liters of 0.2% cetyldimethylbenzylammonium chloride solution in dichloromethane was added at 5 ° C and the two phases were mixed for five minutes. The dichloromethane phase was separated and added to 3.75 liters of sodium iodide solution (0.4%) at 5 ° C. The two phases were mixed for five minutes, then the aqueous phase was separated and freeze-dried. The dry solid was extracted with dry acetone to dissolve the excess sodium iodide and the aqueous solid was dried in vacuo. 2.87 g of a leather-colored powder were obtained. The overall yield of MM 4550 (complex) was 6% at this stage. The calculated cleaning effect with respect to the culture filtrate was 160 times.

A column 63 mm in diameter was packed with cellulose powder ("Whatman CC 31") in isopropanol-water (7: 3 V / V) at a height of 300 mm. 2.0 g of the leather-colored solid powdery substance were dissolved in 5 ml of isopropanol / water (7: 3 V / V) and chromatographed in the same solvent at flow rates of 3 ml / minute. The fractions containing the MM 4550 (complex), determined by test against Klebsiella aerogenes, were combined, concentrated by evaporation in vacuo below 30 ° C. and freeze-dried. 207 mg of a brown solid substance were obtained. The yield of MM 4550 (complex) at this stage was 51% and the cleaning effect was five times.

A 16 mm diameter column was packed with cellulose powder (Whatman CC 31) in propanol / water (4: 1 V / V) to a height of 300 mm. 198 mg of the brown solid substance was dissolved in water / n-propanol (1: 1) and chromatographed in n-propanol / water (4: 1 V / V) at a flow rate of 0.5 ml per minute. Fractions of 6 ml each were collected, tested in a bioassay against Klebsiella aerogenes and the UV spectrum of each fraction measured. Fractions 23 to 28 with UV maxima at approximately 305 nanometers contained the disodium salt of MM 13902. These fractions were pooled, concentrated in vacuo and freeze-dried to give 30 mg of solid. This substance showed only one zone of antibacterial activity at Rf 0.77 in thin layer chromatography and using Eastman Kodak cellulose plates with n-propanol / water (4: 1 V / V) as eluent. The zone was determined by bioautography against Bacillus subtilis. Fractions 29-40 with a UV maximum at approximately 285 nanometers contained MM 4550. These were pooled, concentrated under vacuum, freeze-dried and gave 53 mg of a solid substance containing a salt of MM 4550 contaminated with a small amount of a salt by MM 13902.

Description 12

Organisms

The ability to produce MM 4550 (complex) was first discovered on cultures ATVV 21379, ATCC 21380, ATCC 21381 and ATCC 21382, which were isolated from soil samples obtained from Spain, New Zealand, South Africa and Israel. In the laboratory these cultures appeared identical and all were identified as Streptomyces olivaceus by Dr. Bousfield, the Actinomycet expert of the National Collection of Industriai Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland, using Hütter's widely recognized 1967 classification (Systematics of Streptomycetes, S. Karger, Basel, 382 pp). The production of MM 4550 has now also been found in other Streptomyces species, as can be seen from Table 4.

S. olivaceus was first described by Waksman 1919 (Cultural Studies of Species of Actinomyces, Soil, Sei., 8, 71-215). In 1960, a group of Russian investigators described a species with very similar characteristics, which they named Actinomyces (Streptomyces) fulvoviridis (Kuchaeva et al., Trud. Inst. Mikrobiol., Akad Nauk. SSSR., 8, 226-253, I960).

Microorganisms of the genus Streptomyces are extremely different in their morphological and physiological characteristics depending on the conditions under which they have grown. Descriptions of some species were published before the existence of this extreme diversity was recognized, which subsequently led to duplications and synonym names. Hütter (1967) lists 25 species as synonyms with S. olivaceus including S. fulvoviridis. An international Streptomyces project was started in 1962 to eliminate confusion in nomenclature and classification, Shirling

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and staff, Int. J. Syst. Bacteriol, 18, 69-189 (1968). Collaborators in this project have conducted a series of studies that are suitable for establishing accurate descriptions of Streptomyces species under standard conditions. In this scheme, standard descriptions were produced of types of strains from some 400 named species, including Streptomyces olivaceus and Streptomyces fulvoviridis.

The I.S.P. description of S. olivaceus and S. fulvoviridis differs only in two characters: 1. in the form of the sporophores and 2. in the ability to use inositol as the only carbon source in the rearing.

S. olivaceus is described as follows: Spore chain morphology: Spiral sections with open spirals divided by curved spore chains as an indication of rectiflexibile sections. The mature spore chains are generally long, often with more than 50 spores per chain.

Carbon uptake from D-glucose, L-arabinose, D-xylose, i-inositol, D-mannitol, D-fructose and rhamnose takes place during rearing. There is no rearing or only trace growth on sucrose and raffinose.

S. fulvoviridis is described as follows: Spore chain morphology: Rectiflexibiles sections. The ripe spores are moderately short with 10 to 50 spores per chain. Carbon uptake: D-glucose, L-arabinose, D-xylose, D-mannitol, D-fructose and rhamnose are suitable for rearing. The intake from D-sucrose is doubtful. No growth or only trace growth on i-inositol or raffinose.

Characterization of a number of cultures designated S. olivaceus or S. fulvoviridis or related species was determined using the standard methods and media recommended in the International Streptomyces Project (ISP) by Shirling et al., Int. J. Syst. Bacteriol. 16, 313-340, (1960).

The cultures were derived from various sources. ATCC 21379, 21380, 21381, 21382 were isolated from soil based on the production of MM 4550 (complex). The other strains were obtained from different culture collections for comparison purposes.

The results of the experiments for the production of MM 4550 (complex), color of the aeromycelium, sporophore shape, color of the substrate mycelium, production of soluble pigment, melanin production and utilization of the carbon source are summarized in Tables 4 to 8. The spore surface of all these strains is smooth, as can be seen in the electron microscope.

From the ISP description it is clear that the spiral growth of the sporophores in S. olivaceus is variable in character. Real spirals were observed with various frequencies in the species S. olivaceus ATCC 3335. The majority of the sporophores were long. No real spirals were observed from cultures ATCC 21379, 21380, 21381 or 21382, although the sporophores were long and showed a tendency to spiral, on a background of Rectiflexibiles types. Meanwhile, in two S. olivaceus strains, NCEB 8238 and NCIB 8509, the majority of the sporophores were of the rectiflexibiles type. In NCIB 8238 they were of medium length, whereas those of NCIB 8509 were long.

The sporophores observed in ATCC 15863, i.e. S. fulvoviridis species were shorter than those seen with isolates ATCC 21379, 21380, 21381, 21322 and 31126 and were only rectiflexibiles type. In view of these characteristics, the isolates ATCC 21379, 21380, 21381, 21382 and 31126 are probably good, but not exactly the same as the S. olivaceus description.

The isolates ATCC 21379, 21380, 21381, 21382 and 31126

show a certain variability with regard to inositol exploitation, which is the other differentiating characteristic according to the ISP type description to differentiate between S. olivaceus and S. fulvoviridis (Table 8). ATCC 31126 and ATCC 21380 consume inositol, which is characteristic of S. olivaceus, whereas the other strains use only dubious or negative consumption. However, it is generally not enough to use the ability to consume a single carbohydrate to separate different species. Such a difference can arise from a single gene mutation and is often only attributable to one strain significance. The difference in sugar consumption by S. olivaceus strains NCIB 8138, NCIB 8509, ATCC 21549, ATCC 12019 and ATCC 3335 leads to the assumption that some authorities do not regard it as species significance. ATCC 21379, 21380, 21381 and 21382 and 31126 are actually referred to as S. olivaceus.

Without further studies revealing more significant differences between the cultures currently called S. olivaceus and S. fulvoviridis, it is possible that they may be internationally recognized as one and the same species. In this case, the correct name will be S. olivaceus for priority reasons, and it will also include those cultures that Hütter has listed as a synonym for S. olivaceus.

Testing the culture filtrates of strains of S. olivaceus and related species for the production of MM 4550 (complex) can be carried out in the following manner:

Culture filtrates of the strains listed were applied in spots on “Whatman No. 1” paper strips with 20 ml and chromatographed in n-butanol / isopropanol / water (7: 7: 6) overnight in the cold. Another set of strips was run in n-butanol / glacial acetic acid / water (12: 3: 5) also overnight. A partially cleaned sample of MM 4550 (complex) was run in both systems at the same time as a brand.

Alternatively, it is possible to extract 25 ml of a clarified broth with 12.5 ml of a 0.2% cetylbenzylammonium chloride solution in dichloromethane, phase separation, separation of the organic phase, addition of 2.5 ml sodium iodide solution (0, 5%), shaking, separating the phases, separating the aqueous phase and its chromatographic application, for example by applying 5 μl at a time on thin-layer chromatography strips and developing in n-butanol / isopropanol / water (7: 7: 6).

Table 5

Enabling cultures of Streptomyces olivaceus, Streptomyces fulvoridis and related species to produce MM 4550 (complex)

Culture MM 4550 (complex)

production

Streptomyces olivaceus

ATCC 21379

+

Streptomyces olivaceus

ATCC 31126

+

Streptomyces olivaceus

ATCC 21380

+

Streptomyces olivaceus

ATCC 21381

+

Streptomyces olivaceus

ATCC 21382

+

Streptomyces olivaceus

NCIB 8238

+

Streptomyces olivaceus

NCIB 8509

+

Streptomyces flavovirens

ATCC 3320

+

Streptomyces flavus

ATCC 3369

+

Streptomyces fulvoviridis

ATCC 15863

+

Streptomyces argenteolus

ATCC 11009

+

5

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626 628 12

Table 5 (continued)

Kuimr

MM 4550 (complex) '

production

Streptomyces sioyaensis

ATCC 13,989

+

Streptomyces lipmanü

NRRL3584

+

(Streptomyces olivaceus ATCC 21549, ATCC 12019 and ATCC 3335 did not produce an MM 4550 (complex); ATCC 15863 was also deposited under the name RIA 660).

Table 6

Streptomyces olivaceus, Streptomyces fulvoviridis and related species - color and morphology of the mature sporulating mycelium on ISP media after 14 days of rearing

Culture

SS

YM

GA

OM

Sporophoric morphology and size

ATCC

21379

gray gray gray / white gray langRF

ATCC

31126

gray / brown gray / brown gray gray long RF

ATCC

21380

gray gray gray gray langRF

ATCC

21381

gray gray light gray gray / white long RF

ATCC

21382

gray / white gray gray gray langRF

NCIB

8238

gray / white gray gray gray medium RF

NCIB

8509

gray gray gray gray langRF

ATCC

21549

gray gray gray gray long S

ATCC

12019

gray gray gray gray long RF / S

ATCC

3335

gray gray gray gray long RF / S

ATCC

15863

gray / white gray gray / white gray medium RF

ATCC

3320

gray / blue gray gray / green gray long RF

ATCC

3369

gray gray gray light gray / brown medium RF / RA

ATCC

11009

light gray gray / brown gray / white white / green medium RF / RA

ATCC

13898

white / gray white white / yellow gray / white short S

SS = inorganic salts - starch agar (ISP Medium 4) YM = yeast extract - malt extract agar (ISP Medium 2) GA = glycerin-asparagine agar (ISP Medium 5) OM = oatmeal agar (ISP Medium 3) RF = rectiflexible RA = Retinaculiaperti S = Spirales

Table 7

Streptomyces olivaceus, Streptomyces fulvoviridis and related species - color and substrate mycelium seen from the back

Culture

SS

YM

GA

OM

ATCC 21379

olive olive brown gray / brown olive green

ATCC 31126

olive brown olive brown brown olive brown

ATCC 21380

olive olive green olive brown olive yellow

ATCC 21381

dark brown dark brown olive green olive green

ATCC 21382

olive olive brown olive green

NCIB 8238

brown olive / brown brown olive yellow

NCIB 8509

brown olive olive brown olive yellow

ATCC 21549

leather colors / black brown / black brown / black leather colors / gray

ATCC 12019

leather colors leather colors leather colors leather colors

ATCC 3335

brown brown brown brown

ATCC 15863

brown / gray dark brown olive brown olive yellow

ATCC 3320

yellow / brown olive / brown olive / brown orange / brown

ATCC 3369

leather colors / gray yellow / brown leather colors / green yellow

ATCC 11009

black black / yellow black / yellow gray / green

ATCC 13989

orange yellow yellow colorless

13 626 628

Table 8

Streptomyces olivaceus, Streptomyces fulvoviridis and related species - production of pigments in the culture medium

Culture

soluble non-melanoid pigments

* Melanin

SS

YM

GA

OM

production

ATCC

21379

_

_

+

-

weak

yellow

ATCC

31126

-

-

-

-

-

ATCC

21380

-

-

-

-

-

ATCC

21381

-

-

-

-

ATCC

21382

-

-

-

-

-

NCIB

8238

+

-

+

-

weak

weak weak

brown

brown brown

NCIB

8509

-

-

-

+

-

weak

yellow

ATCC

21549

_

+

4-

-

weak weak weak

brown red / brown brown

ATCC

12019

-

-

-

-

-

ATCC

3335

-

-

-

-

-

ATCC

15863

-

-

-

-

ATCC

3320

+

_

-

-

-

weak

pink

ATCC

3369

-

-

-

-

-

ATCC

11009

-

+

-

-

-

weak

brown

ATCC

13989

-

+

-

-

-

weak

orange

+ = Pigment produced - = pigment not produced

* = tested on peptone yeast iron agar (ISP6), tyrosine agar (ISP17) and trypton yeast broth (ISP1)

Table 9

Streptomyces olivaceus, Streptomyces fulvoviridis and related species - hydrocarbon uptake experiments

0)

O

<L> cn o

a>

8th

O

O

0>

O

Culture it

S

ë

* c3

CÖ

■ s

03 CO

Inosito o s

O

Xylose or

0

1

Mannil

.5

■ 8 • 3

ATCC 21379

+

-

-

±

+

+

+

+

+

ATCC 31126

+

-

-

+

+

+

+

+

+

ATCC 21380

+

-

-

+

+

+

+

+

+

ATCC 21381

+

-

-

-

+

+

±

+

+

ATCC 21382

+

+

-

-

+

+

+

+

+

NCIB 8238

+

-

-

-

+

+

+

+

+

NCIB 8509

+

±

-

+

+

+

±

+

ATCC 21549

-

-

-

+

+

+

+

+

+

ATCC 12019

+

+

+

+

+

+

+

+

+

ATCC 3335

+

-

German

+

+

+

+

+

+

ATCC 15863

+

-

-

-

+

+

+

+

+

ATCC 3320

+

-

-4 ~

-

+

+

+

+

+

ATCC 3369

+

-

±

±

+

+

+

+

+

ATCC 11009

+

-

-

+

+

+

+

+

+

ATCC 13,989

-

+

+

+

+

+

+

+

-

+ means: the connection is used - means: the connection is not used ± means: consumption doubtful or very low

Example 3 Preferred Isolation Process for the Production of MM 13902 4s A stick of spores from S. olivaceus ATCC 31126 was stored in tubes on dry soil in a closed container with a desiccant at a temperature of 20 ° C. A small portion of the soil (approximately 20 mg) was transferred aseptically into a 500 ml alder-50 meyer, which contained 100 ml of the following media:

component

Proportion (g / liter)

Glucose monohydrate

20.0

Soy flour

10.0

deionized water

1 liter

6o The pH was adjusted to 6.5 before sterilization. The soy flour was "Arkasoy 50" supplied by British Arkady Co. Ltd., Old Stafford, Manchester, England.

The Erlenmeyer flask was incubated on a rotary shaker (240 revolutions per minute) for about 30 hours at 28 ° C. 2 ml of the obtained crop were subsequently used as inoculum on a solid agar layer in a Roux vessel. The agar medium had the following compositions:

626 628

14

component

proportion of

"V-8 vegetable juice"

20.0 ml

Agar

20.0 g of deionized water

1 liter

The pH was adjusted to 6.0 before sterilization ("V-8 juice" is available from Campbell's Soups Ltd., Kings Lynn, Norfolk, England).

The inoculate was spread on the agar surface by tilting the bottle, after which the latter was incubated upright at 30 ° C. After two days of incubation, the excess liquid in the bottle was pipetted off and the incubation continued for a further four days.

It has previously been shown that the development of actinophages does not occur in the culture when this method of preparation is used in the Roux vessel.

50 ml of sterilized deionized water, which contained 0.02% "Tween 80", was introduced into the Roux culture vessel and the spores were suspended therein by shaking.

This spore suspension was then added as an inoculum to 75 liters of a sterilized germ medium in a 100 liter stainless steel fermenter. The composition of the seed medium was as follows:

component

Proportion (g / liter)

Soy flour («Arkasoy 50»)

10.0

Glucose monohydrate

20.0

Tap water

1 liter

To control foam formation, 50 ml of a 10% by volume solution of "Pluronic L81" in soybean oil was added to the fermentation medium before sterilization.

The medium was steam sterilized in the fermenter for 20 minutes at 120 ° C. The seed culture was stirred at 140 revolutions per minute with a 19 cm paddle stirrer and aerated at 75 liters per minute through an open-ended inlet tube.

The temperature was controlled at 28 ° C and, after incubation under these conditions, the contents of the vessel were added as an inoculum to 1500 liters of a sterile fermentation medium in a stainless steel fermenter with a surface area of 2000 liters. The fermentation medium had the following composition:

Part component (g / liter)

Soy flour («Arkasoy 50») 10.0

Glucose monohydrate 20.0 lime (precipitated calcium carbonate) 0.2

Cobalt chloride (CoCh-ómO) 0.001

Sodium sulfate (anhydrous) 1.0

Tap water to 1500 liters

The pH was adjusted to 6.0 with sodium hydroxide before sterilization. Three liters of a 10% solution of "Pluronic L81" in soybean oil were added before sterilization to prevent foaming. After sterilization, the pH was readjusted to 7.0 with sterile solution of sodium hydroxide. The fermentation mixture was stirred at 106 revolutions per minute using a 47 cm diameter paddle stirrer. The temperature was regulated to 30 ° C and an air flow of 1200 liters per minute. The fermentation product was harvested after 60 hours and clarified by centrifugation.

An activity of 340 units per ml was arbitrarily attributed to the material so produced. Tests were carried out as set out in Description 2 above.

1050 liters of the culture filtrate with 340 units per ml were extracted at 10 ° C and the pH 8 with 310 liters of dichloromethane at 10 ° C, which contained 1200 g of cetyldimethylbenzylammonium chloride, by flowing the two liquids in predetermined flow rates through an inflow Mixer were pumped. The phases were separated in a Sharples centrifuge after being mixed for two minutes. The dichloromethane phase in an amount of 300 liters was back extracted with aqueous sodium iodide. The back extraction was carried out in four batches using a total of 7 liters of water containing 210 g of sodium iodide. The phases were separated. The aqueous phase was adjusted from pH 7.7 to pH 7.0 with hydrochloric acid, followed by filtering. The 7 liters of sodium iodide extracts contained 21,900 units per ml.

An ion exchange column was prepared by packing "QAE Sephadex A25" (obtained from Pharmacia Ltd.) in a 0.5 molar phosphate buffer of pH 7 containing 0.3 m sodium chloride, using a column of 10 cm in diameter and a height of 40 cm was applied. The sodium extract in the amount of 7 liters was percolated at 5 ° C by the «QAE Sephadex» with a flow rate of 50 ml per minute. The column was eluted with a 0.7 molar solution of NaCl in 0.05 molar phosphate buffer with a pH of 7, the procedure also being carried out at 5 ° C. and a flow rate of 25 ml per minute being observed. Two liters of the eluate were discarded and 90 100 ml fractions were collected. The fractions were checked in an ultraviolet spectrometer and those which had absorption maxima at around 305 nanometers were pooled. Its pH was adjusted to 7 (fractions 50 to 62, total volume 1440 ml, 71,000 units per ml). It had previously been shown that the fractions with a maximum in this region contained the substance MM 13902.

5 g of sodium chloride in 100 ml of water was added to the pooled fractions, followed by percolation at 5 ° C through a 6.3 cm diameter column packed with "Amberlite XAD 4" resin (supplied by Röhm and Haas) at one level of 30 cm, whereby a flow rate of 20 ml / minute was observed. MM 13902 was adsorbed on the resin under these conditions, whereas the inorganic impurities did not. The antibiotics were eluted at room temperature with 200 ml of distilled water, followed by 50% aqueous methanol. The 1 liter eluate was evaporated below 30 ° C under reduced pressure to 70 ml, whereupon the pH was adjusted to 7 and freeze-dried. 1.62 g of a brown solid substance were obtained, which were a partially purified salt of MM 13902 with an activity of 37,000 units per mg.

The partially purified MM 13902 disodium salt (1.0 g, 37,000 units / mg) was chromatographed on a cellulose column 3.8 cm in diameter and 30 cm in length on “Whatman CC30 cellulose”. Elution was carried out with n-propanol / water (4: 1 V / V) at a flow rate of 2.5 ml / minute. 170 ml of eluate were discarded and 100 fractions of 15 ml each were collected. The fractions containing MM 13902 as disodium salt (No. 37 to 43), determined by UV absorption, were combined and gave 89 ml. Evaporation below 30 ° C. under reduced pressure to remove n-propanol and freeze-drying led to a yield from s

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219 mg of a yellowish powder with an activity of 73,000 units per mg.

The above material showed an ultraviolet absorption maximum at about 308 nanometers with an EJ®, of about 343. This material showed the IR and NMR spectra of FIGS. 2 and 3. The antibacterial activity of the material is shown in Table 10. Elemental analyzes of the product indicated that i.a. Contained nitrogen, sulfur and sodium, probably in the ratio N: S: NA = 2: 2: 2. The positive and negative maxima of the circular dichroism curves for disodium MM 13902 were determined on a Cary-61 recording spectropolarimeter at concentrations of 0.37 mg / ml and with a layer thickness of 1 cm. The results were as follows:

k (nm)

AE / m

186 221 286 323

+ 67.24 X10-4 -67.24 X10-4 - 3.30 X10-4 -8.20 XlO "4

Table 10

Antibacterial activity of the disodium salt of MM 13 902 (microtiter method - strong inoculum, 1/100 overnight)

organism

Bacillus subtilis A Staph. aureus oxford staph. aureus Russell io Strep. Faecalis

Enterbacter cloacae N1 E. coli 10418 E. coli JT410 adhesive, aerogenic A is Proteus mirabilis 13 Proteus vulgaris WO 90 Prov. stuartii Ps. aeruginosa A Salmonella typhimurium CT 10 20 Serratia marcescens US 39 Shigella sonnei Haemophilis influenzae P6 Neisseria gonorrhoeae

MIC (ng / ml)

0.15 0.30 0.60 3.10 25 0.15 5

0.03 0.60 0.60 0.07 50 0.30 2.50 0.6-0.3 0.08 0.08

From the data listed above it follows that the new antibiotic MM 13902 has the following structural formula I:

(I)

C - NH - CO - CIL

3 sheets of drawings

Claims (4)

626 628 1" 15 20th 25th 30th 35 40 45 50 55 60 65 626628 1. It is highly soluble in water, soluble in methanol and essentially insoluble in chloroform, diethyl ether and hydrocarbons. 1. Process for the preparation of the new antibiotic MM 13902 and its salts with the following approximate Rf values of the pure disodium salt in thin layer chromatography on cellulose with the following running systems a) n-butanol / isopropenol / water 7: 7: 6 V / V Rf = 0.72 b) Isopropanol / water - 7: 3 V / V Rf = 0.85 c) n-Butanol / ethanol / water - 7: 7: 6 V / V Rf = 0.81 d) n-propanol / water - 4: 1 V / V Rf = 0.75; and an iso value of the pure disodium salt between 0.001 ng / ml and 0.0001 | xg / ml against the ß-lactamase from Escherichia coli B 11, characterized in that an MM 13902-producing strain of Streptomyces is cultivated and MM 13902 or a salt separated from it from the culture. 2. In aqueous solution, it shows a characteristic ultraviolet spectrum with absorption maxima, one of which is at about 305 nm. 2. The method according to claim 1, characterized in that that a strain of Streptomyces olivaceus or an organism related to it is cultivated. 2nd PATENT CLAIMS 3rd 626 628 Magnesium, aluminum, and the usual substituted ammonium ions and the like. Particularly suitable salts are the alkali metal salts such as the sodium or potassium salt, e.g. the disodium or dipotassium salt. Suitable preparations of MM 13902 and its salts, as can be produced according to the invention, have a purity of at least 50%, yes, degrees of purity between 70 and 80 and even up to 100%. Pharmaceutical preparations can be produced from the new antibiotic MM 13902 or its salts with pharmaceutically suitable carrier substances. Here too, the active compound is expediently in the form of the disodium or dipotassium salt. Such preparations can be made in a form suitable for oral, topical or parenteral use. These are tablets, capsules, creams, syrups, powders and sterile preparations that are suitable for injection or infusion. Such preparations can contain customary pharmaceutically acceptable materials, such as diluents, binders, colors, flavors, concentrating agents, stabilizers and the like, as is customary in pharmaceutical practice and is known to the person skilled in the art when formulating antibiotic drugs, such as penicillins or cephalosporins. MM 13902 or its salts can be contained in the pharmaceutical preparations either as the only therapeutic agent or together with a β-lactam antibiotic. Suitable ß-lactam antibiotics include those which are known to be subjected to the action of β-lactamase, as well as those which have their own stability towards β-lactamase. Such ß-lactam antibiotics include Ampicillin, amoxycillin, benzylpenicillin, phenoxymethylpenicillin, propicillin, cephaloridine, cefoxitin, cephalothin, cephalexin, carbenicillin, ticarcillin and in vivo hydrolyzable esters of such compounds as the phenyl, tolyl or indanyl ethyl or diecyloxillin ethyl or pacyloxynyl or dieethyl acyloxynyl or tethylcilloxin or from carbenicyloxyl diecyloxycyloxin or tethylbenzylin or from ethylbenzylcyloxynyl or tethylcillinoxin or from carbenicoxymethyl diecyloxynyl or dieethylcilloxin or from ethylbenzene acyloxillin or from carbenic acid, pacyloxynyl or tethylcillin or from ethylbenzyl acyloxylin, of carbenicyloxyl or ticarcoxylin or from carbenic acid, pacyloxylin or dieethylcylinoxin or tethylcillin or from ethylbenzene, of carbenicoxin or phthalide esters of ampicillin, benzylpenicillin, amoxycillin, cephaloridine, cephaloglycin and the like. The ratio of MM 13902 or a salt thereof to the β-lactam antibiotic is expediently in the range from 10: 1 and 1:10, e.g. between 3: 1 and 1: 3. As a rule, the proportion of antibacterial agent in the dosage forms is normally between 50 and 1500 mg, more precisely between 100 and 1000 mg. Preferably, the dosed preparations according to the invention are used one or more times a day, e.g. administered two to four times a day in the treatment of diseases of the urinary tract, respiratory tract, soft tissue and the like. The preparations can be used to treat diseases such as bronchitis, gonorrhea, otitis media, mastitis and the like. A particular aspect of the invention is the preparation of MM 13902 or a salt thereof by chromatographic separation of a solution of MM 4550 (complex), as described below, in fractions consisting essentially of a solution of MM 13902 or a salt of which and fractions of other fractions exist, whereupon MM 13902 or a salt thereof is isolated from the solution. By "fractions consisting essentially of a solution of MM 13902 or a salt thereof" it is meant that either the only antibiotic material in the solution is MM 13902 or a salt thereof, or, that if any other antibiotic material is present, it will be in a smaller proportion than is the case with MM 13902 or a salt thereof. The proportion of MM 13902 or its is suitable Salt at 70 to 80% and preferably at 90 to 100% of the antibiotic material in the fraction. It has been found that a suitable method for determining the fractions, which are essentially a solution of MM 13902 or salts thereof, is the recording of an ultraviolet absorption spectrum of each sample. Those fractions which show an ultraviolet spectrum similar to that of Fig. 1 normally contain MM 13902 or a salt thereof essentially free of other antibiotic material such as MM 4550 as described below. As a rule, those fractions which have a pronounced ultraviolet absorption maximum at approximately 305 nanometers are of the desired purity. Usually the above-mentioned process is used to isolate MM 13902 as a dibasic salt. If a monobasic salt of MM 13902 or the free acid MM 13902 itself is desired, these substances can be prepared by acidifying the dibasic salt of MM 13902, since in general the monobasic salts of MM 13902 and the free acid MM 13902 are not sufficiently stable to isolate them from the mixtures with MM 4550 (complex) of the type described. The monobasic salts and the free acid are not easy to produce due to their low stability. As stated above, it is believed that the chromatographic isolation of MM 13902 is best performed using a salt of MM 13902, such as the disodium salt. Salts of MM 13902 are more soluble in aqueous solvents than in highly lipophilic solvents, which is why it is preferred to use aqueous solvent systems in the chromatographic purification according to the invention. It has been found that aqueous solutions of electrolytes, which are buffered approximately to the neutral value, are suitable in connection with polar carrier materials, such as basic ion exchange resins. Thus, an aqueous solution of sodium chloride, buffered to about pH 7, with a typical buffer, such as a phosphate buffer, can be used together with a carrier material which has tertiary amino groups or quaternary ammonium groups. It has been shown that basic ion-exchanging celluloses and basic ion-exchanging cross-linked dextran are suitable carrier materials and that in particular “QAE Cephadex A25” is suitable as a carrier, in particular if the solvent system consists of 0.7 m sodium chloride in a pH 7 phosphate buffer ("Cephadex" is a registered trademark). On the other hand, solvent systems consisting of water and water-miscible organic solvents and lower alkanols have been found to be suitable, when used together with inert carrier materials such as silica gel or cellulose. A particularly suitable solvent system for use with cellulose is a mixture of water and isopropanol, in particular in a ratio of 7: 3. In the meantime, the product obtained in the aforementioned processes using ion exchange resins often contains very high proportions of sodium chloride, so that it is favorable to desalt the combined chromatography solutions. Desalting can be accomplished by passing the solution through a column containing a lipophilic material to which the MM 13902 is adsorbed but which does not adsorb the sodium chloride. Suitable column materials are polystyrenes, such as «Amberlite XAD 4». Alternatively, gel filtration can also be used, using filter agents such as cross-linked dextrans, e.g. "Sephadex G25" and polyacrylamide gels, e.g. "Biogel P2" (Amberlite, Sephadex and Biogel are registered trademarks). The antibiotic can be from the column 5 3. In a concentration of 0.4% by weight in a freshly produced KBr disk, it shows a characteristic IR spectrum with absorption maxima at 3450, 2950, 1750, 1620, 1510, 1400 and 1260 cm-1, among others. 4. It shows a characteristic NMR spectrum when recorded in D2O, which i.a. a) has a pair of low field doublets centered at 2.85 T and 4.00 T with coupling constants of approximately 14 Hz; b) a doublet centered at approximately 8.55 T and c) a strong singlet at approximately 8.00 T. 5. It has antibacterial activity against various species, including Strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Salmonella typhi and Pseudomonas aeruginosa. 6. When mixed with ampicillin, it shows synergistic activity against organisms such as strains of Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Proteus morganii and Staphylococcus aureus Russell. The pure disodium salt of MM 13902 is a β-lactamase inhibitor and has an iso value (as defined below) between 0.001 [ig / ml and 0.0001 μg / ml compared to the β-lactamase from Escherichia coli B 11. The pure disodium salt of MM 13902 shows the following Rf values in thin layer chromatography on cellulose with the eluents specified below: a) n-butanol / isopropanol / water - 7: 7: 6 v / v; Rf = 0.72 b) isopropanol / water 7: 3 v / v; Rf = 0.85 c) n-butanol / ethanol / water - 7: 7: 6; Rf = 0.81 d) n-propanol / water - 4: 1; Rf = 0.75 In another aspect, MM 13902 can be characterized as a sulfur-containing antibacterial agent, the salts of which are produced during the cultivation of Streptomyces olivaceus ATCC 31126 and which in the form of an aqueous solution of 45.2 [ig / ml of its disodium salt, ultraviolet absorption maxima at approximately 305 nanometers and at approximately 225 nanometers, essentially as shown in FIG. 1. Streptomyces olivaceus ATCC 31126 was deposited on February 18, 1975 with the American Type Culture Collection, 12301 Park-lawn Drive, Rockville, Maryland, USA. Furthermore, MM 13902 can be characterized as an antibacterial agent which, in the form of its practically pure disodium salt, has an IR absorption spectrum essentially as shown in FIG. 2 when it is taken up in a freshly produced KBr disk which contains the substance in a concentration of Contains 0.4% by weight. In addition, the disodium salt shows an NMR spectrum essentially as shown in FIG. 3 when taken up in a freshly prepared solution in D2O. The peak marked X is due to an impurity in the solvent (HOD). The material MM 13902 tends to be unstable when it is in acid. Accordingly, the production of the salts of MM 13902 is preferable. Suitable salts of MM 13902 are dibasic. The salts with pharmaceutically acceptable ions, such as sodium, potassium, calcium, s 10th IS 20th 25th 30th 35 40 45 50 55 60 65 3. The method according to claim 1 or 2, characterized in that Streptomyces olivaceus ATCC 31126 or a mutant thereof is processed. 4. The method according to claim 1, 2 or 3 for the preparation of the new antibiotic MM 13902 and its salts, characterized in that a solution of MM 4550 complex is chromatographically separated into fractions consisting essentially of a solution of MM 13902 or a salt the same as well as other groups, on what MM 13902 or the salt thereof is isolated from the solution. 5. The method according to claim 4, characterized in that a dibasic salt of MM 13902 is produced .; 6. The method according to claim 4, characterized in that a sodium or potassium salt is produced. It has been found that a new antibiotic can be obtained by fermentation of certain strains of Streptomyces olivaceus and related organisms. In addition to its powerful antibiotic activity, this new product, which was designated MM 13902, shows synergistic effects with penicillins and cephalosporins. British Patent No. 1,363,075 states that a β-lactamase inhibitor can be obtained by fermentation of certain strains of Streptomyces olivaceus. Until the present invention, it was believed that this product, described in British Patent No. 1,363,075, was essentially pure. However, it has been shown that this is not the case and that a smaller proportion of a substance with strong antibacterial activity can be separated from it. This new product is designated MM 13902 and is believed to be responsible for at least a portion of the antibacterial activity in the product according to British Patent No. 1,363,075, although it is only responsible for a very small proportion of the β-lactamase inhibitory Effectiveness of this product can be held responsible. Of course, it is not the intention of the present application to claim the material claimed in British Patent No. 1,363,075 as such again here. Other β-lactamase inhibitors can be produced from Streptomyces strains, e.g. those described in German Patent No. 2,340,005. However, these materials do not show the high antibacterial activity that MM 13902 has. The process according to the invention for producing the new antibiotic MM 13902 and its salts is characterized in that a strain of Streptomyces producing MM 13902 is cultivated and MM 13902 or a salt thereof is separated from the culture. MM 13902 is a solid carboxylic acid, which has the following characteristics in the form of the essentially pure sodium salt: 4th are eluted using water, aqueous methanol or the like. The dibasic salt of MM 13902 can be obtained in solid form from solutions obtained by the aforementioned processes by means of separation from the solution under mild conditions. Evidence has shown that a suitable method for maintaining such solid material is evaporation under reduced pressure and freeze-drying of the combined fractions. If desired, the aforementioned process can be carried out in two or more process steps. For example, a solution of MM 4550 (complex) can be separated into fractions which contain MM 13902 or a salt thereof and which are contaminated to their own weight by other antibacterial agents, whereupon the fractions can be freeze-dried and give a material which e.g. contain about 50 to 60% MM 13902 or its salt, whereupon this material can be chromatographed to give a material containing 90 to 100% MM 13902 or a salt thereof. In the present specification, the term "MM 4550 (complex)" is used to describe the material as originally designated MM 4550 in British Patent No. 1,363,075. MM 4550 (complex), prepared by reworking the examples of British Patent No. 1,363,075, is an impure material which contains, in various proportions, salts of MM 4550 (as described below), salts of MM 13902 and substantial proportions of other material. MM 4550 (complex) does not show the characteristic ultraviolet spectrum of MM 13902. The iso value (as defined below) of MM 4550 (complex), made by reworking the examples of British Patent No. 1,363,075, is normally below 0.0004 µg / ml. The ratio of salts of MM 4550 and salts of MM 13902, as present in MM 4550 (complex), is likely to be highly variable and is likely to depend on certain factors such as the strain or organism used and / or the isolation technique used . However, it has been shown that the complex generally contains more MM 4550 than MM 13902. The production of MM 4550 (complex) is described below under the section entitled "Descriptions". In the present document, the expression “MM 4550” is used to describe an essentially pure compound which is a β-lactamase inhibitor and which, to a certain extent, also has antibacterial activity. The properties of MM 4550 are described below under the section entitled "Descriptions".