DE1180894B - Production and extraction of the antibiotic pristinamycin - Google Patents

Description

Manufacture and Obtainment of the Antibiotic Pristinamycin The present The invention relates to the production of an antibiotic, which will be used in the following No. 7293 R.P. and is called "Pristinamycin".

The antibiotic 7293 R. P. consists essentially of two components, those with certain main components of already known antibiotics, such as streptogramin, Staphylomycin or antibiotic PA 114 (cf. in particular D. C. H o b b s and W. D. C e 1 m e r, Nature, Vol. 187, p. 598 [1960]) are identical. The antibiotic 7293 R. P. possesses the same antibacterial properties as the different ones antibiotics given above.

It has now been found that the antibiotic 7293 R.P. can be cultivated a microorganism identified more fully below, which so far has not yet been described, and with "strain 5647" or Streptomyces pristinaespiralis can produce under artificial conditions.

The said microorganism was found in the Northern Regional Research Laboratory stored where he received the registration number NRRL 2958.

The antibiotic 7293 R.P. is in methanol, ethanol, acetone, ethyl acetate and chloroform soluble. It is slightly soluble in water and insoluble in petroleum ether. There are in the reactions according to Tollens, according to Millon, according to Sakaguchi and according to S e 1 i w a n o f f, with Fehling's solution and with ninhydrin negative tests and with the ferric chloride reaction, the reaction after E h r 1 i c h and the reaction after F o 1 i n - D e n i s positive tests.

The antibiotic 7293 RP contains carbon; Hydrogen, oxygen and nitrogen. Its elemental composition is as follows: C = 61.95 to 62.25%, H = 6.8 to 7%, O = 20 to 20.5%, N = 9.25 to 9.40 % .

The antibiotic has the following physical properties. Appearance. . . . . . . . pale yellow amorphous powder Melting point (Bloc Maquenne) 151 to 153 ° C rotation capacity .. [x] 2D5 = -130 ° (c = 0.50 /, in methanol) [x] δ5 = -1550 ° (c = 1 % in chloroform ) The chromatography of the antibiotic 7293 RP on paper under various conditions shows that this antibiotic contains two active substances that can be isolated by countercurrent distribution. The investigation of these two substances has shown that they are identical to the compounds PA 114 A and PA 114 B described by WD C e 1 mer and BA S obin (Antibiotics'Annual, 1955/1956, p. 437).

The antibiotic efficacy of 7293 R.P. on infections with gram-positive Sprouting is similar to that of streptogramins, staphylomycins and antibiotic PA 114 identical. The two components isolated from the antibiotic 7293 R.P. proved, as already shown for the compounds PA 114 A and PA 114 B, that they have a synergistic effect.

The organism producing the antibiotic 7293 R. P. was from a Earth sample taken from San Carlos (Cordoba) in Argentina isolated. The isolation method is the following: The soil sample taken is immersed in distilled sterile water suspended, and the suspension is diluted to various concentrations. A small volume of each dilution is applied to the surface of Petri dishes, which contain an Emerson nutrient medium or any other suitable medium. After incubation for a few days at 26 ° C, the colonies of the microorganisms that you want to isolate are transferred to agar slants in order to obtain more abundant cultures. in the the following are the morphological characteristics of this new strain, the Strain 5647 is indicated. The breeding characteristics and the biochemical Properties of strain 5647 were determined on commonly used nutrient agar media and examined in commonly used nutrient bouillons. The observations made are given in the table below; concern without any other precise information they are cultures that have been incubated for 2 to 3 weeks at 26 ° C and are at a good stage of development achieved. Microscopic examination The trunk 5647 has the usual structure of Streptomyces. This trunk forms elongated spores with oval to cylindrical ones Shape with more or less rounded ends, the dimensions of which are about 0.4 to 0.6 #t to be 0.9 to 1.2 #t. The spore threads are twisted and bend the most of the time at their end in the form of a hook, or they also form a ring or they roll along with the formation of a very loose, wide-open spiral a single turn or occasionally multiple turns. This type of sporulation was able to do it on agar according to B e n e t t, on agar with oat flour and tomato extract P r i d h a m as well as on glucose-asparagine agar. You allowed this Streptomyces strain in the retinaculum aperture division of the classification of P r i d h a m (Applied Microbiology, 1958, Vol. 6, pp. 52 to 79). Developing temperature Good development at 30 and 37 ° C. Earth odor None or extremely weak. Culture on milk Pure milk At 30 ° C: ring of yellowish-white vegetative mycelium. No mycelium exposed to air. Partial peptonization in 14 days, pH 6.4 to 7.8 going.

At 37 ° C: vegetative mycelium in a yellowish ring. No air exposed Mycelium. Almost complete peptonization in 14 days. pH going from 6.4 to 7.8. Milk with bromocresol At 30 ° C: Vegetative mycelium in a white-gray ring. No air exposed Mycelium. Violet coloration of the bromocresol. The pH ranges from 6.4 to 8.1. Partially Peptonization in 14 days. At 37 ° C: vegetative mycelium in a grayish ring. No air-exposed mycelium. Almost complete peptonization in 14 days. Violet coloring of bromocresol. pH goes from 6.4 to 7.6. Milk with methylene blue: At 30 ° C: Very sparse development. Yellowish ring. No mycelium exposed to air. No change the appearance noticeable. No reduction of the blue. pH goes from 6.4 to 7.1 in 14 days. At 37 ° C: sparse yellowish ring. In 14 days partly flokkung and Start of peptonization. The blue turns green. pH goes from 6.4 to 6.6. Nitrate bouillon Glucose nitrate broth according to W a k s m a n At 30 ° C: White vegetative mycelium in the Ring and parted veil. No mycelium exposed to air. No soluble pigment. Testing of nitrites: strongly positive.

At 37 ° C: white vegetative mycelium in the ring and faint haze; some sedimented flakes. No mycelium exposed to air. No soluble pigment. Testing of nitrites: strongly positive (determination on the 14th day). Synthetic bouillon according to C z a p e k broth with glucose according to C z a p e k At 30 ° C: vegetative white Mycelium in the ring and white veil. No mycelium exposed to air. Soluble pigment: Traces yellowish. Testing of nitrites: positive.

At 37 ° C: white vegetative mycelium in a weak ring and some Colonies on the surface. Very weak yellowish soluble pigment. test of nitrites: positive. No mycelium exposed to air. Broth according to C z a p e k with Sucrose At 30 ° C: White vegetative mycelium in the ring and a thick veil. No air-exposed mycelium. Pale yellowish soluble pigment. Testing of nitrites: Positive (on the 2nd day of breeding).

At 37 ° C: white vegetative mycelium in the ring. Some colonies at the Surface and some sedimented flakes. No mycelium exposed to air. Weak light yellow soluble pigment. Testing of nitrites: strongly positive (on the 2nd day of cultivation). Tryptone Broth At 30 and 37C: Very sparse growth limited to a few sedimented flakes. No soluble pigment. Gelatine Pure gelatine (aqueous 12% solution) At 30 ° C: good development. Veil of greyish-white vegetative Mycelium. Moderate greyish-white mycelium exposed to air. A few flakes numerous penetrate the agar. Light yellow brown soluble pigment in a small amount. Complete Liquefaction in 7 days.

At 37 ° C: ring and some isolated colonies on the surface. Greyish white vegetative mycelium. No aerial mycelium or some faint whitish traces. Yellow light brownish soluble pigment in very small quantities. Complete liquefaction in 7 days. Gelatine with meat extract according to W a k s m a n At 30 ° C: ring of yellowish-white vegetative mycelium. No aerial mycelium. Some submerged flakes. Intense light yellow soluble pigment. Complete liquefaction in 7 days.

At 37 ° C: white-yellowish to dark-yellow ring. Some went into hiding Flakes. No mycelium exposed to air. Intense light yellow soluble pigment. Complete Liquefaction in 7 days. Glucose gelatine according to W a k s m a n Bei 30 'C: ring of yellowish to pink vegetative mycelium. Some flakes will sediment. No mycelium exposed to air. Yellow soluble pigment. Complete liquefaction in 7 days. At 37 ° C: ring of yellowish to pink vegetative mycelium. Some flakes sediment. No mycelium exposed to air. Yellow soluble pigment. Complete Liquefaction in 7 days. Potato Yellow, very folded and crumbling vegetative Mycelium. Moderately whitish air-exposed mycelium. Yellow soluble pigment on the cone of the potato and the liquid underneath. Agar with starch agar with strength A after W a k s m a n At 30 'C: Fairly good growth. Colonies with fairly regular, very little raised edges. Some radial folds from the edge to the center of the colony. Underside brown to black. Gray exposed Mycelium with small white borders. Hydrolysis of starch very weak around the colonies hereabouts. No soluble pigment.

At 37 ° C: growth is not as good as at 30 ° C. Colonies are not very raised, with some radial folds. Underside light brown to dark brown or blackish. Little developed white to grayish air-exposed mycelium only on certain Colonies. A certain number are free of it. Hydrolysis of starch is not very pronounced.

Nutrient agar At 30 ° C: Little abundant development. Pretty big Colonies. Yellow-brownish, very little thick vegetative mycelium with ray-shaped and concentric folds. Underside light yellowish brown. The air-exposed mycelium absent or present in whitish traces on some colonies. Not soluble Pigment.

At 37'C: much the same appearance, but much more abundant growth. Traces of gray sporulation on certain colonies. Agar according to B e n nett at 30 ' C: Strong, irregularly bordered colonies, folded surface, but none radial fold. Beige-yellow vegetative mycelium. Yellowish beige underside to light brown or grayish. Whitish very little developed air exposed Mycelium on some colonies. Gray sporulation and white border on other colonies. No soluble pigment.

At 37 ° C: poorer development than at 30 ° C. Colonies with very wavy surface. Light-brownish yellowish vegetative mycelium. Little developed whitish to grayish airborne mycelium absent from most of the colonies. No soluble pigment.

Glucose-Peptone-Agar Glucose-Peptone-Agar A according to W a k s m a n At 30 ' C: Good development. Large raised colonies with irregular margins that are not press into the agar. Folded vegetative mycelium, tannin-colored. Yellowish underside. No soluble pigment. Neither airborne mycelium nor sporulation.

At 37 'C: As at 30' C, but the edges are even more irregular. Yellowish underside. No soluble pigment. . No mycelium exposed to air. No Cracking. Glucose peptone: Agar B according to W a k s m a n At 30 ° C: large size, in the center raised colonies with fairly regular sharp edges that are not in press in the agar, with radial folds. Light yellowish vegetative mycelium. Light yellowish to light yellowish brown underside. Certain colonies without exposure to air Mycelium; others with white air-exposed mycelium and light gray sporulation in the center. No soluble pigment.

At 37 ° C: similar appearance, but more sparse. No cracks. Agar with tyrosine At 30 ° C: very large, flat, colonies. Vegetative mycelium light brown folded in the center of the colonies. Underside light brownish yellow. On certain colonies air-exposed mycelium and light gray sporulation with a white border. A little light brown soluble pigment. Solubilization of tyrosine very 2 to 4 mm around the colony. At 37 ° C: As at 30 ° C. Synthetic agar according to C z a p e k Agar according to C z a p e k with sucrose At 30 ° C: strong colonies. Yellowish to yellowish brown in all Directions of folded and flaking vegetative mycelium. Underside light yellow brown until blackish-brown. Only some colonies exposed to air and pale gray Sporulation. Clear yellow-brown soluble pigment.

At 37 ° C: similar appearance, but less good development.

Agar according to C z a p e k with glucose At 30 ° C: strong colonies. Dark brown vegetative mycelium. Dark brown, almost black underside. Sublime centers. That vegetative mycelium is folded in all directions and has a tendency to tear. White-grayish air-exposed mycelium in traces, especially around the colonies. Clear brownish-yellow soluble pigment.

At 37 ° C: similar appearance, but less good development. Agar with calcium malate At 30'C: medium development. Colonies with frayed Margins. Underside brownish-yellow to blackish-brown. White air-exposed mycelium in successive borders on the perimeter of the colonies. Center of the colonies raised with gray sporulation. No soluble pigment. Good solubilization of calcium malate within 3 to 4 mm around the colonies. At 37'C: As at 30'C. To the method of P r i d h a m and Gott-1 i e b (J. of Bact., 1948, vol. 56, p. 107 to 114), the strain 5647 was found to have the following as the carbon source Substances evaluated moderately or well: xylose, arabinose, rhamnose, fucose, Glucose, galactose, levulose, mannose, lactose, maltose, sucrose, trehalose, Cellobiose, raffinose, dextrin, inulin, starch, glycogen, glycerin, mannitol, inositol. It does not use: sorbose, esculin, glycol, erythritol, adonit, dulcitol, sorbitol, Ethanol, sodium formate, sodium acetate, sodium oleate, succinic acid, calcium gluconate.

As nitrogen sources, he evaluates: sodium nitrate, sodium nitrite, Ammonium sulfate, adenosine, urea, asparagine, glycocoll, alanine, valine, glutamic acid, Arginine, Lysine, Serine, Threonine, Phenylalanine, Tyrosine, Proline, Hydroxyproline, Histidine. It does not use: acetamide, succinamide, benzamide, creatine, creatinine, methionine, Betaine.

The antibiotic 7293 R.P. is produced by application known methods and consists essentially in the strain 5647 or to grow its mutants on a suitable medium and under suitable conditions and then to separate off the antibiotic formed in the course of the cultivation.

The cultivation of the 5647 strain can be carried out after any aerobic surface cultivation or immersion growth, but the latter is for convenience to prefer.

The fermentation medium is primarily intended to be an assimilable source of carbon, an assimilable nitrogen source and mineral salts and optionally growth factors included, with all of these elements in the form of well-defined products or of complex mixtures such as those found in biological products of various origins encountered, can be supplied. Soy flour and glucose based media are particularly suitable.

The antibiotic 7293 R.P. can be extracted from the fermentation mash customary methods of extraction with solvent can be isolated. It is special It is advantageous to work according to the following procedure: filtering the mash after acidification to about pH 3, neutralizing the filtrate at pH 7, extracting with dichloroethane, Narrowing down and precipitating the raw antibiotic with one the antibiotic bad dissolving solvents such as petroleum ether. The raw antibiotic thus obtained can then be purified by known methods, for example chromatography will.

The following examples illustrate the invention without restricting it. The following is the activity in units per milligram of solid product (U / mg) or expressed per cubic centimeter of solution (U / cm3). The unit is the smallest Amount of product that, in 1 cm3 of a suitable culture medium, stimulates the growth of Staphylococcus 209 P inhibited under certain conditions. Example 1 In an I70-1 fermentation vessel the following components are introduced: enzymatic hydrolyzate of casein .. 1200 g glucose (hydrated). . . . . . . . . . . . . . . . 1200 g meat extract ........................ 360 g tap water q. s. . . . . . . . . . . . . . . . . . 1001 The pH value this medium is adjusted to about 7.2 with 35 cm3 of sodium hydroxide solution. Then will 60 cm3 of soybean oil added.

The culture medium is sterilized by passing it through for 40 minutes of steam at 120 ° C. The pH value is then about 7.1. The medium is then with 250 cm3 of a stirred or shaken Erlenmeyer culture of strain 5647 inoculates. The temperature is kept at 26 to 27 ° C and the ventilation at 5 m3 / hour set. The pH of the medium remains stable for 15 hours, then falls decreases very sharply to reach 4.95 after 22 hours of cultivation and then increases again. The development of the fungus begins around the 15th hour and is 26 hours after inoculation suitable for inoculating the production culture.

The production culture is carried out in an 800-1 fermentation vessel loaded with the following substances: soy flour. . . . . . . . . . . . . . . . . . . . . . 17.75 kg Distiller's Solubles .............. 2.25 kg sodium chloride ................ 4.5 kg tap water q. s ............. 3601 The pH of the medium thus obtained is adjusted to 6.55 with 220 cm 'sodium hydroxide solution (d = 1.33). 1800 cm3 of soybean oil and 2.25 kg of calcium carbonate are then added.

The medium is then made by passing steam through it at 120 ° for 40 minutes C sterilized. After cooling, one sets sterile 501 of a sterile glucose solution, which contains 11.4 kg of glucose hydrate, to the broth.

The pH of the medium produced in this way is 7.1 and the total volume 4501

After adjusting the temperature to 26 to 27 ° C, the medium is inoculated with 401 of the above culture from the 170-1 fermentation vessel. The ventilation is at Maintained 15 m3 / hour.

The pH falls from 7.1 to 6.75 in 4 hours and then rises slowly to reach 6.95 after 24 hours of cultivation. The production of the antibiotic takes place between the 18th and 24th hour of breeding. The fermentation will stopped after 24 hours of cultivation. The activity of the medium is then 1190 U / cm3. Example 2 4201 of the fermentation mash obtained according to Example 1 (Content 1190 U / cm3) are put into a machine equipped with a device for moving Container introduced. The pH is adjusted by adding 6N hydrochloric acid 3 a. Then 18.5 kg of a filter aid are added and the mixture obtained is filtered. The filter cake is washed with 50 liters of water. The filtrate making up 4201 is Adjusted to pH 7 with dilute sodium hydroxide solution and then in a group of countercurrent centrifuges extracted with two extraction stages with 961 dichloroethane. The organic solution is concentrated to 1 l under reduced pressure. The concentrate is made with 101 petroleum ether added, and the resulting precipitate is filtered off, washed and dried. 52.5 g of crude product with an activity of 7600 U / mg are obtained in this way. example 3 50 g of the above crude product (content 7600 U / mg) are dissolved in 2.51 dichloroethane, and the solution thus obtained is filtered and then passed through a column which Contains 0.751 granulated charcoal previously washed with 1.251 dichloroethane. The chromatogram is developed and eluted with 4.51 dichloroethane. The main fraction (2.51) will concentrated under reduced pressure to 250 cm ", the temperature being below 35 ° C is held. The concentrate obtained is filtered and then washed with 3.751 petroleum ether offset. The precipitate formed is filtered off, washed with petroleum ether and dried. This gives 35 g of purified product in the form of an amorphous yellowish yellow Powder with an activity of 8100 U / mg.

Claims (1)

Claim: Process for the production of the antibiotic pristinamycin by biochemical means, d a -characterized that one under usual conditions breeds the microorganism Streptomyces pristinaespiralis (NRRL 2958). Into consideration Drawn pamphlets: "Nature", 1960, p. 598.