HU196844B - Process for producing oligomycin complex and its components by way of fermentation - Google Patents

Abstract

The present invention relates to oligomycin A, wherein R is hydroxy and X is -CH 2 -, oligomycin B - wherein R 1 is hydroxy and X C = O and oligomycin C is \ t wherein, in formula (I), R is hydrogen and X is -CH2- to form an antibiotic-containing oligomycin complex and components thereof by fermentation. Streptomyces diastatochromogenes var. Deposited under the name of NCA1M Β (P) 000366 according to the invention. The sunken culture of the microorganism RO-31 is fermented under aerobic conditions on a medium containing an assimilable carbon and nitrogen source and mineral salts, and the oligomycin complex or components thereof are isolated in a manner known per se from the fermentation broth. -1-

Description

The present invention relates to a process for the preparation of oligomycin-coin complex and its components by the use of Streptomices diastalohromogen, var RO-31.

Oligomycin is known to have been used therapeutically due to the toxicity of the antibiotic oligon iciu, but it is very popular (as a substance in cell metabolism research). wherein in case cftigomicin-A, while oligomycin C, R is hydrogen and R is hydroxy and X -Cllj -C1P X ₂ group, in case of oligomycin B, R is hydroxy and X is C-0 groups - group.

The production of oligomycin by fermentation was first described by Smitli et al., Antibiotics and Chemotherapy 4, 952 (1954). This procedure was improved or refined by Vissner et al., J. of Biochem. And Microbiol. Techn. And Engineering, Vol. 32, 1960 (1960). which is a method not yet applicable to industrial production.

Significant progress was made with Resolution 161,092. The process described in the Hungarian patent. According to the authors, a novel microorganism variant (0 3) was capable of fermentation under appropriate conditions to produce 280,400 pg / ml of oligomycin A component at 125-130 hours. The resulting fermentation broth was able to isolate the desired antibiotic in 50% to 58% yield.

Reviewing the publications dealing with the production of oligomycin, it can be concluded that no process which is economically feasible at the industrial level and which provides high production is known so far.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a process that satisfies the above requirements and enables the production of highly active oligomycin-containing fermentation broths.

• In our experiments, the oligomycin-producing ability of the microorganism Streptomyces diastatochromogenes var 0-3 was examined by varying the components of the medium. Changing several components resulted in an unfavorable effect, however, the surprising fact was that increasing the inorganic phosphate content of the medium not only inhibited the production of oligomycin in the same concentration range as other antibiotics, but also showed a growth inhibitory effect over a certain concentration range.

Thus, it has been found that the inhibitory phosphate concentration has a selectivity effect on productivity, so that maintaining the producing strain in a medium containing 0.1 M or more phosphate does not result in a decrease in productivity as a result of inoculations. observed in all antibiotic producing strains. Moreover, if individual survivors are isolated after plating on growth inhibitory phosphate medium, preferably from 0.1 to 0.2 M, it is highly likely that strains with higher productivity than previously known can be isolated. Thus, the present invention is based on the discovery that, as described above, the known Streptomyces diastatocliromogenesis var. 0-3 microorganisms can be used to produce a highly productive culture. The strain obtained in this manner is Streptomyces diastatochromogenses var. It is named RO-31 and is deposited with NCA1M B (P) 00366 in the National Collection of Agricultural and Industrial Microorganisms.

Streptomyces diastochromogenes var. Morphological and physiological characteristics of the microorganism RO-31 - known from Streptomyces diastatochromogenes var. Compared with the corresponding characteristics of strain 0-3, the following is described.

(a) Morphological and physiological characteristics:

1. Growth on nutrinet medium (composition: Lab-Lemco powder medium 1.0 g / l, yeast extract 2.0 g / l, peptone 5.0 g / l, sodium chloride 5.0 g / l, agar) , 0 g / liter, pH: 7.4) According to Waksman:

003: Medium colonies, rich gray aerial mycelium

RO-31: Medium-sized, tan tan, no aerial mycelium

2. Growth in complete medium containing 0.2 moles of phosphate (composition: 1.0 g / l yeast extract, 1.0 g / l beef extract, 2.0 g / l tryptone, 0.015 g / l iron (II) sulphate, 7.12 g / l disodium hydrogen phosphate, 3.63 g / l potassium dihydrogen phosphate, 10 g / l glucose, 20 g / l agar):

0-3: no growth

RO-31: Brown pigment, no aerial mycelium

3. Gelatin Dispensing (according to Waksman):

0-3: Disburses

RO-31: Disburses

(b) Exploitation of coal

Data on the utilization of different coal sources are given in Table I.

Table I

Coal Source 0-3 RO-31

Glucose +++ ++

Sucrose +++ +++

Glycerin + - +++ ++++

Fructose - ++

Legend to Table I:

- no growth ++ poor growth (colony <20) +++ medium growth (colony 20 200) ++++ strong growth (colony> 200)

Based on the above differences, Streptomyces diastatochromogenes var. Torso of RO-31 Streptomyces diastatochromogenes var. 0-3, particularly with regard to its differential growth in phosphate-containing medium.

The present invention relates to a process for oligomycin A: wherein in formula (I), R is hydroxy and X is -CH ₂ - group - oiigomicin B - wherein R is hydroxy and XC = O in formula (1) - and oligomycin containing components and components of the fermentation complex preparation procedure - oligomycin C - where R is hydrogen and X is -CH ₂ group in formula (I). In accordance with the present invention, Streptomyces diastatochromogeneses var. The submerged culture of RO-31 is aerobically fermented on a medium containing assimilable carbon and nitrogen sources and mineral salts, and the oligomycin complex or components thereof are isolated from the resulting fermentation broth in a known manner.

Streptomyces diastatochromogenes var. The microorganism strain RO-31 is constructed so that Streptomyces diastatochromogenes var. Microorganisms 0-3 are plated on solid medium containing 0.08 0.2 Μ (1-2 wt.) Major wood anion, the surviving individuals are inoculated in at least three consecutive cases on the same medium, and the productivity of the isolates thus obtained is checked in shake cultures and Microorganisms having activity greater than 900 Mg / ml are selected.

Preferably, the known Streptococcus moles diastatochromogenes var. The spores of strain 0-3 were suspended in sterile physiological saline. The suspensions were filtered and homogenized to remove aerial mycelium and media debris, and then diluted with sterile physiological saline.

The diluted spore suspension is plated on yeast, meat extract, tryptone, ferrous sulfate, glucose-containing agar containing increasing amounts of phosphate and incubated.

At the end of the incubation period, inoculate an aliquot of the cultures containing the most phosphate on medium agar made from the same composition as above, and then incubate for the necessary time.

50 ml of medium was sterilized in duplicate inoculum in an 300 ml Erlenmeyer flask containing organic nitrogen and carbon sources and inorganic salts, and incubated with parallel inoculation at + 5 ° C until use. Cultivation on a shaking table at 28 ° C for 5 days. This culture contained an oligomycin content of 980 mg / ml. The drug content of the broths was determined by agar diffusion biological measurement. Aspergillus nigeT strain is used as test organism.

The broths are known in the art, e.g. as described in U.S. Pat.

The advantages of the process of the invention may be summarized as follows;

1) The present invention provides economically efficient antibiotic production that is easily reproducible on an industrial scale.

2) The use of the present invention does not require extremely large and laborious strain selection and breeding experiments to produce high antibiotic content fermentation broths.

3) The process according to the invention produces a fermentation broth with a significantly higher concentration of antibiotics than hitherto known and maintains a high productivity of the microorganism used in a controlled manner over a long period of time.

4) Because of its high antibiotic content, the fermentation broth obtained by the process of the present invention is easier and more economical to process than the previous processes.

The process of the invention is illustrated by the following example.

Example

(a) preparation of Streptomyces diastatochromatogenes var RO-31;

Streptomyces diastatochromatogenes var. 0-3 in physiological sodium chloride solution containing 10 ⁷ spore suspension 5xl0 ⁷ viable spores per ml of the following composition is cast into Petri dishes onto solid medium (sign; I) plated on:

Yeast extract (Oxoid brand) 0.1%

Meat extract (Oxoid brand) 0.1%

Tripton 0.2%

Iron (II) sulfate 0.00003%

Glucose 1.0%

Agar 2.0%

The medium was adjusted to pH 7.0 before sterilization. After sterilization, add the separately sterilized phosphate buffer to the still hot medium, prepared as follows:

KHjPO ₄ M and 1 M Na ₂ HPO _{4 are} mixed so that the pH of the buffer is checked before instrument sterilization. This solution is sterilized and added to the hot agar medium. By this method, the following phosphate concentrations are prepared: 0.01 M, 0.02 M, 0.05 M, 0.2 M, 0.5 M, control.

The solidified medium is plated on Streptomyces diastatochromatogenes var. 0-3 spore suspension and incubated at 28 ° C for 5-7 days. Well-developed colonies are inoculated on inclined agar of the same composition and incubated for 5 to 7 days.

Inoculation of the overgrown cultures is repeated two more times using the above procedure. One copy of the isolates obtained on the following 4-4 slant agar was inoculated with 50 ml of medium (mark II) in a 300 ml Erlenmeyer flask:

Hazelnut flour 1%

Hydrolyzed casein (10%) 1%

Sodium chloride 0.45%

Calcium carbonate 0.5%

Glucose (sterilized separately) 3.0%

If necessary, the pH of the medium is adjusted to 7.0 with sodium hydroxide solution and sterilized by autoclaving at 121 ° C for 20 minutes. After inoculation, cultures are incubated at 28 ° C on a rotary shaker with a rotation of 6 cm in a circular motion and a sterile sample is taken for biological values at 96 and 120 hours. The results are shown in Π. in Table. II. M represents the molar concentration M. II. The data presented in Table 1 are for a sample taken at 120 hours. All 4 colonies were isolated from 0.2 M phosphate medium, and colonies with less phosphate medium were randomly isolated.

Table 11

Phosphate content of culture medium (M) Outgrown Isolated Average colonies colonies production number Mg / ml Maximum production Mg / ml 0 3x 10 ⁷ 36 200 280 0.01 2 x 10 ⁷ 34 230 280 0.02 4x 10 ⁴ 46 350 420 0.05 3x 10 '52 680 760 0.2 4x10 ° 4 980 1200 0.5 0 0 0 0

196 844

(b) Preparation of fermentation broths containing the oligoinicin complex in an experimental fern:

The isolated and assayed Streptomyces diastatochromogenes var. RO-31 strain (MNG 00366) was inoculated with 250 ml of tap water sterilized in a 750 ml Erlenmeycr flask (Medium Mark; III);

Hazelnut flour 1%

Hydrolyzed casein (10%) 1%

Sodium chloride 0.45%

Calcium carbonate 0.5%

Glucose (sterilized separately in 50% solution) 1.5%

The pH of the medium was adjusted to 6.87.0 with sodium hydroxide solution and sterilized in an autoclave at 121 ° C for 30 minutes. The culture was performed on a flat shaker at 28 ° C for 48 hours. The resulting vaccine is a microscope! a rich, filamentous increase in the formation of colonies can be observed. The 250 g of the resulting inoculum was sterilized with 5 liters of medium (denoted IV) for 15 minutes at a temperature of 121 liters in a laboratory test flask with a total volume of 7.5 liters:

Hazelnut flour 1%

Hydrolyzed casein (10%) 1%

Sodium chloride 0 45%

Calcium carbonate 0.5%

Glucose (sterilized separately in 50% solution) 4.5%

Poly (propylene glycol) 0.4%

The medium was adjusted to pH 6.8 to 7.0 with sterile sodium hydroxide solution before sterilization.

At 120 ° C, agitation at 400 rpm and aeration at 7.5 liters / min was carried out for 120 hours. Oligomycin production was then determined by biological assay using the Aspergillus niger test organism.

Result: 960 µg / ml.

The cells of the microorganism are separated from the oligomycin-containing culture medium by centrifugation. The supernatant is substantially free of active ingredient and can be discarded. The wet mycelium (about 800-1000 g in weight) is washed thoroughly several times with tap water (about 22-25 liters for 5 liters of fermentation broth) and filtered under a water jet vacuum. The wet mycelium was then extracted with 500 ml of acetone in a known manner with vigorous stirring for about 1 hour and allowed to stand overnight. The mycelium is then filtered and, preferably, extracted at least two more times with 500 ml to 500 ml of acetone. The aether extracts were combined and concentrated under reduced pressure at 40 ° C to an aqueous residue. The aqueous residue was extracted with ethyl acetate (4 x 300 mL). The ethyl acetate extracts were combined, treated with 1% charcoal and dried over sodium sulfate, and the aqueous residue discarded. The clarifying carbon and sodium sulfate were filtered off, the filtrate was concentrated under reduced pressure to a syrup density at 40 ° C and crystallized from diethyl ether (5 mL) with hexane (300 mL). The precipitated crystals were allowed to stand overnight at room temperature, then filtered and washed with a little hexane. In this way, 2.9-3.1 g of a 95% pure crystalline oligomycin complex was obtained from 5 liters of fermentation broth (according to HPLC):

Oligomycin-A 70-75%

Oligotnicin-B 10-15%

Oligomycin-C 10-15%

The total composition may vary slightly during fermentation.

c) Separation of the individual components of the oligomycin complex:

The individual components of the oligomycin complex are separated by chromatography according to the method of Nasamune et al., J. Am. Chem. Soc. 80, 6092 (1958). 2 kg Hyflo supercel is used as the column fill.

The mixture of 13.2 liters of n-hexane, 4.8 liters of dioxane, 1.5 liters of absolute ethanol and 1.7 liters of distilled water is shaken vigorously in a separatory funnel and after 2 hours the lower (aqueous) phase is discarded. Approximately 2 liters of the upper phase is mixed with the column pack and then filled into a chromatography column. 7.1 g of oligomycin complex is applied to the column and the column is eluted with the remainder of the upper phase at 250 ml / 10 min. The eluates were collected in 250 ml fractions and analyzed by thin layer chromatography. Oligomycin C is shown in Figures 21-30. oligomycin A in fractions 35-55. Fractions 1 to 20, 31 to 34, 58 to 60. Fractions 118120 and 118120 do not contain antibiotics. Fractions containing the same components are pooled and the individual oligomycin components are evaporated from the pooled fractions. The following products are obtained:

Oligomycin A: q _ 54.2 (c = 4.4%, dioxane, literature value: -54.5 ^11). The product is homogeneous by HPLC and is identical to the authentic sample (Sigma standard).

Oligomycin B: ^{[α Ρ} n _o = -46.5 ⁰ (c = 0.76%, dioxane, literature value: -46.5 °). The product is homogeneous by HPLC and is identical to the authentic sample (Sigma standard). Oligomycin-C: [α] ²⁰ D = -80.4 ° (c = 3.7%, in dioxane, liter: -80 /) · The product is homogeneous by HPLC and is identical to the authentic sample (Sigma standard) .

d) Strepto, myces diastatochromogenes var., maintained on phosphate medium. RO-31 Productivity Stability Test:

Inclined agar cultures prepared as described in a) were stored at + 4 ° C and inoculated with the incubated medium from each incubator every two months using the procedure described in b) in an experimental fermentor. Cultured as in (b) and isolated the oligomycin complex as described in (b).

The results are shown in Table III. in Table.

III. spreadsheet

Shelf life (month)

Yield g / fermentor 3.05 2.80 3.12. 3.08 __ 3.10

Inclined agar cultures prepared according to the procedure described in a) are stored at + 4 ° C and inoculated once a month into the maintenance medium of the composition indicated in a).

196,844 of the breeding stock described in the previous paragraph. The results are shown in Table IV. in Table.

Table IV

Number of inoculations Yield g / fermenter 1st generation 2.90 2nd generation 2.80 3rd generation 3.12 4, generation 3.05 5th generation 2.85 6th generation 2.95

Claims

Claims (2)

Claims A process for the preparation of oligomycin-A, wherein R in the formula (I) is hydroxy and X is -CH 3 -, oligomycin-B, in which R in the formula (I) is hydroxy and XC = O, and oligomycin-where O is O. ) for the preparation of an oligomycin complex containing hydrogen and X -CH ₂ - components and their components by fermentation, characterized in that Streptomyces diastatochromogenes var., deposited under NCAIM Β (O) 000366. The submerged culture of RO-3I is aerobically fermented on a medium containing assimilable carbon and nitrogen sources and mineral salts, and the oligomycin complex or components thereof are isolated from the resulting fermentation broth in a known manner. Process according to claim 1, characterized in that the fermentation is carried out at a temperature between 26 ° C and 30 ° C.