SE438163B - Method for producing sisomicin by cultivating Micromonospora danubiensis and this microorganism - Google Patents

Abstract

The invention is a new method of producing sisomicin and then possibly transforming the sisomicin base obtained in this manner into a pharmaceutically acceptable salt thereof by culturing Micromonospora danubiensis in a nutrient-rich medium containing carbon and nitrogen sources that can be assimilated as well as mineral salts.

Description

aoonvus-7 p É 2 10 15 20 25 30 35 40 Pical and biochemical properties: Morphology Macroscopic observations: a 10-day-old culture incubated at 37 ° C on Czapek's agar shows a fairly good to moderate growth, no aerial mycelium, the colonies are elevated with regular round shape, they are light reddish brown. Apart from the colonies, no secretion of soluble pigments can be observed.

Microscopic observations: long, branched wires without transverse walls, the wires diameter 0.5 μm, spores are located in simple sporophones with a diameter of 1 - 1.5 μm. The spores have an ovoid-spherical shape.

Biochemical and physiological properties: Micromonospora danubiensis MG-00171 grows quite well at temperatures between 28 and 37 ° C, but no growth is observed at temperatures of 44 ° C and above.

The utilization of the carbon source has been studied on the following nutrient media: yeast extract 0.5%, carbon source 1.0%, CaCO 3 0.1%, agar 1.5% in distilled water. The results are summarized in Table 1.

Table 1 Carbon source Mikromonospora danubiensis MNG 00171 Arabinos weak (-) Ribos moderately (+) Xylos weak (-) Ramnos weak (-) Fructose fairly good (+++) Galactose moderately (+) Glucose fairly good (+++) Lactose weak (-) Sucrose quite good (+++) Raffinose weak (-) Dulcitol weak (-) Mannitol quite good (+++) Inositol weak (-) Starch quite good (+++) The utilization of the nitrogen source was studied on the following nutrient media: glucose 1%, agar 1.5% in distilled water. The results obtained are summarized in 10 15 20 25 30 35 40 ........_...._ in __...... _ __, 3 8004746-7 table 2.

Table 2 Nitrogen utilization Nitrogen source Micromonospora danubiensis MNG 00171 0.5% yeast extract fairly good (+++)% NZ-amine, type A% asparagine fairly good (+++) moderately-weak (1)% glutamic acid weak (-)% KNO3 weak (-) A growing colony of Micromonospora danubiensis hydrolyzes gelatin, flour -ÅJ-1A reduces nitrate to nitrite. The microorganism tolerates a maximum of 2% sodium chloride in the culture medium.

Culture characteristics Table 3 summarizes the culture characteristics of Mikromonospora danubiensis JMS-3. (In describing color formation for these observations, the following references have been used: Baumann's Color Chart Atla II., Paul Baumann, Aue I-SA 87350 LAu 302, GFR).

Table 3 Cultivation characteristics Medium Micromonospora danubiensis MNG 00171 Bennett agar weak growth, healthy orange color Oc 97 - 98 Czapek's agar fairly good growth, reddish brown color O 142 - 145 Emerson's agar no growth Glucose asparagine agar. no growth Glucose-yeast extract- fairly good growth, lively orange agar color, Oc 99 Nutrition agar quite good growth, burgundy, some places yellow-brown color, Ro 193, respectively Or 165 - 166 Peptone iron agar no growth or only in traces, small colonies, pale orange, Oc 97 and CO 69, no diffusible pigment rasa Qfzaxlzfrsï, 800117464 10 15 20 25 30 35_ 40 »b Table 3 (continued) Cultivation characteristics Medium Micromonospora danubiensis MG 00171 Tyrosine agar growth in traces, no color could be determined diffusible pigment Potato cube without CaCO3 no growth Potato cube with CaCO3 moderate growth, reddish brown color, O 136 and O 145 - 147 Based on the above facts, it has been established that the Micromonospora strain designated JMS-3 differs from literature known Micromonospora strains. When comparing the listed taxonomy characteristics with the Micromonospora system according to Luedemann, the strain JMS-3 qualifies as a new species named after the isolation site "Micromonospora danubiensis species nova".

According to a preferred embodiment, Micromonospora danubiensis is grown in submerged culture under aerobic conditions.

The culture medium in the production of sisomycin can include many sources of carbon and nitrogen, which can be assimilated by Micromonospora danubiensis, inorganic salts, trace elements and antifoam additives. Starch, soluble starch, dextrin, glucose, sucrose, cornmeal, soybean meal, hydrolyzate of soybean meal, hydrolyzate of casein, barley casting liquid, yeast extract, etc. were used as sources of carbon, nitrogen and energy.

Several ammonium, iron, zinc, manganese, magnesium, sodium, potassium and cobalt salts can be used as inorganic salts. To improve the buffer capacity of the culture medium, calcium carbonate is suitably used and various vegetable oils (for example palm oil, sunflower oil, soybean oil) are added as anti-foaming agents.

By using different nutrient medium components, different nutrient media can be produced. The nutritional need for inoculum production differs from that used in the main fermentation. For the production of sisomicin, the nutrient medium containing soybean meal, barley casting liquid, cornmeal, starch, sucrose, calcium carbonate, cobalt chloride and palm oil has proven to be one of the most advantageous.

The temperature during fermentation can vary between 25 and 37 ° c, with za - 33 ° c being most favorable.

The stirrer is rotated according to its design at 100 - 600 revolutions per minute. Aeration is suitably carried out with a ratio between the volume units of 1: 1 per minute.

The activity of the antibiotic produced during fermentation (sisomicin and small by-products) is determined in comparison with a standard product of sisomicin using the agar diffusion method and a test organism, namely Staphylococcus epidermidis.

During 96 - 120 hours of fermentation, the total activity of the fermentation broth reaches a value of 400 - 450 U / ml (1 U = 1 μg of sisomicin base activity). Sisomicin activity accounts for about 85% of the total antibiotic activity in the fermentation broth.

Isolation of sisomycin can be accomplished in known manner. If desired, the resulting sisomicin base can be converted to a pharmaceutically acceptable salt.

The amount of sisomycin produced by Micromonospora danubiensis is a multiple of that produced by hitherto known methods. Under laboratory conditions it exceeds 450 U / ml and in a culture tank in a pilot plant 350-400 U / ml. In addition to sisomycin, the fermentation broth contains only 10 - 15% accompanying antibiotics.

The method according to the invention is illustrated in the following examples.

Example 1) In a 500 ml Erlenmeyer flask, 100 ml of a sterile culture medium of the following composition was charged and inoculated under sterile conditions with 1 ml of a mycelium (vegetative microbes 107 - 10 ”/ ml) of the supercooled strain of Micromonospora danubiensis JMS-3 (MNG O0171): POOR QUALITY 10 15 20 25 30 35 40 8004746-7 6 Starch 25.0 g Grain casting liquid 7.0 g (NHu) zS0u 3: 5 9 Nacl '5.0 g CaCO 3 8.0 g Water tap water 1000 ml The pH of the culture medium was adjusted to 7.5 before sterilization. Consequently, the pH after sterilization was 7.0-7.2.

The culture medium was incubated for 3 days at 28 ° C on a flat shaking table (220 beats per minute, deviation 7.5 cm). With the culture liquid thus obtained, 6 l of a sterile nutrient medium were inoculated into a 10 l glass culture vessel under sterile conditions. The culture medium had the same composition as indicated above for the inoculum medium.

The culture in the glass container was carried out at 28 ° C with stirring at 400 rpm and aeration 1 unit of volume per 1 volume per minute. Palm oil was used as an antifoaming agent, if required.

The culture medium developed in 40 hours was used to inoculate the nutrient medium for the fermentation. The amount of inoculum was 600 ml, the amount of culture medium was 6 l, the pH was 7.0 - 7.2 and the volume of the culture vessel was 10 l. The composition of the culture medium was as follows: Soybean meal 50.0 g Maize starch 10.0 g Sucrose 30.0 g CaCO 3 .0 g Grain casting liquid 5.0 g CoCl2.6H2O 4.5 mg Water tap water 1000 ml The cultivation was carried out at 3100, the stirrer was turned at 400 rpm, the aeration amounted to 6 l per minute and palm oil was used as antifoaming agent§ Antibiotic production started at the fermentation 28 - 34 hours and the highest value was reached after 110 hours of fermentation. At this time point, the total antibiotic content of the fermentation broth was determined to be 400 U / ml relative to a standard isomicin for which Staphylococcus epidermidis was used as the test organism and the agar diffusion method. (J.S, Simpson: Analytical Microbiology, Acad. Press, New York, pp. 87-124, 1963). 2) The following nutrient medium was inoculated with a vegetative culture prepared from Micromonospora danubiensis JMS-3 (MNG 00171) on a flat shaking table in the manner set forth in Example 1): Soybean meal 10.0 g Starch 10.0 g Sucrose 10.0 g CaCO 3 4.0 g Water tap water 1000 ml The pH of the nutrient medium after sterilization was 7.0 - 7.2, the amount of medium was 6 l (in a 10 l glass culture vessel). The cultivation was carried out at 31 ° C, the stirrer was rotated at 400 rpm and the aeration was carried out at 6 l per minute. Palm oil was used as an antifoaming agent, where applicable. 600 ml of the inoculum developed in 36 hours was used to inoculate the culture medium with the following composition: Soybean meal 30.0 g Maize flour 25.0 g cocoa '5.0 g CoCl2.6H2O 10.0 mg Water tap water 1000 ml Nutrient pH after the sterilization was 7.0 - 7.2. The culture was carried out at 33 ° C, the stirrer was turned at 600 rpm and the aeration was carried out at 6 l per minute. Palm oil was used as an antifoaming agent.

The production of antibiotics started between 26 and 30 hours of fermentation and the highest value was reached after 100 hours of fermentation. At this time, the total level of antibiotic in the fermentation broth was 420 U / ml compared to a standard sample of sisomycin. ___..__..-...___..._....._... í ..._ __.... .. ....._... .._..... .We , _. ...-....., _.._._ POOR QUALITY 10 15 20 25 30 35 40 8004746-7 3) The following nutrient medium was inoculated with a vegetative culture prepared with Micromonospora danubiensis JMS-3 (MNG 00171) on a flat shaker table in the manner set forth in Example 1: Starch 25.0 g soybean meal hydrolyzate 15.0 q (NHu) 250u 2: 0 9 NaCl 3.0 g ZnSO 4 0.5 g CaCO 3 8.0 g Water tap water 1000 ml Nutrient pH after sterilization was 7.0 - 7.2, the quantity 6 l (in a 10 l glass culture vessel). The hydrolysis of the soybean meal was performed with a bacterial protease (NOVO) at 60 ° C for 30 minutes and at pH 7.5.

After inoculation, the culture was carried out at 28 ° C, the stirrer was rotated at 400 heat per minute, the aeration was carried out at 6 l per minute and palm oil was used as antifoaming agent. 600 ml of the inoculum developed over 40 hours was used to inoculate the following culture medium: Soybean meal hydrolyzate 50.0 g Starch 10.0 g Dextrin 25.0 g CaCO 3 5.0 g Sucrose 10.0 g CoCl 2 .6H 2 O 4.5 mg Water tap water 1000 ml The pH of the nutrient medium after sterilization was 7.0 - 7.2, the quantity 6 l (in a 10 l glass culture vessel). The hydrolysis of the soybean meal was performed with bacterial protease (NOVO) at 60 ° C for 30 minutes and at pH 7.5.

After inoculation, the culture was carried out at 31 ° C, the stirrer was rotated at 600 rpm, the aeration was carried out at 6 l per minute and palm oil was used as antifoaming agent.

The production of antibiotics started between the 26th and 30th hour of fermentation and the highest value reached 10 100 20 25 30 35 40 9 aoo4746-7 was reached after 100 hours of fermentation. At this time, the total level of antibiotics in the fermentation broth was 430 U / ml compared to a standard sample of sisomicin. 4) The following culture media in an amount of 200 l (300 l stainless steel pre-culture tank) were inoculated with a vegetative culture prepared with Micromonospora danubiensis JMS-3 (MG 00171) in a glass culture vessel in the manner set forth in Example 2: Soybean meal 10, 0 g Starch 10.0 g Sucrose 10.0 g CaCO 3 4.0 g Water tap water 1000 ml The pH of the nutrient medium after sterilization was 7.0 - 7.2. Palm oil was used as an anti-foaming agent.

The culture was carried out at 31 ° C, the stirrer was rotated at 180 rpm and the aeration was carried out at 200 l per minute.

The inoculum developed over 36 hours was used to inoculate a fermentation medium having the following composition (volume 2000 of the medium): Soybean meal 30.0 g Maize flour 7 25.0 g CaCO 3 5.0 g CoCl 2 .6H 2 O 10.0 mg Water tap water 1000 ml The pH of the nutrient medium after sterilization was 7.0-7.2. The cultivation was carried out at 33 ° C, the stirrer was rotated at 220 rpm, the aeration was carried out at 2 mA per minute and a mixture of soybean oil and palm oil (50% - 50%) was used as antifoaming agent.

The production of antibiotics started between the 26 and 30 hours of fermentation and the highest value was reached after 100 hours of fermentation. At this time, the total level of antibiotic in the fermentation broth was 380 U / ml compared to a standard sample of sisomicin.

The fermentation was completed and 288 g of pure sisomicin base were obtained in a manner known per se, which also contained bound water. ~ š;% ë ()] ïi <2¶} 3 & §¿ï: aoo474s-7 w Characteristics of the product: melting point 198 - zo1 ° c, [e] D + 1ss ° (e = 0.3, 1 water) .

Elemental analysis: 7% C% H% N calculated for monohydrate: 49.80 8.2 14.95 Found: 49.15 8.37 15.15 IR spectrum (KBr): vOH, NH 3170 - 3360, vCH = COC 1690, vCOC 1060 cm_1.

PMR spectrum (n2o) = a1.2o (Me-4 ", e, sn), 62.50 (Me-N-3", S, 3H), 32.56 (HB ", d, J2II, 3II = 10 Hz, 10 1H), 63.17 (H-6 ', bs, ZH), 63.30 (hex--5 ", d, Jgem = 12 Hz, 1H), 63.80 (H-2", then, J2 ", 3" = 10 Hz, J1 fl, 2fl = 4 Hz, 1H), 64.04 (He-5 ", d, Jgem = 12 Hz, 1H), 64.88 (H-4 ', bt , 1H), 65.09 (H-1 ", d, J1II, 2II = 4 Hz, -1H), 155.35 (H-1 ', d, J1., 2. = 2 Hz, 1H) ppm .

Mass number of the characteristic ions (m / e): 447, 332, 304, 160, 145, 127, 118, 110, 100.

Preparation of sisomicin sulphate 20, 15 g of pure sisomicin base were dissolved in 60 ml of ion-free water and to the solution was added dropwise such an amount of 5N-H 2 SO 4 that the pH became 4.3. Then 1.5 g of activated carbon was added to the solution and the mixture was stirred for 30 minutes, after which it was filtered on a Seitz sheet. The filter was washed with ion-free water (3 x 50 ml) and the combined filtrate was added to 1 l of methanol with continuous stirring. The solution containing precipitate was allowed to stand in a cold room, after which the precipitate was filtered off.

The filtered sisomycin sulfate was washed with methanol (3 x 50 mL) and dried in vacuo at 50 ° C over Pgos to constant weight. but in this way obtained 22 g of sisomicin sulphate, 40

Claims (8)

11 8004746-7 Patent claims Method of preparing sisomycin and pharmaceutically acceptable salts thereof, characterized in that a Micromonosponadanubiensis strain MNG 00171 (NCIB 11566) is fermented in a nutrient medium containing assimilable carbon and nitrogen sources as well as mineral salts, since the sisomicin base has been isolated from the fermentation broth. 2. A method according to claim 1, characterized in that starch, dextrin, sucrose, corn flour, soy flour, barley casting liquid and hydrolyzed soy flour are suitably used as sources of carbon and nitrogen. 3. A method according to claim 1, characterized in that the cultivation is carried out at a temperature between 25 and 37 ° C, preferably between zs and 33 ° c. 4. A method according to claim 1, characterized in that ammonium, cobalt and zinc salts are used as mineral acid salts. 5. A method according to claim 1, characterized in that the sisomicin base isolated from the fermentation broth is converted to the sulphate. Actinomyces species Micromonospora danubiensis. Actinomyces species Micromonospora danubiensis MNG-00171 (NCIB 11566). 8. A method according to claim 1, characterized in that a nutrient medium containing soybean meal, barley casting liquid, corn flour, starch, sucrose, calcium carbonate, cobalt chloride and palm oil is used.