CH657374A5 - METHOD FOR THE MICROBIOLOGICAL PRODUCTION OF NEBRAMYCIN-5 '. - Google Patents

Description

The invention relates to a method for the microbiological production of culture broths which exclusively contain nebramycin-5 '.

Streptomyces tenebrarius is known to produce a multi-component mixture of antibiotics called nebramycin (Stark, Hoehn and Knox, Antimicrobial Agents and Chemotherapy, 1967, p. 314; GB-PS 1 178 489 and US-PS 3 691 279) . In addition to minor components, the antibiotic mixture contains the components nebramycin-2 (apramycin), nebramycin-4 (6 "-0-carbamoyl-kanamycin B) and nebramycin-5 '(6" -0-carbamoyl-tobramycin) [Koch, Davis and Rhoades, J. of Antibiotics 26, 745 (1973)]. US Pat. No. 3,853,709 describes a microorganism which exclusively produces nebramycin-2 and nebramycin-7. In US Pat. No. 4,032,404, Japanese authors describe the production of Nebramycin-2 and Nebramycin-5 'by cultivating the Streptoalloteichus hindustanus strain. According to the process of HU-PS 176 103, the aerated, submerged fermentation of the Streptomyces tenebrarius MNG-169 and -MNG-170 strains can be used to ferment broths which are either Nebramycin-2, Nebramycin-5 'and Nebramycin-4 , or containing nebramycin-2 and nebramycin-5 '. The antibiotics can be isolated from them according to HU-PS 174315.

The tobramycin (brulamycin) produced by hydrolysis of the nebramycin-5 'are broad-spectrum antibiotics used in human medicine, which are primarily of importance for combating infections caused by polyresistant Pseu domonas strains (Preston and Wiek, Antimicrobial Agents and Chemotherapy , 1970, p. 322).

With the method of HU-PS 176 173 an antibiotic mixture of the following composition is obtained.

Table 1

tribe

Nebramycin-2

%

Nebramycin-4

Nebramycin-5 '

MNG 196 *

77

2nd

21st

MNG 170 *

91

traces

9

* Strain MNG 169 was deposited on December 28, 1977 and strain MNG 170 on April 5, 1978 in the National Strain Collection of the Hungarian National Institute for Health Care (Orszägos Kôzegészségûgyi Intézet) under the names MNG 169 and MNG 170. Both strains have been accessible to everyone since February 28, 1980.

Apparently the strains mainly biosynthesized nebramycin-2. To separate nebramycin-5 'from the other components present in obesity requires complicated and costly methods. The composition of a biosynthesized antibiotic mixture is primarily determined by the genetic information stock of the microorganism cell and only to a small extent by fermentation conditions.

Ferment broths with a predetermined component ratio, which is in each case determined by market conditions, cannot be produced using the currently known methods. Furthermore, in the production of nebramycin 5 ', this antibiotic must be separated from the nebramycin-2 present in the overweight by expensive procedures. Trunk refinement attempts to increase production capability are extremely labor-intensive and time-consuming in these multi-component systems. The amount of the individual components must be individually biologically determined after separation by thin-layer chromatography. It would be extremely cheap to carry out the master finishing tests in a one-component system that enables easy determination. On the other hand, it seems more likely to be able to increase the productivity of such a strain that produces a single antibiotic, since in this case all of the biosynthetic capacity is used to form a single compound.

The aim of the invention is the development of a new, simple fermentation process, by means of which fermentation broths which contain 30 exclusively nebramycin-5 'can be obtained.

In our experiments, it was surprisingly found that the 35 Streptomyces tenebrarius MNG-169 strain pretreated with ethidium bromide (2,7-diamino-10-ethylphenylphenanthridinium bromide) had no nebramycin-2 at all, only traces of nebramycin- 4 and Nebra-mycin-5 'as the main component biosynthesized. This finding is unexpected and all the more surprising since ethidium bromide has never been used as a mutagenic compound.

To obtain a strain that biosynthesizes an antibiotic mixture with a changed component ratio, the spores of Streptomyces tenebrarius MNG 169 were grown in a liquid nutrient medium. The 45-grown culture was divided into five parts and 0.0.05, 0.5.5 and 50 μg / ml ethidium bromide were added. Cultivation was continued for 24 hours, then part of the diluted culture was layered on agar plates, while the other part was treated with an equal amount of ethidi-50 umbromide and grown for a further 24 hours. This procedure was repeated, the new, liquid cultures obtained were layered on agar plates after 24 hours, some isolated colonies were isolated and their productivity was checked using a method summarized in the examples.

Between the several hundreds of strains isolated from Streptomyces tenebrarius MNG 169, there were two mutants that did not produce nebramycin-2 but little nebra-60 mycin-5 'and traces of nebramycin-4. In order to increase the productivity of these strains producing nebramycin-5 ', strain selection work and nutrient medium optimization tests were carried out. During this work, one of the tribes was lost, while the other's productivity was increased fivefold. This strain was included in the National Collection of Strains of the Hungarian National Institute for Health Care (Orszägos

3rd

657374

Kôzegészségûgyi Intézet) deposited under the designation MNG 204.

Based on these observations, the invention relates to a process for the production of fermentation broths containing exclusively nebramycin-5 'by using Streptomyces tenebrarius MNG 204 and preferably in a nutrient medium containing organic carbon and nitrogen sources, inorganic salts, trace elements, animal and / or contains vegetable oils and / or fats, in submerged, aerated fermentation culture, at temperatures of 33 ° C. to 40 ° C., preferably at a temperature of 37 ° C.

For the production of fermentation broths that contain only nebramycin-5 'in addition to traces of nebramycin-4,

the procedure is followed in that Streptomyces tenebrarius MNG 204 or its mutant, variant or recombinant is bred. During the fermentation, in addition to 2100-2600 ng / ml nebramycin-5 '- due to fermentation parameters - also 10-180 (ig / ml nebramycin-4) are enriched. Nebramycin-5' is obtained from the culture in a single chromatographic step of Nebramycin-4 and separated from other impurities, converted to tobramycin in a manner known per se, and the tobramycin isolated.

The microorganism is advantageously grown in a nutrient medium containing organic carbon sources, e.g. Starch, glycerin, fructose and similar compounds, preferably glucose, organic nitrogen sources, e.g. Peanut flour, corn porridge, peptone, yeast extract or similar preparations, preferably soy flour, casein, amino acids, especially glutamic acid, inorganic nitrogen sources, preferably ammonium chloride, ammonium nitrate and ammonium sulfate, inorganic salts, preferably magnesium sulfate, zinc sulfate, cobalt nitrate and calcium carbonate, animal fats and / or animal fats and / or / or oils, e.g. Contains palm oil, linseed oil, sunflower oil, rapeseed oil, pork fat or other fats, preferably palm oil and linseed oil in a weight ratio of 1: 1, as well as trace elements essential for cultivation, in submerged, aerated culture, in a temperature range that promotes the cultivation of the microorganism, preferably between 33 ° C and 40 ° C, grown until significant amounts of the antibiotic accumulate in the culture broth. The antibiotic is isolated using methods known per se.

The trace elements are mostly contained in the nutrient medium as impurities, but can also be added separately.

The amount of air supplied for cultivation is selected according to the composition of the nutrient medium and the technical data of the fermenter. In general, it is advantageous to design the oxygen supply to the cells so that the CCh concentration in the exiting gas mixture does not exceed 1.0%, and furthermore that the reducing sugar content of the nutrient medium is consumed between the 96th and 110th hour of fermentation.

The culture media are steam sterilized at 121 to 123 ° C for 30 to 60 minutes. Before sterilization, its pH is suitably adjusted to 7.2 to 7.4.

During fermentation, no additives are required to suppress foaming, since the nutrient medium contains vegetable and / or animal fats and / or oils as a source of nutrients, which also inhibit foaming.

The cultivation can be carried out in shake flasks as well as in laboratory, small business and large business fermentors.

The antibiotic component composition of the cultures is determined by thin layer chromatography and subsequent bioautography. 0.1 to 0.5 ltg / ml of the antibiotic complex are applied to silica gel (Merck, Darmstadt) thin-layer plates, and the plates are developed in a 1: 1: 1 system of ethanol-methylethylketone-25% ammonium-5 umhydroxide. After drying completely, we put a culture medium inoculated with Bacillus subtilis ATCC 6633 on the plate and incubate at 37 ° C for 16 hours. The size of the inhibition zones created is compared with that of the standard. This io determination is suitable for both qualitative and quantitative evaluation, accuracy ± 10%.

The main advantage of the method according to the invention is that the Streptomyces tenebrarius MNG-204-15 strain which produces practically a single antibiotic can achieve higher production than with the earlier method. Further stem refinement experiments promise quick results, since in this case the entire biosynthetic capacity of the cell is used for the production of a single antibiotic. The biological activity determination of culture broths, which contain a single antibiotic component, is quick, easy and exact, the isolation of the active ingredient does not require costly cleaning procedures.

The invention is explained in more detail with reference to Example 25 below, without restricting the scope of protection to this example.

Example a) Preparation of culture 30 containing nebramycin-5 'in laboratory fermentors.

The spores or the vegetative culture of Streptomyces tenebrarius MNG 204 are used to inoculate slanted agars of the following composition, prepared with distilled water:

Dextrin yeast extract

Casein hydrolyzate (10%) * meat extract 40 cobalt dichloride hexahydrate agar

* hydrolysed enzymatically

1.0%

0.1%

2.0%

0.1%

0.001%

2.5%

The pH of the culture medium is adjusted to 7.2 for 4 s with sodium hydroxide before sterilization and then the culture medium is sterilized at 121 ° C. for 20 minutes.

The cultures are incubated at 37 ° C for 4 days. Then the spores are separated from the surface of the nutrient medium with sterile, distilled water, and inoculated nutrient media of the following composition, prepared with tap water and inoculated with 2 x 100 ml, sterilized in 500 ml Erlenmeyer flasks, are then inoculated with the spore suspension obtained:

so

55

Soy flour

2.0% 3.0% 0.1% 0.3% 0.3% 0.5%

Casein hydrolyzate (10%) *

Ammonium nitrate ammonium chloride calcium carbonate 60 magnesium sulfate heptahydrate mixture of palm oil and linseed oil in a weight ratio of 1: 1 3.0%

Glucose (sterilized separately as a 50% solution) 2.0%

* hydrolysed enzymatically

65

The pH of the nutrient medium is adjusted to 7.2 with a sodium hydroxide solution, then it is sterilized in an autoclave at 121 ° C. for 20 minutes.

657374

4th

The cultivation is carried out on a shaking table with a frequency of 280 minutes and an amplitude of 2.4 cm for 14 to 18 hours at 37 ° C.

5 ml of the main fermentation medium of the following composition, sterilized with tap water and sterilized for 45 minutes at 121 ° C. in a laboratory fermentor (working volume 10 liters), are inoculated with 200 ml of this mature inoculum:

Soy flour 3.0%

Casein hydrolyzate (10%) * 5.0%

Ammonium nitrate 0.1%

Ammonium chloride 0.5%

L-glutamic acid 0.8%

Calcium carbonate 0.5%

Magnesium sulfate heptahydrate 0.5%

Cobalt dinitrate hexahydrate 0.001% mixture of palm oil and linseed oil in a weight ratio of 1: 1 3.0%

Glucose (sterilized separately as a 50% solution) 4.2%

* hydrolysed enzymatically

The pH of the nutrient medium is adjusted to 7.2 with aqueous ammonium hydroxide before sterilization.

The fermentation itself is carried out at 37 ° C, with a stirrer speed of 360 / minute and a sterile air supply of 3 liters / minute.

After 144 hours, 100 ml of the culture broth contains 9 mg of nebramycin-4 and 260 mg of nebramycin-5 '. No nebramycin-2 is detectable.

Culture media of different compositions can also be used as the main fermentation medium. One can proceed according to a), with the difference that a main fermentation medium of the following composition is used:

Soy flour 3.3%

Ammonium chloride 0.6%

Magnesium sulfate heptahydrate 0.7%

Calcium carbonate 0.5%

Cobalt dinitrate hexahydrate 0.001%

Palm oil 2.5% glucose (sterilized separately as a 50% solution) * 2.1%

* Depending on the geometry and components of the fermentor used for breeding, the glucose concentration in the nutrient medium can be increased up to 4.0 ° î>.

After 144 hours, 100 ml of the culture broth contains a little nebramycin-4 (maximum 3 mg) 180 mg of nebramycin-5 '. No nebramycin-2 is detectable.

b) Preparation of culture broths containing nebramycin-5 'in industrial fermenters of 1000 liters.

The procedure according to a) is followed for the production of culture broths which contain large amounts of nebramycin-5 '. Spores or vegetative mycelium from the Streptomyces tenebrarius MNG-204 strain are used to inoculate 100 ml of the inoculum culture medium and the microorganism is grown. After 14 to 18 hours, 100 liters of the inoculum medium are inoculated with this culture and grown at 37 ° C., an air supply of 100 liters / minute and a stirrer speed of 360 / minute. After 16 hours, 50 liters of this inoculum are used to inoculate 1000 liters of the main fermentation medium of the composition specified in a) in a stainless fermenter. The fermentation is carried out with an air supply of 500 liters / minute, a stirrer speed of 200 / minute at 37 ° C. The antibiotic composition during fermentation is shown in Table 2.

Table 2

Fermentation time in hours

Nebramycin component in [ig / ml 2 4

5 '

24th

0

0

50

48

0

0

400

72

0

5

1200

96

0

30th

1740

120

0

40

2320

144

0

60

2650

c) Decarbamoylation of nebramycin-5 'and isolation of tobramycin.

22 g of sodium hydroxide are added to 3 liters of culture broth containing 2600 ng / ml nebramycin-5 'and 90 (ig / ml nebramycin-4. The culture broth is heated to 90-100 ° C. with stirring and at this temperature for 25 minutes After the reaction has ended, the mixture is cooled to room temperature and its pH is adjusted to 2.0 with 50% strength sulfuric acid, 2.0 g of oxalic acid are added to this acidic culture broth with constant stirring, its pH value using a concentrated ammonium hydroxide The solution was adjusted to 7.0 and the mixture was stirred for 30 minutes, then filtered and the mycelium washed with 2 × 600 ml of water, the filtrate and washing solution were combined and made up of 20 ml of Amberlite CG-50 (NH4 +) (Rohm and Haas , Co., Philadelphia, USA) The column is washed with 400 ml of ion-free water and then sequentially with 1000 ml of 0.05 N, 2000 ml of 0.1 N and 3000 ml of 0.15 N ammonium hydroxide in Fractions of 250 ml each eluted Fractions containing ramycin (13 to 18) are combined, their pH is adjusted to 7.0 with 50% strength sulfuric acid, and the mixture is chromatographed on 120 ml of Amberlite CG-50 (NHt +). The ion exchanger is washed with 200 ml of ion-free water, and the antibiotic is subsequently eluted in sequence with 1000 ml 0.05 N, 2000 ml 0.1 N, 2000 ml 0.15 N and finally with 2000 ml 0.2 N ammonium hydroxide in fractions of 60 ml each. Fractions 45 to 46 are combined and evaporated under reduced pressure. The distillation residue is dried over phosphorus pentoxide and contains 0.7 g (70%) kanamycin B, 12% component N-13 (6'-N-carbamoyl-tobramycin), 10% unknown component and 8% tobramycin. When fractions 57 to 58 are evaporated, a distillation residue containing 60% tobramycin, close to 40% kanamycin B and 0.1 to 0.2% component N-13 is obtained. Evaporation of fractions 59 to 69 leads to a tobramycin preparation (5.7 g) which contains 0.1 to 0.15% component N-13. When this crude product is recrystallized from 96% ethanol, 4.2 g of pure tobramycin are obtained.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

B

Claims (4)

657 374 1. A process for the microbiological production of culture broths which exclusively contain nebramycin-5 ', characterized in that Streptomyces tenebrarius MNG 204 is used for the production of the culture broths. 2. The method according to claim 1, characterized in that one uses the spores or the vegetative mycelium of the exclusively Nebramycin-5 'producing Streptomyees tenebrarius strain MNG 204. 2nd PATENT CLAIMS 3. The method according to any one of claims 1 or 2, characterized in that with a nutrient medium, the organic carbon source of starch, glycerol, fructose, preferably glucose, and as an organic nitrogen source peanut meal, corn porridge, peptone, yeast extract, preferably soybean meal or casein , Amino acids, especially glutamic acid, is worked. 4. The method according to any one of claims 1 or 2, characterized in that the cultivation of the microorganism is carried out at a temperature of 37 ° C.