DE1184901B - Manufacture of the antibiotic 1415 - Google Patents

Description

Manufacture of antibiotic 1415 C The invention relates to the manufacture, extraction and purification of a new antibiotic designated 1415. This antibiotic is obtained from the culture of Streptomyces Aetnaensis nov. spee. 1415 (NRRL 2836). The cultivation takes place in a fermentative way under aerobic conditions. The antibiotic is obtained by extraction from the mycelium or by adsorption or extraction from the nutrient solutions. The species of Streptomyces was isolated from a volcanic soil sample from Mount Etna. The strain has been deposited under number NRRL 2836 with the United States Department of Agriculture in Peoria, Illinois, USA.

The Streptomyces strain Aetnaensis nov. spec. 1415 shows the following properties: Morphological properties Morphological analysis on platelets in liquid Czapek agar and nutrient broth on inclined Petri dishes. Culture: 24'C. Color: crystal violet.

The vegetative mycelium shows on both Czapek agar and nutrient broth wavy or straight hyphae, with connections at the ends being very rare no tendency to form bundles of the parallel hyphae.

The hyphae of the vegetative mycelium show particularly on Czapek medium some interruptions in the youth stage; later these interruptions will not observed more. The aerial mycelium on both media shows hyphae that are short bundles form with a number of ticks.

When the above conditions are met, spore formation is slow. The spore shape is round to oval.

The morphological and physiological properties are shown in Table I. Table 1 Morphological and physiological properties of Streptomyces aetnaensis nov. spee. 1415 Medium Growth Vegetative mycelium Air mycelium Soluble 1 Pigment* Agar acc. To Czap ek + -, + pink to violet gray-pink (l / II GE 6) pink-violet Oat agar + yellow-pink gray-pink pink (l / II EC) Potato glycose agar + + + pink (l / VI NC 6) gray-pink pink Asparagine agar ++++ pink (l / IV GC 5) gray- pink; Surface pink pink - excretion Yeast agar +++ nut brown pink, light gray pink, nut brown pink wrinkled (l / VIII NN 5) secretion drops colored Meat agar ++ colorless to cream-colored scanty - whitish pure Glucose agar ++ + wrinkled, like yeast agar gray-pink, nut-brown-pink Secretion drops pink After 7 days at 27'C. Table I (continued) Medium Growth Vegetative mycelium Air mycelium Soluble 1 Pigment* Starch agarl + cream to pink gray pink light pink Ca-Malate-Agar- ' + + T ereme to pink light gray pink light pink (l / 11 EC 4) Agar + + hazelnut-brown-pink gray-pink, pink Secretion drops pink Cellulose agar + colorless gray-pink none Gelatine-Agar3 + cream (yraurosa none Agar "Roux" potatoes -Li ereme whitish none »Roux« carrots + -i- + - none white to gray-pink Meat broth5 + pink light gray none Coagulated serum + nut brown light gray light nut brown After 7 days at 27'C. Gelatin degradation +. HydroIysis potato state unmodified. Malate reduction broth - Stand clear broth, sediment. Table II Biochemical properties of Streptomyces aetnaensis 1415, NRRL 2836 Reduced nitrates to nitrites.

Coagulates milk. Peptonized milk. Liquefies gelatin. Dismantling of caleium malate.

Hydrolysis of starch.

The isolated Streptomyces aetnaensis strain 1415 is similar to Streptomyces vinaceus, Streptomyces violaceus, Streptomyces californicus and Streptomyces griseoflavus, but these show the following differences: Streptomyces vinaceus red-blue soluble pigment, dark red-blue on glucose agar.

Gelatin: Fast liquefaction, no soluble pigment.

Strength: Vegetative mycelium: wrinkled, dry, white, limited hydrolysis.

Streptomyces violaceus nitrates: No reduction. Strength: weak Hydrolysis.

Weak aerial mycelia on all substrates. Open spirals.

Hyphae of the air mycelium elongated.

No or little growth on cellulose agar.

Streptomyces californicus No soluble pigment on agar according to C za p e k. On glucose agar: vegetative mycelium inhibited, creamy sulfur yellow.

Formation of vancomycin. Open spirals. Streptomyces griseoflavus A soluble yellow pigment forms on Czapek agar.

Soluble pigment does not form on potato agar, but on gelatin on the other hand a yellowish pigment.

Milk is not coagulated. Streptomyces aetnaensis nov. spec. 1415 prevents the development of some gram-positive germs on a solid medium (nutrient agar). In liquid substrates that contain an organic or inorganic nitrogen source, a source of carbon, mineral salts and possibly growth factors and trace elements contain, forms Streptomyces aetnaensis nov. spee. 1415 (with stationary or stirring or shaking culture) a complex of antibiotic substances that are resistant to various gram-positive germs are effective.

Antibiotic 1415 differs in terms of its chemical, physical and biological properties of other already known biotics.

The procedure for preparing antibiotic 1415 is as follows before: The inoculation can both by means of its spore suspension, which comes from a culture was obtained on solid medium, as well as by skimming the trunk or on the way of a pre-inoculation with a liquid, different length of time under fermentation standing culture. The fermentation takes place at one temperature between 20 and 35'C, the optimum being around 27'C.

The course of the fermentation itself is largely dependent on the Aeration, stirring or shaking, the composition of the nutrient substrates and the capacity and the shape of the fermentation vessels. Extracting must take place at the moment when the broth has reached the highest activity. Strictly speaking, this point in time results from the fermentation methods used.

The effective complex called antibiotic substance 1415 is thereby obtained by extracting the broth using water-immiscible solvents.

The complex can, however, also be obtained directly from the broth by absorbing it onto activated carbon with subsequent elution with organic solvents. All known organic solvents with the exception of petroleum ether, gasoline and ligroin can be used for this purpose. After evaporation of the solvent, the crude residue can be purified using chromatography on various solvents (carbon or aluminum oxide, activated silica [Celid], which is saturated with a suitable stationary phase, etc.), but also by recrystallization from suitable solvents, such as Diethyl ether, carbon tetrachloride, etc. Mixtures of different solvents. The effective fraction can also be precipitated by dissolving it in a solvent in which the complex is soluble and precipitating it by adding a solvent in which it is insoluble, such as petroleum ether, gasoline, ligroin, water. By paper chromatography with ascending mixture according to Z a ff aroni and B urton (RB B urton, A. Zaffaroni and E. H. Keutmann, Paper Ehromatography of steroids, II, Corticosteroids and related Compounds, J. Biol. Chem., 188 [1951 ], p 763) in a 24-hour process to 25 - (4,5 pre-impregnated with formamide strips in airtight column diameter 5 cm, height 40 cm), and by seeding with poly (Wathman paper No.1); the complex can be broken down into eight biologically active constituents recognizable by autobiography on agar inoculated with Bacillus subtilis (see FIG. 1).

The antibiotic activity of the complex is illustrated in Table III below. Table III Antibiotic effectiveness of the complex of strepto- myces Aetnaensis 1415 (Triple pass on agar according to Waksmann-Roilly: Compare SA Waksmann and H. C. Roilly, Ind. Eng. Chem. Anal., Ed. 17, p. 556 [19451) Test organism dilution agar 1 mg / ccm Bacillus subtilis ........ 0.25 to 0.10 Baeillus anthracis ...... 0.25 to 0.10 Sarcina lutea .......... 0.50 to 0.25 Difeo Sareina lutea ATCC 9341 0.50 to 0.25 (bacto- Micr. pyogenes var.aureus 114 ...... 5.0 to l'o * beef) Micr. pyogenes var.Oxford ......... 5.0 to 1.0 * Micr. pyogenes var. ATCC 6538 ..... 5.0 to 1.0 * riptose Klebsiella pneumoniae ATCC 6538 ......... 1.0 to 0.5 * T Streptococcus haerno- Difeo liticus ** ............ 5.0 to 2.5 Streptococcus pyogenes * * 50.0 to 25.0 (bacto- Streptococcus faecalis ** 100 beef) Streptococcus iTriptose faecalis ATCC 1054 100 Streptococcus viridans ** 100 Corynaebaeterium equi 100 Corynaebaeterium xerose ** ............ 100 Difco Corynaebacteriumcutis ** 100 (bacto- Escherichia coli ........ 200 beef) Serratia marcenses marcescens .......... 200 Concentration of the stripped solution. Tribe of the Istituto sieroterapico rnflanese collection »S. Belfanti (i. The incubation time is 24 hours at 37'C. The toxicity in white mice with an average weight of 20 g: LD5, = 400 mg / kg iv, 600 mg / kg i. p. (Live weight).

Example 1 The cultivation of Streptomyces Aetnaensis nov. spee.1415 (NRRL2836) is made in Roux bottles containing glycerine-asparagine agar at a temperature of 27'C; good spore formation is achieved after several days of incubation. A few 300 ml Erlenmayer flasks are inoculated with a spore suspension which has been grown in the aforementioned medium. Each flask contains 60 ml of broth of the following composition: Peptone ........................... 2.5 g Liebig's meat extract .............. 2.5 g Corn steep liquor (400 /, solid phase) .. 10 g Glucose .......................... 10 g NaC1 ............................ 5 g Tap water .................... 1.00 ml The broth is neutralized by adding NaOH and then stirred in a shaking apparatus (108 strokes per minute, range 6.5 cm). Temperature: 27'C. After acidification in the first few hours of fermentation, the pH rises to around 7.5 in 3 days. The broth reaches a value of 1600 dilution units for Bacillus subtilis.

Example 2 used a mixture which, as described in Example 1, fermented 24 hours, and was inoculated as above for inoculation of some 20 1 of the same broth containing fermentation vessels. After aeration with 20 1 of air per minute and stirring (320 rotations per minute) and at a temperature maintained at 27'C, the activity of the broth dilution to 1600 units in Bacillus subtilis rises to 11 /, days fermentation.

EXAMPLE 3 The fermentation broth obtained according to the procedure as described in Example 2 is centrifuged, the mycelium is extracted by treating twice with boiling acetone and the extract is added to a clear broth, which in turn was extracted with an amount of ethyl acetate equal to the initial volume of broth. The addition takes place slowly in small amounts with prolonged stirring.

After the extraction has been carried out, the solvent is dried and concentrated. As soon as a volume of about 11 is reached, what has been extracted with ethyl acetate is allowed to drip through a column which contains 10 g of charcoal and 10 g of silica (Celite) intimately mixed.

The column is first washed with ethyl acetate until the solvent becomes active on Bacillus subtilis, then with acetone, which takes away any remaining activity. The eluates are evaporated in vacuo, and a solid crude product is obtained which can be dissolved in benzene and precipitated with 10 volumes of petroleum ether.

EXAMPLE 4 To 11 of the broth as obtained according to the instructions given in Examples 1 and 2, 5 g of activated charcoal are added with vigorous stirring over the course of 1/2 hour. This broth is then filtered by suction and the charcoal is washed with water until it flows off clear and colorless. The residue is dried and eluted with acetone until the solvent is active against Baeillus subtilis. The solvent is evaporated to dryness in vacuo, whereupon it is possible to treat the residue as described in Example 3. Example 5 A sample of the antibiotic substance, which is obtained by the procedure described under Examples 3 and 4, is taken up with benzene, separated from the insoluble fraction in the solvent and precipitated again with 10 volumes of petroleum ether, then dissolved in carbon tetrachloride while warm and filtered, then allowed to crystallize.

In this way, a substance is obtained with a semi-solid surface which, after separation from the liquid below and drying in a vacuum drying cabinet, forms a pale yellowish solid substance with an antibiotic spectrum as shown in Table III, with UV and IR spectra, such as they in Fig. 3 and 4 are shown. In Fig. 4 the percent crossings are shown in the ordinates, the wavelength in (-t) in the lower abscissae and the frequency (cm-) above.

The substance has a specific rotation [X] -21 D 31 ' (C = 111 /, in methanol), it decolorizes a permanganate solution, absorbs bromine in chloroform and has the following composition: C = 52.39, 52.270 /,; H = 6.570 / 0; N = 1.55 to 0.20 / 0th

An even greater purity of the antibiotic complex can be achieved if it is treated as described in Example 6 below. EXAMPLE 6 1 g of the solid substance obtained according to the procedure given in Example 5 is dissolved warm in carbon tetrachloride (200 ccm) and allowed to cool; the solid surface phase is separated off by centrifugation and the liquid below is introduced into a column 65 cm high and M cm in diameter containing 60 g of activated charcoal (Dareo G 60) and 60 g of Hyflo-Hypercel, intimately mixed and pressed.

After eluting with benzene until the antibiotic activity disappears, concentration to a small volume of about 100 ccm by centrifugation and precipitation with petroleum ether, a white solid substance forms which softens at about 90 to 95 ° C and has a specific rotation of [ix] - 'D' = -34 '(C = 10/0 in methanol).

Composition: C = 59.44 to 59.570 / "H = 7.98 to 8.040 / 0, N = 2.040 /,.

The corresponding UV and IR. # Spectra are shown in FIGS. 5 and 6 ; In the latter, the percentage crossings can be read off the ordinate, the wavelength (cm-) is indicated on the abscissa at the bottom and the frequency (cm-) at the top.

Using paper chromatography, according to the procedure already mentioned, was it is possible to use the eight antibiotic constituents in the compound obtained ascertain.

The complex is stable for at least 5 days in aqueous solution, which is obtained by dissolving it in a very small amount of acetone and diluting it with water; when dissolved in aqueous 511 /, hydrochloric acid at room temperature, it is stable for at least 6 hours and with buffers pH = 2 at 100'C for at least 3 minutes.

If you buffer to pH = 9, the activity remains in the cold for at least 6 hours and at a temperature of 100 ° C for 3 minutes; In sodium hydroxide solution of 5% strength, rapid inactivation occurs even when cold, with the solution turning an intense yellow.

The nihydrin reaction, negative in itself, becomes positive after 48 hours acid hydrolysis at 100'C. With diethyl ether and diethyl acetate it is possible to do some extract yellow or reddish substances that have not been identified. The aqueous phase contains six amino acids, which are determined by means of two-dimensional paper chromatography are detectable.

First direction. Normal butanol - acetic acid - water 4: 1 : 5 v / v; second direction. phenol saturated with water + "Versene" Watman paper # 1 V (. s F i g. 2) by developing with Nihydrin.

(S. Figure), the spots which appear are: A glycine, alanine B, D proline, valine F, E Phenylatanin (this was fixed on carbon by one uties of M. J stemming technique in J. L oise 1 eur, cited "Techniques de laboratoire", 2nd edition, p. 652, Masson & Cie., Paris, 1954, and washed out with water saturated with hydrogen sulfide and ethyl acetate), G Leucine, C most likely belongs to the aminobutyric acid group and was eluted in the same way as γ-aminobutyric acid.

The identity of various amino acids was confirmed by repeating Chromatography in the presence of solutions of known samples.

Claims (1)

Claim: Process for the production of the antibiotic 1415 by a biochemical route through aerobic conditions, characterized in that the strain Streptomyces Aetnaensis nov. spec. 1415 (NRRL 2836) is used.