GB2078714A - Micromonospora Rosea Species, Cultivation Thereof and Antibiotics Produced Thereby - Google Patents

Abstract

This invention relates to microorganisms of the species Micromonospora rosea and to a nutrient medium containing such microorganisms, in particular strain MNG 00182. The invention further relates to a process which comprises cultivating Micromonospora rosea under aerobic conditions in a nutrient medium containing assimilable sources of carbon and nitrogen and inorganic salts whereby antibiotics, in particular the antibiotic sisomicin, may be produced.

Description

SPECIFICATION Micromonospora Rosea Species, Cultivation Thereof and Antibiotics Produced Thereby This invention relates to a new species of Micromonospora, viz. Micromonospora rosea, and to its cultivationto produce antibiotics, in particularly the hitherto known antiobiotic sisomicin.

Sisomicin (or Antibiotic 66-40) is (0-2,6-diamino-2,3,4,6-tetradeoxy--D-glyero-hex-4- enopyranoyl/1 44/0-(3-deoxy-4-C-methyl-3-(methylamino)-p-L-arobinopyranosyl/1 6fl-2-deoxy-D- streptamini.

Sisomicin is a broad spectrum antibiotic, which has an adverse effect upon the growth of Grampositive and Gram-negative bacteria.

There are several microbiological methods known for its production. It is produced for example as main product by cultivating Micromonospora inyoensis NRRL 3292 (USP 3.932.286 and USP 3.907.771) or together with verdamicin by cultivating Micromonospora grisea NRRL 3800 (USP 3.951.746) or together with Antibiotic G-52 by the cultivation of Micromonospora zionensis NRRL 5466 (USP 3.956.068) or together with gentimicin by cultivating Micromonospora purpurea var.

nigrescens MNG 00122 (Hungarian Patent 1 68.778).

According to one aspect of the present invention we therefore provide microorganisms of the species micromonospora rosea.

According to a further aspect of the invention we provide a nutrient medium containing microorganisms of the species Micromonospora rosea, preferably of the strain Micromonospora rosea MNG 00182 or of a strain derived therefrom.

According to a further aspect of the present invention we provide a process which comprises cultivating Micromonospora rosea under aerobic conditions in a nutrient medium containing assimilable sources of carbon and nitrogen and inorganic salts.

According to a yet further aspect of the present invention we provide sisomicin or a pharmaceutically acceptable salt thereof whenever produced by a process as defined above.

The novel microorganism used according to this invention for the production of sisomicin was isolated from a Hungarian soil sample. This strain, which is different from any other microorganism and has the ability to produce sisomicin has been classified as a new species of Micromonospora and it has been named Micromonospora rosea species nova. From this microorganism the strain S1/109 was obtained with various breeding procedures.

It will be noted that the microorganisms of the invention can be distinguished from those found in nature by virtue of having been cultivated, for example in nutrient media other than natural soil.

Further, some strains of the microorganisms according to the invention have been mutated or modified from the naturally occurring strain. They will in general be substantially free from other microorganisms such as those coexisting in the original soil sample.

A culture of the living organism, Micromonospora rosea S1/109 has been deposited on 12th December 1 978 at the National Collection of Microorganisms in Budapest, where it has been assigned accession number MNG 00 1 82. By its cultivation a fermentation medium of high sisomicin content is achieved.

As mentioned earlier the invention relates to a novel process which comprises cultivating Micromonospora rosea, advantageously the strain MNG 00182, in an aqueous nutrient medium containing assimilable sources of nitrogen and carbon and inorganic salts under submerged aerobic conditions. If desired, the process of the present invention may further comprise the isolation of the sisomicin thereby produced, the separation thereof from the accompanying antibiotics, the purification thereof and/or the conversion thereof to a pharmaceutically acceptable salt.

The deposited microorganism, Micromonospora rosea S1/109 has the microscopic, macroscopic and bio-chemical properties set forth below.

Morphology a/ Macroscopic observations of 10 day old culture incubated at 370C Czapek's Agar show fair growth with no aerial mycelium, no diffusible pigment, well developed, regular, round colonies, orangeblack colour.

b/ Microscopic observations of the organism show long, branched, reguiar filaments without dividing wall which are 0,5 y in diameter. Spores are produced on simple sporophores of 1-1,5 u in diameter and are spherical to ovoid in shape.

Biochemical and Physiological Properties The strain Micromonospora rosea S1/109 shows good growth at 28--37 OC and no growth occurs at 440C.

The utilization of carbon source was tested in a nutrient medium consisting of 0,5% yeast extract, 1% carbon source, 0,1% calcium carbonate, 1,5% agar all in distilled water.

In Table I there are set forth observations on carbon utilization: Table I Carbon Utilization Carbon source Micromonospora rosea Sol/109 Arabinose poor Ribose fair Rhamnose poor Xylose good Fructose good Galactose moderate Glucose good Lactose poor Sucrose good Raffinose moderate-poor Dulcitol moderate-poor Mannitol poor Inositol poor Starch good The nitrogen utilization was tested in a nutrient medium consisting of 1% glucose,1,5% agar and a nitrogen source as indicated in Table II, all in distilled water. In Table II nitrogen utilization is set forth.

Table II Nitrogen Utilization Nitrogen source Micromonospora rosea S 1/109 0,5% Yeast extract good 1% Asparagine moderate-poor 1% Glutamic acid poor 1% Ammonium nitrate poor 1%NZAmine good A growing colony of Micromonospora rosea will hydrolyse gelatin, milk and starch and reduce nitrate to nitrite. The microorganism will tolerate a maximum of 2% sodium chloride in a growth medium.

Cultural Characteristics In Table Ill there are set forth culture characteristics of Micromonospora rosea S1/109 (In describing the color formations for this observations the following reference is employed: Baumanns Farbtonkarte Atlas II. Paul Baumann, Aue 1-SA 87350 LAu 302, GFR).

Table Ill Cultural Characteristics Medium Micromonospora rosea Sol/109 Glucose asparagine agar no growth Pepton iron agar Growth poor; dirty yellow, turning into grey; Co 40. Little spore-layer: Co 63. No diffusible pigment Emerson's agar Growth poor; 1-2 colonies, color: orange, vivid, slightly brownish, 0 126, or Oc 117 Glucose-yeast extract agar Growth: fair; color: vivid orange, turning into black, strongly plicate colonies; Oc 98--0e 120, and later 8 Table Ill (cont.) Cultural Characteristics Medium Micromonospora rosea S1/109 Nutrient agar Growth: fair; color: vivid orange, turning into black; Oc 98-Oc 120, and later 8 Tyrosine agar Growth: very poor; color: grayish white, in some places black; 8, pink soluble pigment, Oc 101 Czapek's agar Growth: fair; color: vivid orange and later black; Oc 97-Oc 120, and later 8 Bennet's agar Growth: fair; color: pale orange, turning into brown; Oc 97Oc 119 or 120, weak brown soluble pigment Potato piug Growth: in traces (without calcium carbonate) color: orange; Oc 96 Potato plug Growth: in traces or (with calcium carbonate) very poor; color: orange; Oc 96-97 and 99 By comparison of the above identifying characteristics of the strain S1/109 with Leudemann's Micromonospora system the strain can be classified as a new species of Micromonospora.

According to a preferred embodiment of the process of the present invention the microorganism Micromonospora rosea S1/109 is cultivated under submerged conditions.

The nutrient medium can contain various assimilable sources of carbon and nitrogen, inorganic salts, trace elements and antifoam agents. Exemplary of assimilable carbon and nitrogen sources, respectively energy sources are soybean meal, soybean hydrolysate, casein hydrolysate, corn steep liquor, yeast extract and different carbohydrates, such as glucose, starch, soluble starch, dextrin and the like. Preferred inorganic salts are for example ferrous, magnesium and cobalt salts. To increase the buffer capacity of the nutrient medium preferably calcium carbonate is added. As antiform agents plant oils can be used (for example palm oil, sunflower oil and soybean oil).

By combination of the various components of the nutrient medium different nutrient broths can be obtained. The medium used for shake flask or inoculum fermentation is different from that used for the main fermentation.

The microorganism is grown most advantageously in a nutrient medium consisting of soybean meal, pepton or casein hydrolysate, potato or corn starch, dextrin or soluble starch, glucose, calcium carbonate, ferrous and magnesium sulfate, cobalt nitrate and palm oil.

Production of sisomicin can be effected between 25--400C, preferably at 28-330C. Agitation is between 200-400 r.p.m. Air input is preferably 1/1 v/v pro minute.

In order to assay the total antibiotic activity (sisomicin and minor antibiotics) during the fermentation Staphylococcus epidermidis is employed as test organism. The assay is run against a standard preparation of sisomicin using agar diffusion method.

The total activity of the fermentation medium in 100-1 30 hours is about 600-700 U/ml (1 U= 1 y9 activity of sisomicin base.) About 85% of the antibiotic material produced in the fermentation medium is sisomicin.

When peak antibiotic activity is attained, the sisomicin may be isolated e.g. by a combination of the steps known in the art. The sisomicin base obtained thereby is, if desired, converted to a pharmaceutically acceptable salt.

The quantity of the sisomicin produced under laboratory conditions is over 700-800 yg/mi and on the pilot plant (a 1 m3 fermentation tank) it is 600-700 yg/ml.

The antibiotic concentration is more than three times that described in USP 3.832.286 which has hitherto been considered the best result. The fermentation medium contains only 15% or less of accompanying antibiotics besides sisomicin.

The following Examples illustrate preferred embodiments of the present invention without serving to limit the scope of protection sought therefor.

Example 1 Add 1 ml of the vegetative mycelia (stored deep-frozen at-200C) of the strain Micromonospora rosea S1/109 (MNG 00182) under aseptic conditions to a 3000 ml Erlenmeyer flask containing 800 ml of the following sterile medium: Tryptone (Oxoide) 5g Soybean meal 10g Soluble starch 209 Glucose 1g Calcium carbonate 29 Tap water to 1000 ml Prior to sterilizing the medium, adjust its pH to 7.0. Incubate the fermentation medium for 3 days at 280C on a rotary shaker (260 r.p.m., 10 cm stroke).With the medium obtained, inoculate 100 liters of the following sterile medium under aseptic conditions: Soybean meal 10g Casein hydrolysate (dry weight) 5g Potato starch 229 Glucose 109 Calcium carbonate 29 Palm oil 29 Tap water to 1000 ml Casein is hydrolysed with pancreatine. Adjust the pH of the medium, prior to sterilizing, to 7.0.

Aerobically ferment for 36-48 hours at 300C, while stirring at 400 r.p.m. with air input 1/1 v/v. Palm oil is used as antifoam agent. Use 10 liters of the medium obtained for the inoculation of 100 liters of a fermentation medium of the following composition: Soybean meal 40g Pepton 10g Potato starch 10g Dextrin 259 Glucose 5g Calcium carbonate 49 Magnesium sulfate (7H20) 29 Ferrous sulfate (7H20) 0.29 Cobalt nitrate (6H20) 0.8mg Palm oil 29 Tap water to 1000 ml Prior to sterilizing the aforedescribed medium, adjust the pH to 8.0. Sterilize for 1 hour at 1 21 "C, under 1.3-1.4 atmospheres pressure with stirring. Use a fermentation tank of stainless steel. Stir at 400 r.p.m. with air input at 1/1 v/v.Use palm oil as antifoam agent.

Antibiotic production starts after 35-40 hours and reaches its maximum after 11 0-1 20 hours of fermentation. The time for finishing the fermentation is determined by a fast turbidimetric procedure applying Klebsiella pneumoniae (F. Kavanagh: Dilution Methods of Antibiotic Assay, analytical Microbiology, Acad. Press, New York, pp. 12 5--1 40, /1963/).

The quantity of the antibiotic produced reaches at the end of the fermentation 650 U/ml (1 U is equivalent to the standard efficiency of 1 y9 sisomicin base as determined with Staphylococcus epidermidis test organism by the agar diffusion method (J.S. Simpson: Analytical Microbiology, Acad, Press, New York, pp. 87-1 24,/1 963/).

Example 2 Under aseptic conditions, add 1 ml of the vegetative mycelia (stored deep-frozen at -200C) of the strain Micromonospora rosea S1/109 (MNG 00 182) to a 3000 ml Erlenmeyer flask containing 800 ml of the following sterile medium: Tryptone (oxoide) 5g Soybean meal 10g Soluble starch 209 Glucose ig Calcium carbonate 2g Tap water to 1000 ml Prior to sterilizing the medium, adjust its pH to 7.0. Incubate the fermentation medium for 3 days at 280C on a rotary shaker (260 r.p.m., 10 cm stroke).With the medium obtained, inoculate 100 liters of the following sterile medium under aseptic conditions: Soybean meal 10g Sucrose 10g Potato starch lOg Calcium carbonate 49 Palm oil 29 Tap water to 1000 ml Adjust the pH of the medium prior to sterilizing to 7.0. Aerobically ferment for 48-60 hours at 300C, while stirring at 400 r.p.m. with air input at 1/1 v/v. Use palm oil as antifoam agent.With the medium obtained inoculate 1 m3 of a fermentation culture medium of the following composition: Soybean meal 409 Pepton 10g Potato starch 10g Dextrin 259 Glucose 5g Calcium carbonate 4g Magnesium sulfate (7H20) 29 Ferrous sulfate (7H20) 0,2g Cobalt nitrate (6H20) 0,8mg Palm oil 29 Tap water to 1000 ml Prior to sterilizing the aforedescribed medium, adjust the pH to 8.0. Sterilize for 1 hour at 1 21 OC, under 1.3-1 ,4 atmospheres pressure, with stirring. Use a fermentation tank of stainless steel. Stir at 400 r.p.m. with air input at 1/1 v/v. Use palm oil as antifoam agent.

Antibiotic production starts after 35-40 hours and reaches its maximum after 1 10--120 hours of fermentation. The time for finishing the fermentation is determined by the fast turbidimetric procedure applying Klebsiella pneumoniae.

The quantity of the antibiotic produced reaches at the end of the fermentation 600 U/ml (agar diffusion method was used).

Example 3 Transfer 100 liters of the inoculum according to Example 1 to 1 m3 of a fermentation medium of the following composition: Soybean meal 40g casein hydrolysate (dry weight) 10g Potato starch 10g Dextrin 259 Glucose 5g Calcium carbonate 49 Magnesium sulfate (7H20) 29 Ferrous sulfate (7H20) 0,29 Cobalt nitrate (6H20) 0,8mg Palm oil 2g Tap water to 1000 ml Hydrolyse casein with pancreatine. Adjust the pH of the medium, prior to sterilizing, to 8.0.

Sterilize for 1 hour at 121 OC, under 1,3--1,4 atmospheres pressure with stirring. Use a fermentation tank of stainless steel. Stir at 400 r.p.m. with air input at 1/1 v/v. Use palm oil as antifoam agent.

Antibiotic production starts after 35 40 hours and reaches its maximum after 11 0-120 hours of fermentation. The time for finishing the fermentation is determined by a fast turbidimetric procedure applying Klebsiella pneumoniae.

The quantity of the antibiotic produced reaches at the end of the fermentation 650 U/ml (agar diffusion method was used).

Example 4 Transfer 100 liters of the inoculum medium according to Example 1 to 1 m3 of a fermentation medium of the following composition: Soybean meal 40g Casein hydrolysate (dry weight) 10g Corn starch 50 g Glucose 5g Calcium carbonate 49 Magnesium sulfate (7H20) 29 Ferrous sulfate (7H20) 0,2g Cobalt nitrate (6H20) 0,8mg Palm oil 29 Tap water to 1000 ml Hydrolyse casein with pancreatine. Adjust the pH of the medium, prior to sterilizing, to 8.0.

Sterilize for 1 hour at 121 OC, under 1 ,3-1 ,4 atmospheres pressure with stirring. Use a fermentation tank of stainless steel. Stir at 400 r.p.m. with air input at 1/1 v/v. Use palm oil as antifoam agent.

Antibiotic production starts after 35 40 hours and reaches its maximum after 110-120 hours of fermentation. The time for finishing the fermentation is determined by a fast turbidimetric method applying Klebsiella pneumoniae.

The quantity of the antibiotic produced reaches at the end of the fermentation 680 U/ml (agar diffusion method was used).

At the end of the fermentation, pure sisomicin is isolated by steps known in the art.

M.p.: 198--2010C, (a)=+ 1 880(c=0,3 water) IR spectrum (KBr): vOH, NH 3170-3360, PCH=COC 1690, PCOC 1060 cm-1 PMR spectrum (D20): 81,20 (Me s, 3H), S2,50 (Me-N-3", s 3H), 2,56 (H-3", d, J2,,3,,=1 0 Hz, 1 H), #3, 17(H-6', bs, 2H), S3,30 (HaX5", d,Jgem=12 Hz, 1H), #3,80 (H-2", dd, J2,310 Hz, J1,2=4 Hz, 1 H), #4,04 (H@-5", d, Jgem=12 Hz, 1H), #4,88 (H-4', bt, 1H),#5,09 (H 1",d, J1,,2rn-4 Hz, 1H),5,35 (H-11, d, J1',2'=2 Hz, 1H) ppm.

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

Example 5 Production of Sisomicin Sulfate Dissolve 15 g of pure sisomicin base in 60 ml of deionised water and adjust the pH to 4.3 with 5N sulfuric acid. Stir the solution with 1.5 g of activated charcoal for 30 minutes and filter with Seitz sheet. Wash the filter three times with 5 mi of deionised water and add the combined filtrates to 1 liter of methanol under vigorous stirring. Store the solution in a cool room and filter the resulting precipitate. Wash the obtained sisomicin sulfate three times with 50 ml of methanol and dry it in vacuum at 500C over phosphorous pentoxide obtaining 22 g of sisomicin sulfate.

Claims (21)

Claims

1. Micromonospora rosea species.

2. Micromonospora rosea MNG 00182.

3. Strains of Micromonospora rosea derived from Micromonospora rosea MNG 00182.

4. A nutrient medium containing microorganisms of the species Micromonospora rosea.

5. A medium as claimed in claim 4 containing assimilable sources of carbon and nitrogen and inorganic salts.

6. A medium as claimed in claim 4 or claim 5 wherein the said microorganisms are of the strain Micromonospora rosea MNG 00182.

7. A process which comprises cultivating Micromonospora rosea under aerobic conditions in a nutrient medium containing assimilable sources of carbon and nitrogen and inorganic salts.

8. A process as claimed in claim 7 wherein the Micromonospora rosea is of the strain Micromonospora rosea MNG 00182.

9. A process as claimed in claim 7 or claim 8 further comprising the isolation from the said medium of antibiotics, including sisomicin, produced by the cultivation.

1 0. A process as claimed in claim 9 wherein sisomicin is separated from the said antibiotics.

11. A process as claimed in claim 10 wherein the sisomicin separated thereby is purified.

12. A process as claimed in claim 10 or claim 11 further comprising the conversion of the said sisomicin to a pharmaceutically acceptable salt thereof.

13. A process as claimed in claim 10 or 11 further comprising the conversion of the said sisomicin to the sulfate salt thereof.

14. A process as claimed in any one of claims 7 to 13 wherein the said carbon and nitrogen sources comprise substances selected from the following: starch, dextrin; sucrose; corn meal; soybean meal; corn steep liquor; and soybean meal hydrolysate.

1 5. A process as claimed in any one of claims 7 to 14 wherein the said inorganic salts comprise salts selected from ferrous, magnesium and cobalt salts.

1 6. A process as claimed in any one of claims 7 to 13 wherein the said nutrient medium comprises: soybean meal; pepton or casein hydrolysate; potato or corn starch; dextrin or soluble starch; glucose; calcium carbonate; ferrous and magnesium sulfates; cobalt nitrate; and palm oii.

17. A process as claimed in any one of claims 7 to 1 6 wherein the said cultivation is effected at a temperature of from 25 to 400C.

1 8. A process as claimed in any one of claims 7 to 1 6 wherein the said cultivation is effected at a temperature of from 28 to 330C.

19. A process as claimed in any one of claims 7 to 1 8 substantially as herein described.

20. A process for the cultivation of Micromonospora rosea substantially as herein described in any one of Examples 1 to 4.

21. Sisomicin or a pharmaceutically acceptable salt thereof whenever produced by a process as claimed in any of one of claims 7 to 20.