US3408258A - Process for producing oxytetra-cycline antibiotic - Google Patents

Process for producing oxytetra-cycline antibiotic Download PDF

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US3408258A
US3408258A US435336A US43533665A US3408258A US 3408258 A US3408258 A US 3408258A US 435336 A US435336 A US 435336A US 43533665 A US43533665 A US 43533665A US 3408258 A US3408258 A US 3408258A
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oxytetracycline
species
waksman
actinomycetes
vol
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Grein Arpad
Barchielli Riccardo
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Pfizer Italia SRL
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Farmaceutici Italia SpA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P29/00—Preparation of compounds containing a naphthacene ring system, e.g. tetracycline
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/886—Streptomyces

Definitions

  • the present invention relates to a fermentative process for obtaining the antibiotic, oxytetracycline, by means of a new species of St'reptom'yces, named by us Streptomyces henetus or Streptomyces F1. 1782, and which has been deposited at the Institute of Microbiology of the- Rutgers University, U.S.A., where it received the index I.M. 3872.
  • the process of the present invention results in obtaining oxytetracycline in very high yields. These yields are shown to be as high as 11,000 per cc.
  • This new microorganism has been isolated from a soil sample taken in the neighborhood of Ramera (Italy) and shows the following morphological, macroscopic, microscopic and biochemical properties:
  • the vegetative myceliumon culture media of potatoagar and Czapek-agar consists of hyphae 0.8-0.9;t thick, of intermediate length, branched and forming aerial mycelium consisting of hyphae from 1.2 to 1.6 thick and not very long. These hyphae abundantly ramify and form straight conidiophores bearing spiralled hyphae then transforming into spores.
  • the conidia have a smooth surface, are prevalently oval: 1.2 to 1.6, by 1.4 to 1.7a and are disposed first in chains and then free; each chain hearing more than ten spores.
  • the hyphae transforming into spores are attached to conidiophores alternatively or oppositely.
  • the spirals are closed, sometimes forming clews.
  • the microorganism reduces nitrates to nitrites, hydrolyzes starch and gelatin, does not produce hydrogen sulphide, does not coagulate or peptonize milk, and does not produce melanin and digest tyrosine. It produces acids from d-mannose, d-arabinose, glucose, mesoinosite, dfructose, adonitol, maltose and ramnose but not from d-xylose, sorbitol, or saccharose.
  • the microorganism does not form sclerotia and does not grow at C.
  • Potato-A gar 4 Abundant From dirty w bite to light brown grey. Frlonill stbraw-yellow to .do ig t rown. Oats-Agar Mod r Scarce, whitish Frorfi coloaless to do ye OWlS Asparagine-glycerin-Agar 1 do d Colorless, yellowish do Starch peptone-Agar 1 do.. do Slightly light brown do Positive hydrolysis. Tyrosine-Agar 5 do Scarce, dirty wh1te Colorless (ln lMIelanin-Agar Scarce..
  • V etative m From strawellow to From cream-colored to Strong apricotpr From colorless to a From pale Brown-black.
  • the description of the microorganism under examination relates to the genus Streptomyces Waksman and Henrici -(Bergeys Manual of Determinative Bacteriolog 7th ed., 1957, pp. 744745). From examination of the species it may be concluded: In the classification system of Pridham et al. (Appl. Microbiol., 1958, 6, p. 52) the microorganism belongs to the section Spira series White; in the classification system of Baldacci (Giorn. Microbiol., 1958, 6, p.
  • the microorganism belongs to the series Albidofiavus; and in the classification systern of Waksman (The Actinomycetes, vol. H, 1961, p. 123) the microorganism belongs to the series Flavus.
  • Streptomyces F1. 1782 A comparison between the properties of the micro organism Streptomyces F1. 1782 and those of species belonging to the above systematic groups (Taxa) demonstrates that Streptomyces RI. 1782 has quite different properties. Besides the differences listed in Table II, Streptomyces F.I. 1782 has a clearly different morphology from Streptomyces rimosus: The hyphae of the former transforming into spores are alternatively or oppositely attached along straight sporophores; while in the latter the hyphae forming spores are attached to short sporophores in a more or less compact fascicled system.
  • Taxa group A comparison between the properties of the micro organism Streptomyces F1. 1782 and those of species belonging to the above systematic groups (Taxa) demonstrates that Streptomyces RI. 1782 has quite different properties. Besides the differences listed in Table II, Streptomyces F.I. 1782 has a clearly different morphology from Strepto
  • Streptomyces RI. 1782 differs from the species S.
  • griseoflavus because the latter shows a different color of the vegetative and the aerial mycelium, furthermore ecause it produces soluble pigments (Waksman, The Actinomycetes, vol. II, 1961, p. 222); from the species S. flavochromogenes because the latter produces soluble pigments and shows a different color of the vegetative and the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 209); from the species S. alboflavus because the latter does not form spirals and because it shows a different color of the vegetative mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 169); from the species S.
  • alboniger because the latter does not produce spirals and shows a different color of the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1961, p. 170); from the species S. lzachijoenis because the latter does not produce spirals and shows a different color of the aerial mycelium (Waksman, The Actinomycetes, vol. II, 1 961, p. 226); from the species S. eurocidicus because the latter does not produce spirals and because it produces soluble pigments (Waksman, The Actinomycetes, vol. II, 1961, p. 205); from the: species S.
  • cacaoi because the latter produces longand open spirals, forms an aerial mycelium which is colored from light-gray to mouse-gray and reduces nitrates weakly (Waksman, The Actinomycetes, vol. II, 1961, p. 183); from the species S. eryrhreus because the latter produces a cream to purple-red aerial mycelium and wineecolored soluble pigments (Waksman, The Actinomycetes, vol. II, 1961, p. 204) from the species S. galtieri because. the latter produces a cream-colored aerial mycelium, a brown soluble pigment and liquefies gelatin weakly (Waksman, The Actinomycetes,vol. II, 1961, p.
  • Streptomyces El. 1782 should be considered a species different from those known up to the present and therefore a new species which we have named Streptomyces henetzts.
  • the microorganism Streptomyces F1. 1782 may be stored by successive cultivation on a solid medium (potato-agar, Sabouraud agar) or by lyophilization, milk being the suspending medium.
  • the production of the antibiotic Oxytetracycline according to the invention may be performed by cultivating Streptomyces F.I. 1782 in a liquid nutrient medium under aerobic conditions. A temperature of from 24 to 37 C., preferably 28 C., and a period of from 72 to 144 hours have been found satisfactory. The pH may vary from 6.1-6.5 to 8 according to the fermentative media employed.
  • the cultural medium should consist of a carbon source, a nitrogen source, and salts.
  • the carbon source may be, for example, saccharose, glucose, dextrin, starch, soyabean meal, peanut meal, cotton-seed meal, corn steep liquor, distillers solubles, soyabean oil, or lard oil.
  • the nitrogen source besides the above complex substances containing nitrogen, may be meat extract, peptone, casein,
  • hydrolysate of casein and ammonium salts such as ammonium sulphate or diammonium phosphate.
  • the mineral salts useful for the production of the antibiotic vary according to the medium employed. Calcium carbonate is normally present, and sodium, potassium, manganese, iron, copper, cobalt or zinc chlorides, sulphates or phosphates may be added.
  • the fermentation may be carried out in Erlenmeyer flasks and in laboratory or industrial fermenters of various capacity.
  • the quantity of antibiotic present in the broth may be qualitatively assessed by paper chromatography in comparison with a standard sample of oxytetracycline and quantitativelychecked by spectrophotometric or biological methods.
  • the yields obtainable by the process of the invention are as high as 11,000 per cc.
  • the pH of the culture broth is adjusted to 2-2.5, the mycelium is separated by filtration or centrifugation, and the Oxytetracycline may be recovered from the liquid phase and purified.
  • Useful methods of purification are: extraction with an organic solvent, for example butanol, pentanol, hexanol, amyl alcohol, methylethylketone, methyl isobutylketone or butyl acetate; adsorption on a solid substance, for example activated charcoal, and subsequent elution with water or an organic solvent; precipitation of the antibiotic in the form of one of its derivatives or complex salts bymeans, for example, of an amine, a quaternary ammonium compound in the presence of a metal ion, ammonia or sodium carbonate, urea, N,N -malonylurea, sulphonic derivatives or sulphates.
  • Oxytetracycline thus obtained may be then purified by recrystallization or transformed
  • the present invention includes a method of extraction and purification of the oxytetracycline produced, which comprises extracting the acidified culture broth, filtering the product and treating the resulting filtrate with oxalic acid, filtering, making alkali, extracting with a mixture of tricresol and carbon tetrachloride, separating the organic phase, treating it with aqueous hydrochloric acid and acetone and separating the aqueous phase.
  • the culture broth is acidified with a mineral acid, for'exarnple hydrochloric, sulphuric or phosphoric acid, to pH 2-2.5, is filtered and the filtrate obtained is first treated with oxalic acid and then filtered to remove precipitated calcium oxalate; the filtrate is extracted with a mixture of tricresol and carbon tetrachloride adjusting the pH to 8-8.5 by addition of alkali.
  • the tricresol em ployed is that generally known and employed for industrial uses, having a specific gravity 1.0301.038, consisting of a mixture of the three isomeric cresols.
  • a mixture of tricresol and carbontetrachloride in a ratio 2:1 to 1:2 by volume.
  • the phases thus obtained are separated by centrifuging and to the separated organic phase, which contains the antibiotic, a mixture of equal volumes of acetone and aqueous hydrochloric acid, preferably 1 N hydrochloric acid, is added.
  • aqueous phase thus obtained and adjusted to pH 7, by addition of an alkaline material such as an alkali metal hydroxide or carbonate, the antibiotic Oxytetracycline precipitates and is isolated and purified by recrystallization or transformed into a salt with a mineral or organic acid.
  • Each flask is inoculated with 1.5 cc. of a suspension of spores obtained by scraping off, into 5 cc. of distilled sterile Water, the surface of a 20 days old slant of a culture of Streptomyces RI. 1782 grown on the following solid medium: 200 g. of peeled potatoes are boiled for about 20 minutes in 500 cc. of water. The volume is brought to its original value and filtered through gauze. 2% of glucose and 2% of agar are added. The volume is brought to 1000 cc. and sterilized in an autoclave at 120 C. for 20 minutes.
  • the pH of the medium is 6.8-7.0.
  • the flasks are incubated at 28 C. for 24 hours on a rotary shaker with a stroke of 30 mm. at 220 rpm.
  • 1 cc. of a culture thus grown is used to inoculate Erlenmeyer flasks of 300 cc. with 60 cc. of the following productive medium:
  • the sterilization is carried out at 120 C. for 20 minutes.
  • the pH after sterilization is 6770. It is incubated at 8 C. under the conditions described above for the vegetative medium. 4000 per cc. of Oxytetracycline is produced after 96 hours of fermentation.
  • EXAMPLE 2 The operation is as in Example 1, with the difference that the culture to be inoculated is 10 days old and is grown on the following medium:
  • the productive phase differs in-the composition of the medium being as follows:
  • the pH after sterilization is 6.5-6.7.
  • the medium is sterilized in an autoclave at 120 C. for 20 minutes.
  • the quantity of the vegetative medium to be inoculated into the productive medium is 4.3% of the latter.
  • the incubation of the productive phase is carried out for 24 hours at 28 C. and then at 24 C. for 120 hours. 7600 per cc. of oxytetracycline are obtained.
  • EXAMPLE 4 Operation is as in Example 2 with the difference that operation is always at 24 C.
  • the production obtained in 96 hours of fermentation is 6400 per cc. of oxytetracycline.
  • the pH after sterilization is 6.5.
  • the above medium is contained ma 5 liter neutral glass fermentor, having the above-described proper-ties.
  • foaming is checked by adding small amounts ofv silicone antifoaming agent.
  • the highest production of oxytetracyclineobtain ed in 88 hours of fermentation corresponds to 58007 per cc.
  • EXAMPLE 6 Purification
  • the pH of 333 liters of a broth culture having an antibiotic concentration of 4500 per cc., obtained asdescribed in any of the preceding examples, is adjusted to 2 by adding dilute 25% aqueous sulphuric acid.
  • the culture broth is filtered with 1 kg. of infusorial earth and the filtration cake is washed with acidulated water (pH 2- sulphuric acid) so that the volume of the filtrate reaches 45 liters.
  • 2.2 kg. of oxalicacid are added and the mixture is stirred for 30 minutes, filtered and the filter washed with water so that the volume of the filtrate reaches 50 liters.
  • the two'layers are. separated by centrifuging.
  • the heavy layer is extracted twice again with 3 liters-and 2 liters respectively of 1 N aqueousrhydrochloric acid.
  • the pH of the combined aqueous layers (10 liters) is adjusted to 7 by adding with stirring a dilute (20%) aqueous sodium hydroxide.
  • a precipitate separates, which is recovered by filtration, washed with diethyl ether and dried in vacuo at 40 C.
  • the residue is ground, washed again with 'etherand dried at 40 C. 150 g. of crude oxytetracycline are obtained.
  • a process for preparing oxytetracycline which comprises cultivating the new' microorganism Streptomyces henetus Rutgers University Institute of Microbiology I.M.
  • a process for preparing oxytetracycline which comprises cultivating the new microorganism Streptomyces lzenetus Rutgers University Institute of Microbiology I.M. 3872 under aerobic conditions in a nutrient medium containing a source of carbon, nitrogen and mineral salts, at a temperature of from 24 to 37 C., over a period of from 72 to 144 hours, at pH 6.1 to 8, acidifying the culture broth containing oxytetracycline to pH 2-2.5 with an inorganic acid, filtering and treating the filtrate with oxalic acid and filtering to remove calcium oxalate precipitated and making alkaline the filtrate obtained to pH 8-8.5 by adding an inorganic base and extracting with a mixture of tricresol and carbon tetrachloride, and transferring the oxytetracycline from the organic phase into an aqueous phase by a mixture of aqueous hydrochloric acid and acetone, and precipitating the oxytetracycline from the a
  • a process for preparing oxytetracycline which comprises cultivating the new microoganism Streptomyces henetus Rutgers University Institute of Microbiology I.M. 3872 under aerobic conditions in a nutrient medium containing a source of carbon, nitrogen and mineral salts, at a temperature of from 24 to 37 C., over a period of from 72 to 144 hours, at pH 6.1 to 8, acidifying the cuture broth containing oxytetracycline to pH 2-2.5 with an inorganic acid, filtering and treating the filtrate with oxalic acid and filtering to remove calcium oxalate pre cipitated and making alkaline the filtrate obtained to pH 8-8.5 by adding an inorganic base and extracting with a mixture of tricresol and carbon tetrachloride in a ratio of 2:1 to 1:2 by volume, and transferring the oxytetracycline from the organic phase into an aqueous phase by 10 a mixture of aqueous hydrochloric acid and acetone, and precipit
  • a process for preparing oxytetracycline which comprises cultivating the new microorganism Streptomyces henetus Rutgers University Institute of Microbiology I.M. 3872 under aerobic conditions in a nutrient medium containing a source of carbon, nitrogen and mineral salts, at a temperature of from 24 to 37 C., over a period of from 72 to 144 hours, at pH 6.1 to 8, acidifying the culture broth containing oxytetracycline to pH 2-2.5 with an inorganic acid, filtering and treating the filtrate with oxalic acid and filtering to remove calcium oxalate precipitated and making alkaline the filtrate obtained to pH 8-8.5 by adding an inorganic base and extracting with a mixture of tricresol and carbon tetrachloride, and transferring the oxytetracycline from the organic phase into an aqueous phase by a mixture of aqueous 1 N hydrochloric acid and acetone in an equal ratio by-volume, and precipitating the oxyt

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US435336A 1964-02-27 1965-02-25 Process for producing oxytetra-cycline antibiotic Expired - Lifetime US3408258A (en)

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GB1324022A (en) * 1969-10-01 1973-07-18 Squibb & Sons Inc Purification of tetracycline antibiotics
JPS52104551U (en)) * 1976-02-05 1977-08-09
JPS52157444U (en)) * 1976-05-21 1977-11-30
JPS52159953U (en)) * 1976-05-28 1977-12-05
JPS60162423U (ja) * 1983-03-03 1985-10-29 株式会社ラツクス 手動式果実収穫器

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US2516080A (en) * 1949-10-13 1950-07-18 Pfizer & Co C Terramycin and its production

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Publication number Priority date Publication date Assignee Title
US2516080A (en) * 1949-10-13 1950-07-18 Pfizer & Co C Terramycin and its production

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DE1492137A1 (de) 1969-08-07
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JPS499758B1 (en)) 1974-03-06
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