GB1562987A - Process for preparing multhiomycin - Google Patents

Description

(54) AN IMPROVED PROCESS FOR PREPARING MULTHIOMYCIN (71) We, KUMIAI CHEMICAL INDUSTRY COMPANY LIMITED, a Japanease corporation, of No. 4-26, 1-chome, Ikenohata, Taitoh-ku, Tokyo, Japan, do hereby declare the invention, for which we pray that a patent be granted to us and the method by which it is to be performd, to be particularly described in and by the following statement: In the Journal of Antibiotics Vol. XXIII, No. 5, pp. 231-237 (1970), H. Yonehara et al reported the preparation of an antibiotic named multhiomycin from the mycelium of Streptomyces sp. 8446-CCI by aerobic cultivation in the presence of dextrin, yeast, NaC1 and CaCO3. Multhiomycin was extracted with methanol and purified by silica gel chromatography, giving yellow needle-shaped crystals. The morphology of Streptomyces sp. 8446-CCI, and the characteristics of multhiomycin, are given in the above article.

Streptomyces sp 8446-CCI has been deposited with the Fermentation Research Institute of the Agency of Industrial Science and Technology under the Deposit No. FERM-P No.

3284. Samples of the strain are available on application to the Institute.

We have found that the addition of sulphur-containing carboxylic acid, or a derivative thereof, to a culture medium containing Streptomyces sp. 8446-CCI and assimilable sources of carbon and nitrogen can enhance the production of multhiomycin, e.g. by a factor of 2 to 4 times.

Suitable carbon sources for use in the present invention include glucose, maltose, fructose, lactose, molasses, starch and dextrin. Suitable nitrogen sources include soybean flour, peanut flour, cottonseed flour, meat extract, corn steep liquor, yeast, glutamic acid, urea, sodium nitrate, ammonium nitrate and ammonium phosphate.

The amount of the sulphur-containing carboxylic acid is preferably from 0.0005 to 10%, more preferably 0.01 to 5%, by weight. Suitable sulphur-containing carboxylic acids, or their derivatives, include thioglycolic acid, thioglycolic acid amide, thiomalic acid, thiobenzoic acid, methionine, cystine and ammonium, alkali metal or alkaline earth metal salts thereof.

If desired, sodium chloride. sodium phosphate or a very small amount of metal ions may be added to the medium. A culture medium comprising (1) dextrin, dry yeast, methionine, sodium chloride and calcium carbonate, or (2) starch, cottonseed flour, methionine, sodium chloride and calcium carbonate are preferred for the production of multhiomycin.

Although cultivation of the Streptomyces strain can be accomplished using a solid culture, it is more advantageous to employ a liquid culture, particularly a submerged culture, for mass cultivation. Cultivation can be conducted under aerobic or semi-aerobic conditions.

For instance it is possible to carry out cultivation under a flow of germ-free air or by surface culture with no aeration. The cultivation temperature is usually from 10 to 50"C, and preferably 23 to 32"C. but in most cases a temperature of around 27"C. is found to be optimal.

Generally, production of multhiomycin reaches its maximum after about 1 to 9 days cultivation in the case of shaking cultures and in about 1 to 8 days cultivation in the case of aerated tank cultures. In performing the cultivation, a mineral acid such as hydrochloric acid or sulphuric acid or an organic acid such as acetic acid or oxalic acid is added in an appropriate amount to maintain the pH of the culture solution at 4.0 to 9.0, preferably at 5.5 to 8.5, to maximise the production of multhiomycin.

Since multhiomycin is present in both the culture broth and the mycelium-containing solids, collection of multhiomycin from the cultured product after cultivation can be conducted by collecting multhiomycin separately from the filtrate and the solid mass after filtering out the solids and then combining together the collected mass or immediately collecting multhiomycin from the culture solution containing the solids. The collection of multhiomycin can be accomplished by any suitable known methods generally used in separating and collecting the antibiotics from a culture of microorganisms or by combining such methods.

For collecting multhiomycin from the culture broth from which the mycelium-containing solids have been filtered out, it has been generally attempted to extract the product with a single solvent immiscible in water, such as, for example, esters, methanol, ethanol, butanol, amyl alcohol, benzene acetone or methyl isobutyl ketone. We have found that multhiomycin present in the filtrate can be extracted with greater efficiency using a solvent mixture comprising (i) an alcohol e.g. a C1.4alkanol such as methanol, ethanol, isopropanol, n-propanol or butanol, or a celelosolve such as methyl cellosolve and (ii) a halogenated hydrocarbon e.g. a mono- or poly- halogenated saturated aliphatic hydrocarbon such as methyl chloride, dichloroethane, chloroform, carbon tetrachloride, chlorobromomethane, dichloroethane, chlorobromoethane, trichloroethane, dichloropropane or chlorobutane, preferably in a mixing ratio of 1: 0.1 to 1:10, more preferably 1:1 to 1: 7 (for alcohol :halohydrocarbon).

For instance, when a culture broth obtained from cultivation is divided into two portions and one portion is subjected to three successive extractions with butyl acetate and the other is extracted with a 1:4 (v/v) mixture of methanol, it is found that the extracted amount of multhiomycin in the case of the mixed solvent may be four times as high as that in the case of butyl acetate alone.

Further, as an alternative, we have found it very satisfactory to extract multhiomycin from solids using a mixture of a ketone such as acetone or methyl ethyl ketone and an alcohol such as methanol, ethanol, butanol or phenol, preferably in a ratio of 1:1.0 to 1: 20. For instance, the collection rate using methanol in extracting multhiomycin from the fungus-containing solids obtained under the same conditions by three successive extraction operations is 2.62 gr. as measured on a purity basis, whereas the collection rate attained from initial extraction with methanol and additional extractions with a 1:4 (v/v) mixture of methanol and methylene chloride may be as high as 4.46 gr.

In general, it is beneficial to extract multhiomycin from a composition such as mycelium or culture broth in which it was grown by an extraction process comprising the use of a mixture of an alcohol (including aromatic alcohols such as phenol) and a ketone or halogenated hydrocarbon in the extraction or, if there is more than one extraction stage, in at least one of the extraction stages, preferably in at least the final stage. Suitable ratios and materials are given above.

The following Examples illustrate the invention.

Example 1 Methionine, cystine and thioglycolic acid were added at the proportions shown in Table 1 to a culture base having the following composition (percentages by weight): Dextrine 2.5% Dry Yeast 2.0% Sodium Chloride 0.5coo Calcium Carbonate 0.4coo Streptomyces sp. 8446-CCI strain was inoculated in each of the thus-prepared culture media and subjected to cultivation at 28"C, and the multhiomycin potency on the 4th day after the start of the cultivation was measured. The results obtained are shown in Table 1.

Multhiomycin was extracted by a mixed solution of acetone and methanol (4:1 v/v) from the culture mycelium separated centrifugally. and diluted with a 30% acetone-containing 0.1 M phosphoric acid buffer solution (pH 8).

The potency of the solution was determined according to the cylinder gas agar plate method by using Bacillus subtilis as test organism.

Table 1 Amount of additives added to basic culture medium %(w/w) Methionine Cystine Thioglycolic Strength on 4th acid day (y/ml) 0.001 353 0.005 401 0.01 517 0.05 728 0.1 1,012 0.5 869 1.0 470 3.0 329 0.001 305 0.01 634 0.1 842 1.0 540 3.0 423 0.001 282 0.01 336 0.1 578 1.0 719 3.0 318 basic culture medium (no addition) 235 Example 2 100 litres of a culture medium (whose pH had been adjusted to 5.6) containing 2.5% of starch, 2% of cottonseed flour, 0.1% of methionine. 0.56sic of sodium chloride and 0.4% of calcium carbonate was inoculated with a seed culture solution of Streptomyces sp.

8446-CCI. The culture medium was cultured at 27"C. with agitation at 300 r.p.m. and aeration at 15 1/min. As production of multhiomycin reached its maximum after 80 hours cultivation, cultivation was stopped at that point. The mycelium was filtered out to obtain about 48 kg. of wet mycelium and this was then homogenised and divided into 27 groups each of 1 kg., and each group was extracted with 1.5 1. of methanol and filtered. A second extraction of each specimen was carried out using 1.5 1. of various mixed solvents as shown in Table 2. The methanol-extracted solution and each mixed solvent were combined and subjected to vacuum distillation at 60"C. to fractionate each solvent from the extracted solution, and the n 50 c.c. of each remaining aqueous solution was further extracted with 100 c.c. of ethyl acetate. The ethyl acetate was distilled off under vacuum from each ethyl acetate- extracted solution and each of the oily substances remaining was dissolved in one of the mixed solvents as shown in Table 2. n-Hexane, in an amount of 1.5 times as much as that of each of the resultant solutions was added portion-wise to each solution, giving a yellow sediment of multhiomycin. Each sediment was collected on a glass filter and washed once with 10 ml. of methanol to collect crude crystals of multhiomycin. Then the purity of each crude crystal specimen was measured by the cup method (as described in "Basic Knowledge of Antiobiotics", Nakazawa, published by Nanzan-Do, Tokyo, 1966), and the purity-converted total weight (gross activity) was determined from the value obtained and each crystal weight.

Table 2 Converted Crude crystal into purity Mixed solvents for 2nd Weight Purity Total weight extraction (mg) (%) (mg) Chloroform 10: methanol 1 2242 68.6 1538 " 5: " 1 2574 77.3 1990 " 4: " 1 2231 91.5 2041 " 2: " 1 2299 82.4 1895 " 1: " 1 2738 73.3 2007 1,2-dichloroethane 10: " 1 2324 70.5 1638 " 5: " 1 2540 82.0 2083 " 4: " 1 2563 80.2 2056 2: 1 2271 63.5 1442 " 1: 1 1 1919 71.3 1368 trichloroethylene 10: " 1 1913 69.8 1335 " 5: " 1 2414 74.0 1786 " 4: " 1 2515 68.2 1715 " 2: " 1 1822 82.5 1503 " 1: " 1 2000 78.7 1574 Table 2 (cont'd) Converted Crude crystal into purity Mixed solvents for 2nd Weight Purity Total weight extraction (mg) (%) (mg) acetone 10: methanol 1 2072 73.5 1523 5: " 1 2593 72.3 1875 4: " 1 2855 70.5 2015 2: " 1 2263 93.0 2105 1: 1 1 2134 88.3 1884 chloroform 10: phenol 1 2345 63.2 1482 5: " 1 2420 83.6 2006 4: 1 2755 94.5 2603 2: " 1 2642 82.3 2174 " 1: " 1 2015 72.7 1465 acetone 10: " 1 2445 50.7 1240 5: " 1 2721 84.2 2291 4: " 1 2863 91.3 2614 2: " 1 2821 86.4 2437 " 1: " 1 2562 75.6 1937 chloroform 4: ethanol 1 2239 76.3 1708 1,2-dichloroethane 4: " 1 2197 73.7 1619 trichloroethylene 4: " 1 2440 69.8 1703 acetone 4: " 1 2716 81.3 2208 methanol single solvent 2067 55.3 1143 ethanol " 2183 41.5 906 The multhiomycin prepared in Examples 1 and 2 has the same characteristics as the multhiomycin prepared in Journal of Antibiotics Vol. XXIII, No. 5. pp. 231-237 (1970).

For example, the ultra-violet absorption curves in alkaline. neutral and acid methanol, and the infra-red absorption curves, measured in the form of a potassium bromide tablet, are substantially identical.

Claims (10)

WHAT WE CLAIM IS:

1. A method for preparing multhiomycin comprising cultivating Streptomyces sp. 8446 CCI in a culture medium containing assimiable sources of carbon and nitrogen, in which the medium additionally comprises a sulphur-containing carboxylic acid or a derivative thereof

2. A method according to claim 1 in which the sulphur-containing carboxylic acid is a sulphur-containing amino acid.

3. A method according to claim 1 or claim 2 in which the medium contains from 0.0005 to 10% by weight of the sulphur-containing carboxylic acid.

4. A method according to any preceding claim in which, during the cultivation, the pH of the medium is from 4.0 to 9.0.

5. A method according to any preceding claim comprising the additional step of extracting multhiomycin from the medium using a mixture of an alcohol and a halogenated hydrocarbon as extraction solvent.

6. A method according to claim 5 in which the alcohol is an alkanol of 1 to 4 carbon atoms and the halogenated hydrocarbon is a mono- or poly-halogenated saturated aliphatic hydrocarbon.

7. A method according to claim 5 or claim 6 in which the ratio of alcohol to halogenated hydrocarbon is from 1:0.1 to 1:10.

8. A method according to any of claims 1 to 4 comprising the additional step of extracting multhiomycin from the medium using a mixture of an alcohol and a ketone.

9. A method according to claim 1 substantially as described in Example 1 or Example 2.

10. Multhiomycin when prepared by a method according to any preceding claim.