EXTRACTION OF TEICHMONYCIN A2 FROM WHOLE CULTURE FERMENTATION BROTH

Abstract: Teichomycin A₂ is recovered from whole culture fermen­ tation broth of Actinoplanes teichomyceticus sp. or any similar microoganism producing Teichomycin A₂ by extract­ ing the broth with a water miscible solvent, such as acetone, acetonitrile, dimethylsulfoxide, n-propanol, methyl ethyl ketone or mixtures thereof.

Description: The present invention relates to a method of extracting Teichomycin A₂ from a whole culture fermentation broth with a suitable water miscible solvent. Under the nomenclature established in the World Heath Organization Handbook for International Non-Proprietary Names (INN), the name "Teichomycin A₂" has been changed to "Teicoplanin A₂" as the most recent name for this antibiotic factor. United States Patent 4,239,751 discloses the antibiotic Teichomycin A₂ which is obtained by cultivation of strain Actinoplanes teichomyceticus nov. sp. ATCC 31121. Teichomycin A₂ is one of a mixture of antibiotic factors Teichomycin A₁, Teichomycin A₂, Teichomycin A₃, and Antibiotic 8327 Factor B and Antibiotic 8327 Factor C produced by this strain. U.S. patent 4,239,751 teaches a method of isolating the antibiotic factors. In the method described in U.S. Patent 4,239,751, the fermentation broth is filtered in order to remove the mycelial cell mass leaving a mycelial cell cake. The filtered fermentation broth is then mixed with a water immiscible organic solvent, such as, halogenated C₁-C₄ hydrocarbons or C₄-C₆ alkanols, in which the antibiotic mixture is soluble. The water immiscible organic solvent is then separated from the filtered fermentation broth by high-speed centrifugation, concentrated to about 1/10 to 1/20 of its original volume, cooled and allowed to stand until a precipitate (the antibiotic) forms which is recovered by filtration. Additional product may be recovered by extracting the mycelial cake with aqueous acetone. After distillation of the acetone, the aqueous phase is submitted to the same treatment described above for the filtered fermentation broth. Briefly, in accordance with the present invention, Teichomycin A₂ is extracted from whole culture fermentation broth of Actinoplanes teichomyceticus or any similar microoganism producing Teichomycin A₂ containing a mycelial mass by a process comprising mixing the broth containing the mycelial mass with an effective amount of a water miscible solvent to accumulate Teichomycin A₂ in the broth/solvent liquid; separating the broth/solvent liquid containing accumulated Teichomycin A₂ from the mycelial mass; and precipitating and separating Teichomycin A₂ from the broth/solvent liquid. The useful water miscible solvents include acetone, acetonitrile, dimethylsulfoxide, n-propanol, methyl ethyl ketone or mixtures of these solvents. The present extraction process is carried out by mixing an effective amount of a solvent, described above, with whole culture fermentation broth of Actinoplanes teichomyceticus which contains Teichomycin A₂. The solvent and broth are thoroughly contacted for an effective time period whereby the Teichomycin A₂ is accumulated in the liquid solvent/broth mixture. The Teichomycin A₂ is then recovered from this liquid employing separatory and purification techniques. The present invention employs a whole culture fermentation broth of Actinoplanes teichomyceticus containing Teichomycin A₂. Water miscible solvents used to accumulate Teichomycin A₂ in the broth/solvent liquid include acetone, acetonitrile, dimethylsulfoxide (DMSO), n-propanol, methyl ethyl ketone (MEK) and mixtures of these solvents. The preparation of Teichomycin A₂ by the fermentation of Actinoplanes teichomyceticus is described in U.S. Patent 4,239,751 which is incorporated herein by reference. The whole culture fermentation broth containing the cell mass, aqueous nutrient medium and the Teichomycin A₂, is treated according to the present invention after the fermentation is complete. The solvents employed in the practice of the present invention include acetone, acetonitrile, DMSO, n-propanol or MEK. Mixtures of these solvents can also be used. The solvents are employed in amounts between 5 to 80 percent by volume based on the total volume of solvent and broth, i.e., solvent/broth ratio of from 1/19 to 4/1. Preferably, from 10 to 60 percent by volume of solvent is employed. Preferred solvents are acetone and n-propanol. The present invention is advantageously conducted at ambient temperatures and ambient pressures. The mixing is typically conducted with mild agitation sufficient to maintain a thorough contacting of fermentation broth and solvent. The pH of the fermentation broth is not critical to the present process. An acidic pH is preferred and it is especially preferred that the pH of the fermentation broth be in the range of from 3 to 4. The pH of the fermentation broth can be adjusted by the addition of an acid or base. Suitable acids include H₂SO₄, HCl, acetic acid and formic acid. Suitable bases include alkali metal bases, such as, NaOH or KOH. In conducting the extraction process, an effective amount of the herein described solvents is mixed with whole culture fermentation broth of Actinoplanes teichomyceticus , which contains Teichomycin A₂, for an effective time period to accumulate the Teichomycin A in the mixed fermentation broth/solvent liquid. The rate of mixing the solvent and the fermentation broth is not critical. Preferably the solvent is added to the fermentation broth. An effective mixing time period is usually achieved in from 2 to 60 minutes. After completion of the mixing period, the fermentation broth/solvent liquid is separated from the mycelial mass by centrifugation or filtration. The fermentation broth/solvent liquid, separated from the mycelial cake, hereinafter referred to as "supernatant", is then concentrated, for example, to about 1/2 to 1/10 of its original volume, cooled and allowed to stand until a precipitate forms. The precipitate, containing Teichomycin A₂, is then recovered from the supernatant by filtration. Alternatively, Teichomycin A₂ can be recovered from the supernatant by acid precipitation. In a preferred embodiment of the present invention, acetone or n-propanol is added to whole culture fermentation broth of Actinoplanes teichomyceticus which contains Teichomycin A₂ whereby the acetone or n-propanol represents from 20 to 50 percent by volume of the mixture. The mixture is agitated, from 2 to 60 minutes, such as by stirring or shaking to cause a thorough mixing of the acetone or n-propanol with the fermentation broth. The fermentation broth/solvent liquid is separated from the mycelial mass by centrifuging or filtering. Teichomycin A₂ is isolated from the supernatant by concentrating the supernatant to 1/2 - 1/10 its original volume, cooling the concentrate and allowing it to stand until a precipitate forms. The precipitate is Teichomycin A₂ which is collected by standard separation techniques such as filtration. The following examples illustrate the practice of the present invention but should not be construed as limiting its scope. Example 1 Samples (3 ml each) from whole culture fermentation broth (pH = 6.8-7) of Actinoplanes teichomyceticus were mixed with volumes of acetonitrile to give from 20 to 60 percent by total volume mixtures of the acetonitrile in fermentation broth. After a 5 minute mixing period with continuous agitation, the samples were centrifuged and the supernatant was assayed for Teichomycin A₂ employing standard high pressure liquid chromatography (HPLC) methods employing: Column: DuPont ZORBAX® 4.6 mm x 25 cm ODS Flow: 1.8 ml/min. Solvent: 24.5/75.5 acetonitrile/0.03 M ammonium formate at pH 6.0 Absorbance: 254 mm The results are listed below: Example 2 Employing substantially the same procedures of Example 1, various water miscible solvents contemplated by the present invention were used to extract Teichomycin A₂ from samples of whole culture fermentation broth of Actinoplanes teichomyceticus . The fermentation broth had a pH of 6.0. The results are listed below: The supernatant fluids containing high levels of Teichomycin A₂ from Examples 1 and 2 above are then treated to isolate the Teichomycin A₂. The supernatant fluids are concentrated to from 1/2 to 1/10 their original volume, cooled and allowed to stand until a precipitate forms. The precipitate, containing Teichomycin A₂, is recovered by filtration from the supernatant. The Teichomycin A₂ recovered employing the procedures of the present invention is then used for known uses, such as, an antibiotic.

Claims: A method of extracting Teichomycin A₂ from a whole culture fermentation broth containing a mycelial mass comprising (a) mixing the broth containing the mycelial cake with an effective amount of a water miscible solvent to accumulate Teichomycin A₂ in the broth/solvent liquid; (b) separating the broth/solvent liquid containing accumulated Teichomycin A₂ from the mycelial mass; and (c) precipitating and separating Teichomycin A₂ from the broth/solvent liquid. The method of Claim 1 wherein the water miscible solvent is acetone, n-propanol, acetonitrile, methyl ethyl ketone, dimethylsufoxide, or mixtures thereof. The method of Claim 1 wherein the solvent is between 5 to 80 percent of the volume of the broth/solvent liquid. The method of Claim 1 wherein the water miscible solvent is mixed with an acidic fermentation broth. The method of Claim 4 wherein the acidity of the fermentation broth is set to a pH of 3 to 4. The method of Claim 4 wherein the acidity of the fermentation broth is adjusted to a desired pH using sulfuric acid, hydrochloric acid, acetic acid, or formic acid. The method of Claim 1 wherein the mixing of the fermentation broth and the water miscible solvent is carried out for a time of at least 2 minutes. The method of Claim 1 wherein the broth/solvent liquid containing the accumulated Teichomycin A₂ is separated from the mycelial mass by centrifugation. The method of Claim 1 wherein the broth/ solvent liquid containing the accumulated Teichomycin A₂ is separated from the mycelial mass by filtration. The method of Claim 1 wherein the precipitated Teichomycin A₂ is separated from the broth/ solvent liquid by filtration.

Inventors: COLEMAN, JOHN STEVEN, SNIPES, CARL EVAN
Applicants: THE DOW CHEMICAL COMPANY
Application year: 1984
Main classification: C07G11
IPC codes: A61K35, C07K9, C12R1, A61P31, C12P1, C12N1, C12N9