NL8205011A - MICROBIOLOGICAL PROCESS FOR THE PREPARATION OF NEBRAMYCIN-2, NEBRAMYCIN-5 'AND NEBRAMYCIN-4. - Google Patents

Description

- 1 -

N / 31.247-Kp / Pf / vdM

Microbiological method for the preparation of nebramycin-2, nebramycin-5 'and nebraraycine-4.

The invention relates to a microbiological process for the preparation of fermentation scalding, which contains either almost exclusively nebramycin-5 'or a mixture of nebramycin-2 and nebramycin-5' in any desired predetermined ratio, as well as nebramycin-4.

Nebramycin, a complex mixture of various antibiotics, is known to be a biosynthetic metabolite of Streptomyces tenebrarius (Stark, Hoehn and Knox, Antimicrobial Agents and Chemotherapy, 1967, p. 314; British Patent 1,178,489 and U.S. Patent 3,691,279 ). The antibiotic mixture, apart from several minor factors, consists of the following main ingredients! nebramycin-2 (apramycin), nebramycin-4 (6 "-O-carbamoylkanamycin B) and nebramycin-5 '(6" -O-carbamoylto-15 bramycin) (Koch, Davis and Rhoades, J. Antibiotics 2, 745, 1973). U.S. Patent 3,853,709 describes a microorganism that produces nebramycin-7 in addition to nebramycin-2. Japanese researchers describe in U.S. Pat. No. 4,032,404 that nebramycin-2 and nebra-20 mycine-5 'are co-produced by culturing Streptoalloteichus hindustanus. According to Hungarian patent 176,103, Streptomyces tenebrarius MNG 169 and 170, cultivated under submerse, aerated conditions, produces fermentation scalding containing either nebramycin-2, nebramycin-5 '25 and nebramycin-4, or nebramycin-2 and nebramycin-5'.

These antibiotics are isolated in a manner described in Hungarian Patent 174,315.

Nebramycin-2 inhibits the growth of phytopathogenic and zoopathogenic microorganisms (U.S. Pat. Nos. 30 3,691,279, 3,853,709 and 3,876,763), while both kanamycin B obtained by hydrolysis of nebramycin-4 and tobra mycine (Brulamycin), obtained by hydrolysis of nebramycin-5 'are commercial, broad-spectrum antibiotics, which play an important role in human therapy in the treatment of infections caused by poly-8205011

* I

- 2 - resistant bacteria (eg Preston and Wick, Antimicrobial Agents and Chemotherapy 1970, biz. 322).

Using the method according to Hungarian patent 176,103, an antibiotic mixture of the following composition is obtained (Table A):

TABLE A

strain nebramycin-2 nebramycin-4 nebramycin-51

PER CENT

MNG 169 * 77 2 21 10 MNG 170 ** 91 tracks 9 yr

Filed December 29, 1980 with the National Collection of Microorganisms, National Institute for Public Health, Budapest and available to the public since February 28, 1980.

15 Filed April 5, 1978 at the same institute, available to the public since February 28, 1980.

It is clear that nebramycin-2 is the major product of biosynthesis in both strains, while nebramycin-5 'must be separated from the other predominant components using a very time-consuming and costly procedure.

The composition of an antibiotic complex biosynthesized by a microorganism is primarily determined by the cellular genetic information supply and only to a much lesser extent by the fermentation conditions.

In the methods known hitherto, the ratio of the components in the fermentation broth cannot be adjusted to the actual commercial requirements.

Furthermore, if the production of nebramycin-51 is required, it must be separated from the predominant nebramycin-2 by very expensive procedures. In such multicomponent systems, performing stem selection experiments aimed at increasing strain productivity is rather laborious and time consuming. After 8205011 Λ '- 3 - * "* thin layer chromatographic separation, each component is quantified separately using a biological assay. It would be beneficial to be able to perform these master selections with a one component system, allowing for simple analysis procedures Furthermore, the increase in productivity in a strain that biosynthesizes a single antibiotic offers greater promises, since in this case the cells can utilize their entire biosynthetic potential to form a single compound.

The aim of the present invention is to develop a new and simple fermentation process for the preparation of fermentation broth, which comprises either only nebramycin-5 'or a mixture of nebramycin-2 and ne-bramycin-51 in any desired predetermined ratio. contain. .

In the course of our experiments, it was quite surprisingly found that Streptomyces tenebrarius MNG 169 treated with ethidium bromide (2,7-diamino-10-ethylphenyl-phenanthridinium bromide) yields a mutant strain unable to produce nebramycin-2, but, on the other hand, nebramycin-5 'as a main ingredient in addition to spores produces nebramycin-4. This phenomenon is rather unexpected and surprising, since hitherto ethidium bromide has not been used as a mutagen.

For the isolation of a mutant, which biosynthesizes an anti-biotic complex with altered ratios between the components, the spores of Streptomyces tenebrarius MNG 169 were grown in a liquid nutrient medium.

The culture culture was divided into five parts and 0.05.

30 0.5. 5.0 and 50.0 µg / l ethidium bromide was added sequentially. Culturing was continued for 24 h, after which part of the culture was spread on agar plates after dilution, the other part was used for inoculation of liquid media containing equal amounts of ethidium bromide and their culture was continued for an additional 24 h. Subsequently, the above-described plating and grafting procedure was repeated.

The resulting new fluid cultures were replated after 24 hours on plates, the agar plates were incubated, some separated colonies were isolated, and their productivity was tested according to the procedure described in the examples.

Of the several hundred isolated colonies from Streptomyces tenebrarius MNG 169, two mutant strains were unable to produce nebramycin-2, but did produce relatively small amounts of nebramycin-5 'as well as traces of nebramycin-4. To improve the productivity of these two mutants, producing nebramycin-5 ', strain selection and medium optimization experiments were performed. In the course of this, one of the mutants was lost, while the production capacity of the second strain was increased fivefold and could be maintained at this level. This strain was deposited on September 2, 1981 with the National Collection of Microorganisms (National Institute for Public Health, Budapest, Hungary), where it obtained accession number MNG 204.

The invention further relates to a process for the preparation of fermentation scalding which contains a mixture of nebramycin-2 and nebramycin-5 'in any desired predetermined ratio by spores or vegetative mycelia of the original Streptomyces tenebrarius MNG 169 strain and which mix the new Streptomyces tenebrarius MNG 25 204 strain, which does not produce nebramycin-2, in the desired ratio and grow them thereon in a mixed culture. Quite surprisingly, during this mixed culture fermentation, both strains were found to produce higher levels of antibiotics than in fermentations, each strain being grown separately.

On the basis of the above, the invention relates to a process for the preparation of fermentation scalding, which comprises either practically only nebramycin-5 'or a mixture of nebramycin-2 and nebramycin-51 in any desired predetermined ratio, as well as nebramycin- 4. According to the process of the invention a) for the preparation of fermentation scalding, which practically contain nebramycin-5 ', the MNG 204 strain 8205011 I * - 5 - of Streptomyces tenebrarius, or b) for the preparation of fermentation scalding, which use nebramycin -2 and nebramycin-5 'in any desired predetermined ratio, as well as nebramycin-4, contain the 5 spores or vegetative mycelia of a nebramycin complex producing Streptomyces tenebrarius, preferably the MNG 169 strain, and that of a Streptomyces tenebrarius practically exclusively Nebramycin-5 'preferably produces the MNG-204 strain, mixed in the desired ratio and grown in a medium containing organic carbon and nitrogen sources, inorganic salts, trace elements, oils and / or fats from animal and / or vegetable origin, in a submerse, aerated culture at 33-40 ° C, preferably at 37 ° C.

According to a preferred embodiment of the method according to the invention, the production of fermentation scalding containing nebramycin-5 'in addition to spores nebramycin-4 is carried out by cultivating Streptomyces tenebrarius MNG 204 or one of its mutants, variants or recombinants.

In the course of the fermentation, 10-180 µg / ml nebramycin-4 is accumulated in the fermentation broth, in addition to 2100-2600 µg / ml nebramycin-5 ', depending on the culture conditions. Nebramycin-5 'is isolated from the fermentation broth and separated from Nebramycin-4 and various impurities by a single chromatographic step, then hydrolysed to tobramycin by methods known per se. Possibly. Nebramycin-5 'can already be converted to tobramycin in culture and isolated from it.

According to a further preferred embodiment of the method according to the invention for producing fermentation scalding, which in addition to nebramycin-4 contains a mixture of nebramycin-2 and nebramycin-5 'in any desired predetermined ratio, the spores and vegetative mycelia of Streptomyces tenebrarius MNG 169 and that of Strep-35 tomyces tenebrarius MNG 204 are mixed in the desired ratio and grown in a mixed culture. The antibiotics produced are isolated by methods known per se, preferably according to Hungarian patent 174,315. The ratio of the amount of nebramycin-2 to the amount of nebramycin-5 'biosynthesized is almost proportional to the ratio of the number of cells of the Strepto-xnyces tenebrarius MNG 169-, respectively. MNG 205 strains when mixed in identical growth stages.

It is clear to a person skilled in the art that the above extensions are only valid in fermentation systems, in which the mixed cells expand at almost identical rates and in which the fermentation conditions are favorable for the biosynthesis of both components. Otherwise, the ratio can be shifted to either direction. Determining the mixing ratio is no problem for an expert.

According to the present invention, the microorganisms are grown in a nutrient medium containing an organic carbon source, ie starch, glycerol, fructose or other similar compounds, preferably glucose, an organic nitrogen source, ie nut flour, corn liquor, peptone, yeast extract or similar preparations. preferably soybean meal, or casein, amino acids, preferably glutamic acid, an inorganic nitrogen source, preferably ammonium chloride, ammonium nitrate and ammonium sulfate, inorganic salts, preferably magnesium sulfate, zinc sulfate, cobalt nitrate and calcium carbonate, animal and / or vegetable fats and / or oils, ie palm oil, linseed oil, sunflower oil, rapeseed oil, lard or other, preferably a 1: 1 mixture of palm oil and linseed oil, further containing trace elements essential for the growth of the microorganism, including submerse aerated fermentation conditions at a temperature which ensures the growth of the microorganism t, preferably at 33-40 ° C, until significant amounts of the antibiotics have accumulated. The antibiotics are isolated by methods known per se.

The trace elements can either be present in the form of impurities in other components of the nutrient medium or added separately to the medium.

The intensity of the air flow is controlled depending on the composition of the medium, the power of the stirrer and other technical properties of the fermentation device. It is usually beneficial to control the oxygen supply so as to ensure that the CO2 content of the effluent gas mixture is not greater than 1.0% and that the reducing sugar content is consumed completely between the 96th and 110th hours in the medium. .

The flow sterilization of the medium is performed at 121-123 ° C for 30-60 min. The pH of the medium is preferably adjusted to 7.2-7.4 prior to sterilization.

No anti-foaming agent is added during the fermentation, since the oils and / or fats of animal and / or vegetable origin, which have been added as nutrient to the medium, can at the same time prevent foaming of the culture.

Culturing itself can be performed in shake flasks, or laboratory, pilot plant, or industrial scale fermenters.

The antibiotic composition of the cultures is analyzed by bioautography after preliminary thin layer chromatography on silica gel. Amounts of 0.1-0.5 µg / ml of the antibiotic complex are spotted on the silica gel thin layer plates (Merck, Darmstadt, Federal Republic of Germany) and subjected to chromatography in a system of ethanol / methyl ethyl ketone / 25% ammonium hydroxide, 1: 1: 1. After complete drying, a medium containing Bacillus subtilis ATCC 6633 is spread on the surface of the plate and incubated at 37 ° C for 16 h. The size of the braking zones is then compared to that of the 30 standards. This procedure is suitable for qualitative and quantitative analysis, where the standard deviation is + 10%.

The main advantages of the method according to the invention are the following: Streptomyces tenebrarius MNG 204, which practically produces a single component, provides higher levels of antibiotics than the older parent strain. Further selection efforts that can be made on this new strain 8205011-8 promise rapid success, as this strain can utilize its entire biosynthetic potential for the formation of a single antibiotic component. The biological analysis of these fermentation broths, which contain a single antibiotic component, is fast, simple and exact, the isolation of the antibiotic does not require expensive purification procedures. By mixing the cells of two different strains and growing them in a mixed culture, fermentation broths containing nebramycin-2 and nebramycin-5 'in any desired predetermined ratio can be produced in accordance with actual commercial demand and cost -situation. Furthermore, it is an additional advantage that in the course of this mixed fermentation neither strain loses any significant part of its productivity and that both synthesize an overall higher level of antibiotics. ""

The following examples are illustrative, but not limiting, of the scope of the invention. EXAMPLE I

a) Production of fermentation scalding containing nebramycin-51 in laboratory fermenters.

Inclined agars, prepared with distilled water and with the following composition: dextrin 1.0% yeast extract 0.1%. casein hydrolyzate (10%) 2.0% meat extract · 0.1% cobalt dichloride hexahydrate 0.001% agar 2.5% 30% enzymatically hydrolyzed were inoculated with the spores or vegetative mycelia of Strepto myces tenebrarius MNG 204. The medium was adjusted. with an aqueous sodium hydroxide solution at 7.2, prior to sterilization, and then sterilized for 35 min at 121 ° C.

The cultures were incubated at 37 ° C for 4 d, after which the spores of the surface of the medium were rinsed with sterile distilled water and the thus obtained 8205011-9 spore suspension was used to inoculate 2 x 100 ml inoculum medium, prepared with tap water and sterilized in 500 ml Erlenmeyer flasks. The inoculum had the following composition: 5 soybean meal 2.0% casein hydrolyzate (10%) * 3.0% ammonium nitrate 0.1% ammonium chloride 0.3% calcium carbonate 0.3% 10 magnesium sulfate heptahydrate 0.5% 1: 1 mixture of palm oil and linseed oil 0.5% glucose (sterilized separately as a 50% solution) 2.0% dm '15 enzymatically hydrolysed

The pH of the medium was adjusted to 7.2 with an aqueous sodium hydroxide solution and sterilized at 12 ° C in a pressurized vessel for 20 min.

The inoculated cultures were grown at 37 ° C for 14-18 h on a rotary shaker (diameter 2.4 cm, 280 rpm).

200 ml of this fully developed inoculum was used to inoculate a laboratory fermenter with a working volume of 10 l, in which 5 l of the main fermentation medium was prepared with tap water, sterilized for 45 min at 121 ° C and with the following composition: soybean meal 3 .0% casein hydrolyzate (10%) * 5.0% ammonium nitrate 0.1% 30 ammonium chloride 0.5% L-glutamic acid 0.8% calcium carbonate 0.5% magnesium sulfate heptahydrate 0.5% cobalt dinitrate hexahydrate 0.001% 35 L: 1 mixture of palm oil and linseed oil 3.0% glucose (sterilized separately as a 50% solution) 4.2% 8205011 -10- ν enzymatically hydrolyzed.

The pH of the medium was adjusted to 7.2 with ammonium hydroxide prior to sterilization.

Culturing was performed at 37 ° C, at a stirring speed of 360 rpm and at a sterile air flow of 3 1 / min.

In the 144th hour of culture, 260 mg of nebramycin-5 'and 9 mg of nebramycin-4 were determined analytically in 100 ml of the broth. No nebramycin-2 was observed.

Other nutrient media with a different composition can be used for the main fermentation. Thus, similar results were obtained when proceeding according to a), with the proviso that the following medium 15 was used: soybean meal 3.3% ammonium chloride 0.6% magnesium sulfate heptahydrate 0.7% calcium carbonate 0.5% 20 cobalt dinitrate hexahydrate 0.001% palm oil 2 .5% glucose (sterilized separately in a 50% solution) * 2.1%

V

depending on the geometry and tools of the fermenter used for the culture, factors influencing cell metabolism, the glucose concentration can be increased to 4.0%.

In the 144th hour of culture, 100 ml of the fermentation broth contained, in addition to spores nebramycin-4 (up to 3 mg), 180 mg of nebramycin-51. No nebramycin-2 was observed.

b) Production of fermentation scalding containing nebramycin-51 in a 1000 L pilot plant fermenter.

The procedure described under a) 8205011-11 was used for the production of fermentation scalding containing large amounts of nebramycin-5 '. 100 ml of the inoculum medium was inoculated with the spores or vegetative mycelia of Strepto-myces tenebrarius MNG 204, cultivated for 14-18 h, after which 100 l of a sterile inoculum medium was inoculated with this culture and grown at 37 ° C , using an air flow of 100 1 / min and a stirring of 360 rpm. After 16 h, 1000 l of the sterilized main fermentation medium (composition see under a) was seeded in a stainless steel fermenter with 50 l of this inoculum. The main-10 fermentation was performed at 37 ° C, using an air flow of 500 1 / min and at a stirring speed of 200 rpm. The changes in antibiotic content are shown in Table B.

TABLE B

Duration of the fernebramycin component mentation (h) (µg / ml) 2 4 5 '24 0 0 50 48 0 0 400 20 72 0 5 1200 96 0 30 1740 120 0 40 2320 144 0 60 2650 c) Decarbamoylation of nebramycin-51 and isole-25 ring of tobramycin.

Sodium hydroxide in an amount of 22 g was added to 3.0 L of a fermentation broth containing 2600 µg / ml nebramycin-5 'and 90 µg / ml nebramycin-4, the broth was stirred to 90-100 ° C with constant stirring heated and held at this temperature for 25 min. When the reaction was completed, the mixture was cooled to room temperature and its pH adjusted to 2.0 with a 50% aqueous sulfuric acid solution. To this acid fermentation broth, 6 g of oxalic acid was added with constant stirring, its pH was adjusted to 7.0, the mixture was stirred for an additional 30 min, filtered and the mycelium was washed twice with 600 ml of 8205011-12 - water. The filtrate and rinses were combined and the resulting calcium ion-free solution was subjected to ion-exchange chromatography on a column prepared with 200 ml Amberlite CG-50 (NH4 +) (Rohm and Haas Co., Philadel-5 phia, USA). The column was washed with 400 ml of deionized water and eluted first with 1000 ml of a 0.05 N, then with 2000 ml of a 0.1 N and finally with 3000 ml of a 0.15 N solution of ammonium hydroxide. 250 ml fractions were collected in the course of the elution. Fractions 13-18, containing 10 tobramycin, were combined, the pH of the solution adjusted to 7.0 with an aqueous 50% sulfuric acid solution and subjected to ion exchange chromatography on 120 ml Amberlite CG-50 (NH4 +). The column was washed with 200 ml dexonized water and the antibiotic was eluted with first 1000 ml of a 0.05 N, then with 2000 ml of a 0.1 N and then with 2000 rals of a 0.15 N, and finally with 2000 ml of a 0.2 N ammonium hydroxide solution. 60 ml fractions were collected. Fractions 45-56 were combined and evaporated to dryness under reduced pressure. After drying over phosphorus pentoxide, the residue yielded a product containing 0.7 g (70%) of kanamycin B, 12% of component N-13 (61-N-carbamoyltobramycin), 10% of an unknown component and 8% tobramycin. Evaporation of fractions 57-58 gave a residue containing 0.2 g (60%) of tobramycin, almost 40% of kanamycin B and 0.1-0.2% of component N-13. The distillation of fractions 59-69 gave 5.7 g of tobramycin containing 0.1-0.15% of component N-13. Recrystallization of this crude tobramycin from aqueous 96% ethanol gave 4.2 g of pure tobramycin.

EXAMPLE II

Production of fermentation scalding containing nebramycin-2 and nebramycin-5 * at a predetermined ratio.

Using the procedure of a) in Example I, 10 100 ml aliquots of an inoculum medium as described therein were inoculated with a spore suspension of Streptomyces tenebrarius MNG 204 and 10 100 ml aliquots of the same inoculum medium were inoculated with the spore suspension from Streptomyces tenebrarius MNG 169. The microorganisms were 8205011. . "- 13 - cultivated according to a) in Example 1, after which 100 ml portions of a medium sterilized in 500 ml Erlenmeyer flasks and 5 1 portions of a main fermentation medium sterilized in a 10 1 fermenter and of a composition as described in a) in Example I, were inoculated with the two strains, either separately or after mixing in a ratio set forth below.

Culturing was continued for 144 hours under the fermentation conditions described in Example I. Subsequently, the content of the antibiotic components was analyzed by thin layer chromatography, subsequent bioautography and comparison with a standard. The results are listed in Table C.

15 8205011

- 14 TABLE C

nebramycin • component microorganism culture nism 1 (ml) nism 2 (ml) ^ 4 51 5 shake bottle 1 MNG 169 5 ml 5040 180 1310 2 MNG 204 5 ml 0 80 2150 3 MNG 169 4/5 ml MNG 204 0 1.5 ml 5640 175 1280 4 4.0 ml 1.0 ml 5140 195 1370 10 5 3.5 ml 1.5 ml 4740 180 1610 6 3.0 ml 2.5 ml 3140 240 1690 7 2.5 ml 2, 5 ml 2850 170 1840 8 2.0 ml 3.0 ml 2310 19.0 .. 1880 9 1.5 ml 3.5 ml 1320 240 2430 15 10 1.0 ml 4.0 ml 470 120 2340 11 0.5 ml 4 .5 ml 150 95 2340 Fermenter I MNG 169 200 ml 3900 210 870 20 II MNG 204 200 ml 0 90 2300 III MNG 169 175 ml MNG 204 25 ml 4380 240 1130 IV 150 ml 50 ml 3050 340 1530 V 125 ml 75 ml 2450 220 1640 VI 100 ml 100 ml 2320 420 2150 25 VII 75 ml 125 ml 1420 300 2470 VIII 50 ml 150 ml 980 160 2540 IX 25 ml 175 ml 280 140 2310

The data of Table C clearly demonstrate that fermentation broths containing nebramycin-2 and nebramycin-5 'in any desired predetermined ratio can be produced using Streptomyces tenebraius MNG 204 and Streptomyces tenebrarius MNG 169. The antibiotics may are isolated from the fermentation scalding by the method described in Hungarian Pat. No. 174,315 8205011-15.

The bioautography of scalding obtained in the fermenters I-IX is shown in Figure 1.

5 8205011

Claims (4)

A process for the preparation of fixed scalding, comprising either practically exclusively nebramycin-5 'or a mixture of nebramycin-2 and nebramycin-5', in any desired predetermined ratio, as well as nebramycin-4, wherein a) for the preparation of fermentation scalding containing practically exclusively nebramycin-5 ', the MNG 204 strain of Streptomyces tenebrarius, or b) for the preparation of fermentation scalding, containing nebramycin-2 and nebramycin-51 in any desired predetermined ratio, the spores or vegetative mycelia of a nebramycin complex producing Streptomyces tenebrarius, preferably the MNG-169 strain (deposited on December 28, 1980 at the National Collection of Microorgarrsimen, Budapest, Hungary) and that of a Streptomyces tenebrarius, which practically produces nebramycin-5 ' , preferably the MNG 204 strain (deposited on September 2, 1981 with the National Collection of Microorganisms, Budapest, 20 Hungary), are mixed in the desired ratio and grown in a medium containing organic carbon and nitrogen sources, inorganic salts, trace elements, oils and / or fats of animal and / or vegetable origin in a submerged aerated culture, at 33- 40 ° C, preferably at 37 ° C. 25 Method according to claim 1, characterized in that starch, glycerol, fructose, preferably glucose, are used as organic carbon source and nut flour, corn liquor, peptone, yeast extract, preferably soy flour, or casein, amino acids, preferably glutamic acid as an organic nitrogen source. 3. Fermentation broth containing nebramycin-51 or a mixture of nebramycin-2 and nebramycin-5 'in any desired predetermined ratio, as well as nebramycin-4, characterized in that it is prepared using a process according to claim 1 or 2. 4. Nebramycin-51 or a mixture of nebramycin-2 and nebramycin-5 'in any desired predetermined ratio, as well as nebramycin-4, characterized by 82 0 5 0 1 1 ........... ............... 5 - 17 - that it is obtained using a method according to claim 1 or 2. 8205011