DE1180890B - Process for the production of a new growth substance - Google Patents

Description

Process for the production of a new growth substance in patent 1 123 436 new growth substances, the ferrioxamines, are described. You will, among other things obtained by fermentation of actinomycetes. This is usually a mixture obtained from various components, for example from Streptomyces pilosus Ettlinger et a1. NRRL 2857 (ETH 21748) the ferrioxamines A, B, C, Dl, D2, E, F and G.

Surprisingly, new strains of actinomycetes have now been found, which only produce ferrioxamine E. This is a big advantage, because because of their close relationship, the ferrioxamines are relatively heavy from one another to separate. For the sake of exact dosage and repeatability, however, it is desirable for the manufacture of remedies a single compound available.

Ferrioxamine E is a compound of the formula In paper electrophoresis in 0.33 n-acetic acid at 220 V, it migrates 3.9 cm within 41/2 hours (fructose also 3.9 cm). The paper chromatogram in the system n-butanol-glacial acetic acid-water (4: 1: 5) has an Rf value of 0.68, in the system tert-butanol-water-saturated aqueous sodium chloride solution-0.1 N hydrochloric acid (50 : 25: 25: 1), paper impregnated with acetone-water-saturated aqueous sodium chloride solution (6: 3: 1) has an Rf value of 0.59. The partition coefficient in the system n-butanol-benzyl alcohol-water-saturated aqueous sodium chloride solution-0.1 n-hydrochloric acid (200: 100: 300: 60: 3) is 1.593.

Ferrioxamine E is a growth substance for various microorganisms and can therefore be used in the breeding of these organisms to increase the yield. As an example in FIG. 1 shows the growth substance effect of ferrioxamine E on Mikrobacterium lacticum ATCC 8181. The incubation time in hours is plotted on the abscissa and the extinction of the culture solution is plotted on the ordinate. Curve I shows the growth without the addition of ferrioxamine E (control), curves 1I, 111 and IV the growth with the addition of 10, 31.6 and 100 y / 1 ferrioxamine E. It also has pronounced anti-anemic properties and can accordingly be in the form of Preparations are used as remedies in human and veterinary medicine.

The new ferrioxamine-E producing strains are Streptomyces antibioticus (Waksman et Woodruff) Waksman et Henrici ETH 27083 and Nocardia brasiliensis (Lindenberg) Castellani et Chalmers ETH 27413; also strains of the species Streptomyces albus (Rossi-Doria emend. Krainsky) Waksman et Henrici, such as ETH 24454 and ETH 28481. They are in the inventor's laboratories and at the Federal Institute of Technology, Institute for Special Botany, Zurich, kept under the name given.

Streptomyces antibioticus 27083 was obtained from a soil sample from the argentinian gran chaco isolated. The organism has the following characteristics on 1. The spores are smooth, ellipsoidal, 0.7 to 2.2 - 0.4 to 0.8 #L in size; in the electron microscope one sees numerous degenerate, dumbbell-shaped spores, singly or in chains can occur along with healthy spores.

2. The aerial mycelium is initially white-gray or brown-gray in maturity Ash gray condition (cinereus). 3. The spore chains are monopodial branched, with long, straight or more or less wavy branches; there were no spirals observed.

4. Melanin formation occurs on peptone-containing culture media; the Tresner-Danga reaction (T r e sn e r and D a n g a, J. Bacteriol., 76, p. 239 [1958]) is positive.

5. The substrate mycelium is, depending on the nutrient medium, light yellow, yellow-brown, golden yellow to dark brown with a little greenish-brown tint.

For further characterization, the growth of S. antibioticus 27083 described on different culture media.

The nutrient media 1 to 5 were according to W. L i n d e nb ein, Arch. Mikrobiol., 17, p. 361 (1952); Nutrient medium 6 according to H. D. T r e s n e r and F. D a n g a, J. Bacteriol., 76, p. 239 (1958); 7 and 8 according to T. G. P r i d h a m et al., Antibiotics Annual 1956/1957, p.947; 10 according to Manual of Methods of Pure Culture Study of Bacteria, Committee on Bacteriological Technic of S.A. B. Biotechn. Publications, Geneva N.Y. 44-9-10 (1946); Nutrient medium 9 has the following composition: 1 g tryptone, 1 g yeast extract (Difco), 1 g meat extract (Lab-Lemco), 8.5 g sodium chloride, 17 g agar, 1000 ml a. d.

1. Synthetic agar Growth thinly veil-like to wrinkled, yellow-brown to golden yellow; with a little dusty, chalk-white aerial mycelium; The substrate is not discolored. 2. Glucose-asparagine-agar growth thinly veil-like, greenish-yellow to olive-green; Aerial mycelium dusty or velvety, less often not very woolly, light gray or white-yellow; The substrate is not discolored.

3. Gelatin tinge (27 ° C) Initially pustular, light brown surface growth (Pellicle and ring), later basic growth light brown or light yellow; Substrate dark brown discolored and gelatin liquefied up to 25 mm after 10 days; Aerial mycelium is absent.

4. Starch plate Growth thin, veil-like, white-yellow to yellow-brown or light brown; Aerial mycelium dusty, chalk white or milk white; Substrate will not discolored; Starch hydrolysis 2 mm after 5 days, 5 to 15 mm after 10 days.

5. Litmus milk initially white-yellow, later dark brown to blue-black Ring growth; Aerial mycelium is absent; Substrate discolored dark brown; pH after 5 days and after 10 days 8.1.

6. Peptone-Iron-Agar Growth thin, veil-like, white-yellow; no aerial mycelium; very strong positive reaction (black discoloration of the medium).

7. Yeast extract agar growth thin veil-like to slightly wrinkled, dark brown; Aerial mycelium velvety, ash gray; Substrate discolored dark brown.

B. Carvajal's oatmeal agar Growth thinly veil-like or little wrinkled, greenish yellow or light reddish brown; Aerial mycelium velvety or woolly, ash gray; The substrate is not discolored. 9. Chromogen agar growth thinly veiled or pustular, white-yellow, with dusty, chalk-white aerial mycelium; The substrate turns dark brown discolored (melanin formation).

10. Nitrate broth surface growth and flaky sediment, white-yellow; occasionally punctiform, light yellow colonies with little dusty, ash-gray aerial mycelium in the ring; Substrate discolored yellow-brown to light brown; Nitrate reduction pretty weak. Nocardia brasiliensis 27413 was derived from one on the society island of Bora-Bora (French Oceania) collected soil sample isolated.

Morphologically, the trunk is characterized by a strongly branched, wavy, initially not segmented, about 0.6 to 1.1 g, broad substrate mycelium the end. It only develops after a few days - depending on the culture conditions a monopodially branched aerial mycelium that gradually grows into a woolly coating. The substrate mycelium breaks down into parts of different lengths from 1 to 15 l in length and also forms 1.2 to 1.5 u long segmentation pores; in aerial mycelium could last a long time Chains of the same segmentation pores are found.

Of the in W a k s m a n (Bergey's Manual, VII, p. 713 [1957]) and at K r a s s i l n i k o w (diagnostics of bacteria and actinomycetes, Jena, 1959) mentioned species of the genus Nocardia (or Proactinomyces Jensen 1934) are not correct coincides with strain 27413. The trunk, on the other hand, has the characteristic properties of the species Nocardia brasiliensis (Lindenberg) Castellani et Chalmers in 1913 (cf. G o r do n et al., J. Bacteriol., 73, p. 15 [1957]; B o j a 1 i 1 et a1., J. Bacteriol., 78, p. 852 j1959]; B o j a 1 i 1 et a1., Mycopathologia et Mycologia Appl., 11, P. 287 [1957]: B u r n s et a1., Mycopathologia et Mycologia Appl., 11, p.297 [1957]) and is therefore counted among this species.

For further characterization, the growth of Nocardia brasiliensis 27413 described on various nutrient media. The production the nutrient media is the same as given for S. antibioticus 27083 above. 1. More synthetic Agar growth initially thinly veil-like, white-yellow, later somewhat wrinkled and light yellow or yellow-brown; with abundant velvety, at the end woolly, snow-white aerial mycelium.

2. Glucose asparagine agar Growth thinly veil-like, white-yellow to light yellow, later wrinkled and golden yellow; Aerial mycelium woolly, snow-white to partially white-gray.

3. Gelatin tinge (27 ° C) growth superficial, light brown to red-brown; Gelatin is rapidly liquefied (2 cm in 7 days). 4. Starch plate growth point-like, light yellow; Hydrolysis 0.9 cm after 5 days.

5. Litmus milk Point-shaped, colorless to white-yellow ring growth; Peptonization and coagulation; pH 7.25 after 7 days. 6. Peptone Iron Agar Growth thin, veil-like, white-yellow to light yellow; negative reaction.

7. Yeast extract agar growth wrinkled, rarely thin veil-like, golden yellow to light brown; with abundant woolly, snow-white to white-yellow aerial mycelium.

B. Carvajal's Oatmeal Agar Growth thinly veil-like, initially light brown, later light yellow-red and finally red-brown; Aerial mycelium woolly, snow-white to white yellow.

9. Chromogen agar growth thinly veiled, light yellow; no melanin formation.

10. Nitrate broth Initially sediment flakes and white-yellow ring growth, later wrinkled pellicle; Nitrates are reduced to nitrites. Ferrioxamine-E-producing strains of the species S. albus are the strains ETH 28481 and ETH 24454. The allocation of the strains to the species S. albus was based on the five characteristics for species characterization and recognized by the Stockholm Discussion Group (Stockholm 1958, Küster 1959) Species differentiation-1. Spores ellipsoid; 0.6 to 1.4 - 0.4 to 0.9 #L in size, with a smooth surface, rarely a little warty.

2. Aerial mycelium white, light white yellow or light white gray (niveus).

3: Spore chains in short, closed, more rarely in open spirals usually from two to five turns; monopodially branched, occasionally less pronounced Whorls.

4. No melanin formation on peptone-containing culture media and negatives Tresner-Danga reaction. 5. Substrate mycelium mostly white-yellow to light yellow, more rarely yellow-brown or light brown.

More detailed information on the specific characteristics of Streptomyces albus can be found in E. B a 1-d a c c i, Atti Ist. Bot. Pavia Ser. 4, 11, p. 191 (1939); Mycopathologia, 2, p. 145 (1940); J. D u c h 6, Encyclopedie Mycologique, 6, Les Actinomyces du Groupe Albus, Paul Lechevalier et Fils, Paris, 1934; R. H ü t t e r, Arch. Mikrobiol., 38, p. 367 (1961).

The present invention relates to the use of Streptomyces antibioticus ETH 27083, Nocardia brasiliensis ETH 27413 or Streptomyces albus ETH 24454 or ETH 28481 for the biochemical production of ferrioxamine E.

The said strains are in aqueous, a carbon and nitrogen source as well as containing inorganic salts and optionally growth-promoting substances Nutrient solution grown aerobically until it shows a significant ferrioxamine effect and ferrioxamine E isolated thereon. For the cultivation of the mentioned microorganisms as an assimilable source of carbon come carbohydrates, e.g. B. glucose, sucrose, Lactose, mannitol, starch as well as glycerin are in question. As nitrogenous nutrients and, where appropriate, growth-promoting substances may be mentioned: amino acids, peptides and proteins and their breakdown products, such as peptone or tryptone, fine meat extracts, water-soluble fractions of cereal grains, such as maize and wheat, of distillation residues alcohol production, yeast, seeds, especially the rapeseed and soy plants, of the cotton plant, etc., but also ammonium salts and nitrates. From other inorganic The nutrient solution can salt, for example, chlorides, carbonates, sulphates of alkalis, Contain alkaline earths, magnesium, iron, zinc and manganese.

The cultivation takes place aerobically, for example in resting surface culture or preferably submerged with shaking or stirring with air or oxygen in shaker bottles or the known fermenters. A suitable temperature is, for example, between 18 and 40 ° C. The nutrient solution generally shows a significant ferrioxamine effect after 2 to 10 days. 0.1% ferric chloride is added to the culture and the mycelium is separated from the culture filtrate, after which the majority of the ferrioxamine is found in the culture filtrate. Nevertheless, significant amounts of it remain adsorbed on the mycelium. It is therefore advantageous to wash out the latter well. For example, water and / or aqueous organic solvents, such as alcohols, e.g. B. aqueous methanol.

For the isolation of ferrioxamine E from the culture filtrate one can proceed according to methods known per se and z. B. one of the following Use working methods or combinations thereof.

1. One can use adsorbents, e.g. B. activated carbons such as Norit, activated earths such as frankonite, fuller's earth or floridin, or resin adsorbers such as Asmit. The adsorbates are expediently eluted with mixtures of with water miscible organic solvents with water, e.g. B. with water-methanol, water-pyridine, dilute acetic acid-methanol or water = methanol-glacial acetic acid-butanol mixtures. As particularly advantageous for the elution of a frankonite or noritol adsorbate the mixture of water (4 parts by volume) and pyridine (1 part by volume) has proven itself.

2. A second method of separating the ferrioxamine is to that it is adsorbed on cation exchangers, especially those containing acid groups Resins such as Dowex 50 are suitable. The latter can be in the acid form as well can be used in the sodium form, but a mixture of these two has also been found Tried and tested forms. The elution is expediently done with acidic agents, e.g. B. with methanolic hydrochloric acid or acidic buffer solutions.

3. Next, the ferrioxamine can be an aqueous solution by means of organic Solvents are removed. For this extraction process, higher organic alcohols, e.g. 13. Benzyl alcohol or isopropyl alcohol, particularly suitable. Appropriately, the aqueous phase is an inorganic salt, for. B. ammonium sulfate or sodium chloride, added. From the obtained organic extracts that can Ferrioxamine either by evaporation of the solvent or by means of precipitation a suitable organic solvent, e.g. B. ether, petroleum ether or ethyl acetate, can be obtained in enriched form.

4. Another method of enriching the ferrioxamine is in that it is between an aqueous solution and a solution of phenol in Chloroform distributed, both the pH of the aqueous Solution as the phenol content of the chloroform solution can also be varied. Is one understood by that Partition coefficient of ferrioxamines the ratio of concentration in the organic phase for concentration in the aqueous, it is found that the partition coefficient with increasing phenol content of the organic phase increases and with decreasing pH the aqueous phase decreases. Since it is thus possible to use any arbitrary distribution coefficient Setting of ferrioxamines in this system can be achieved by combining Separate a large part of inactive impurities in a few distribution operations.

5. Another method of enriching and / or separating the ferrioxamine represents the chromatography, like adsorption chromatography on various Materials, e.g. B. Norit, aluminum oxide, magnesium silicates, silica gel, calcium sulfate, as well as partition chromatography with cellulose, starch, silica gel, celite and the like. as carrier substances, or chromatography on ion exchange resins.

6. Furthermore, the ferrioxamine can be distributed by countercurrent distribution according to C r a i g can be enriched between two immiscible solvent phases. The following Solvent systems have proven particularly useful: a) Benzyl alcohol - 20% aqueous solution of ammonium sulfate.

b) n-butanol (100 parts by volume) - benzyl alcohol (200 parts by volume) - 1 n-hydrochloric acid (6 parts by volume) - water (300 parts by volume) - aqueous, saturated at 19 ° C Sodium chloride solution (60 parts by volume).

The invention is described in the following examples. The temperatures are given in degrees Celsius. Example 1 The strain Nocardia brasiliensis (Lindenberg) Castellani et Chalmers 27413 is grown in a well-ventilated submerged culture at 27 °. A nutrient solution is used that contains 20 g of soybean meal per liter of tap water (full fat) and contains 20 g of mannitol. The pH is adjusted to 7.8 with potassium hydroxide. After 6 days, 10/0 ferric chloride is added to the crude cultures and added filtered off from celite. 20 g of sodium chloride per liter are added to the culture filtrate. The clear solution is extracted several times with a chloroform-phenol mixture (1 ml: 1 g).

800 mg of chloroform-phenol extract (from 1 1 culture filtrate) are one Countercurrent distribution according to C r a i g subjected. A solvent system is used for this from 31 n-butyl alcohol, 1.51 benzyl alcohol, 4.51 0.001N hydrochloric acid and 0.9 l saturated aqueous sodium chloride solution. The substance is in the first two glasses of the Apparatus filled and the distribution carried out over 60 stages. In the political groups 0 to 2 and 51 to 60 accumulate brown inactive impurities while A uniform red zone develops with a maximum in level 37. The colorimetric Evaluation at 430 m # L gives a curve corresponding to a uniform connection. Antisideromycin activity can be detected in levels 25 to 45 (maximum 36 to 39) (modified Bonifas test). The distribution maximum corresponds to a distribution coefficient K = 1.593.

Fractions 30 to 43 of the distribution are pooled. The lower one Phase is separated off and washed twice with ether. The upper phase staggered with ether and shake out three times with water. The extracts washed with ether are combined with the previously separated lower phase.

Now the combined extracts are saturated with table salt and with extracted from a mixture of equal parts of phenol and chloroform. The extract is dried with sodium sulfate, mixed with three times the volume of ether and shaken out three times with water. The red-brown ferrioxamine E goes completely into the aqueous phase. Practically pure ferrioxamine E can be passed through from this Gain evaporation in vacuo. The residue is in about 20 ml of hot methanol dissolved and evaporated to 4 to 5 ml. When standing in the cold, pure crystallizes Ferrioxamine E in very fine red-brown shiny needles that show their birefringence lose at 280 ° and char on further heating without melting. the Yield is 28 mg. Example 2 The Streptomyces antibioticus strain (Waksman et Woodruff) Waksman et Henrici 27083 is grown in a well-ventilated submerged culture at 27 ° bred. A nutrient solution is used that contains 20 g of rapeseed meal per liter and contains 20 g of malt extract. The pH is adjusted to 7.5 with potassium hydroxide. After 6 days, 10 / ″ ferric chloride is added to the raw culture and added filtered off from celite. The culture fluid is that described in Example 1 Way worked up. It does not contain any other ferrioxamine than ferrioxamine E. This one is, as described in Example 1, isolated. Example 3 The Streptomyces strain albus (Rossi-Doria emend. Krainsky) Waksman et Henrici ETH 28481 is in well-ventilated Submerged culture grown at 27 °. A nutrient solution is used that contains 20 per liter g glucose, 5 g 1-asparagine, 1 g magnesium sulfate, 0.5 g calcium chloride and 1 g secondary Contains potassium phosphate. After 8 days, 10/00 ferric chloride is added to the crude culture and filtered off with the addition of Celite. The culture liquid is in the example 1 described manner worked up. It does not contain any other ferrioxamines than Ferrioxamine E. This is as described in Example 1, isolated.

In the same way, ferrioxamine E is made by cultivating Streptomyces albus (Rossi-Doria emend. Krainsky) Waksman et Henrici 24454 and Streptomyces antibioticus (Waksman et Woodruff) Waksman et Henrici ETH 27083 or Nocardia brasiliensis (Lindenberg) Castellani et Chalmers ETH 27413 on the above synthetic nutrient solution obtain.

Claims (1)

Claim: The use of Streptomyces antibioticus ETH 27083, Nocardia brasiliensis ETH 27413 or Streptomyces albus ETH 24454 or ETH 28481 for the biochemical production of the growth substance ferrioxamine E under aerobic conditions in a common nutrient medium.